Dhingra, H. et al.


Current Topics in Biotechnology & Microbiology

Narayan Hari


Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology

Edited by

Dr. Harish Kumar Dhingra
Ph.D. (Biotechnology & Molecular Biology) Assistant Professor in Science, Mody Institute of Technology & Science Laxmangarh Distt. Sikar Rajasthan, India

Dr. Prabhat Nath Jha
Assistant Professor Dept. of Biosciences Birla Institute of Technology and Science Pilani, -333 031 Rajasthan, India

Ms. Pratima Bajpai
M. Tech, M.Sc. (Biotechnology) Assistant Professor in Science, Mody Institute of Technology & Science Laxmangarh Distt. Sikar Rajasthan, India


Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology

Acknowledgement First of all, I would like to express my sincere gratitude to the almighty who has made me capable for preparing this manuscript. The completion of this book has come through the overwhelming help that came from many people whom mentioning each one may not be exhaustive. I wish to express my sincere gratitude to all the people who offered their kind help and guidance all the time till completion of this manuscript. I deeply appreciate the efforts of co-editors of this manuscript, Dr. Prabhat Nath Jha, Assistant Professor, Dept. of Biosciences, Birla Institute of Technology and Science, Pilani and Ms. Pratima Bajpai, Assistant Professor in Science, Mody Institute of Technology & Science, Laxmangarh, Distt. Sikar, Rajasthan whose help, guidance and experience, has assisted me in production of this book. I would also like to thank all the authors of different chapters included in this book for providing me the data of this manuscript. My sincere acknowledgements are due to Prof. Dr. S. Baijal, Dean FASC, MITS University, for her kindness and inspiration through out the course. My special gratitude also goes to R. K. Gaur (HOD, Department of Science, FASC, MITS University) for his immense support and help. I would like to pay special thanks to my wife for giving me inspiration for the writing of the manuscript. My heart felt gratitude to all my friends who helped me in completing this project. Last but not least, I thank all my family members for all the support during completion of this manuscript.

Dr. Harish Kumar Dhingra


Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology

Welcome to Current Topics in Biotechnology and Microbiology. Our book includes various chapters related to recent branches of Biotechnology and Microbiology like Nanobiotechnolgy, genetic engineering and agricultural as well as medical microbiology etc. We have tried to include the applied part of these brances, how knowledge of these branches can be helpful to the society, to agriculture and to medical fields etc. Science is experimentation to determine the evidences behind every truth and explore these evidences and the results, therein, for the welfare of the society. Exploitation of the biological agents or their components for the welfare of the human being can be evidenced with the advent of Biotechnology and Microbiology. The rapid pace of discovery in the field of Biotechnolgy and in its associated branches makes it most exciting of all the natural sciences. This field is having application in all fields of life viz. medical, agriculture, industry, environmental quality management and waste management. One of the main goals of this book is to fascinate with the living world and their capabilities, to introduce the dynamic nature of science by conveying the passion for biological research and to excite the students about the opportunities in this field. It was not possible to include all the applications and all the recent topics of Biotechnology & Microbiology; however we will try to include more on that in our future publications.

Dr. Harish Kumar Dhingra Dr. Prabhat Nath Jha Ms. Pratima Bajpai


Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology


Chapter No. I.

Title & Authors Name

Page No.



Jola Dubey, B. N. Tiwary and Sudershan Ganguly II. III.

37-45 46-69





Rajesh Mehrotra, Uthra Balasubramaniyan, Purva Bhalothia, Bhavya Ravi and Sandhya Mehrotra VI.











Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology










Manoj Kumar and Subhash V. Kapre XV



H.Dhingra. et al.segmental areas and release the bacterium in hemocoel where it propagates. Guru Ghasidas Vishwavidyalaya. N. biological. involving morphological. The polymerase chain reaction (PCR) in combination with restriction fragment length polymorphism (RFLP) of internal transcribed spacer (ITS) region of ribosomal DNA. It also protects the cadaver from colonization by other micro-organisms. Chhattisgarh 2 EPN Genomics Laboratory.A. Bilaspur. The infective juveniles (IJs) or dauer juveniles containing bacterial symbiont infect the host through natural openings or inter. Gel diffusion and immunoelectrophoresis techniques is used to differentiate Steinernema isolates.. Email: pandeyjola@rediffmail. New Delhi Corresponding Author: Jola Dubey. have been found to be more useful for detailed information about variation within and among nematode species than PCR-RFLP approaches. GERMANY . phylogenetic relationships and molecular evolution. causes septicemia and kills the host within 24-48 h. Division of Nematology. the DNA sequences of ITS region of ribosomal DNA. B. KG. biochemical and molecular parameters.I. Recently. I. is being used in the descriptions of new species of Steinernema and deducing their phylogenetic relationships with other species.com ABSTRACT: Entomopathogenic nematodes belonging to the families Steinernematidae and Heterorhabditidae have tremendous biocontrol potential against several insect pests. Isozymic patterns may prove to be excellent tool for preliminary identification of newly isolated strains during the routine surveys. Unlike other soil nematodes. These nematodes carry mutualistic bacteria of the genus Xenorhabdus for Steinernematidae and Photorhabdus for Heterorhabditidae. Mitochondrial DNA (mtDNA) is also now widely used in studying speciation. DUDWELLER LANDSTR. the taxonomic study of these nematodes is very complex. These nematodes being compatible with recommended doses of several pesticides and non target microorganisms can suitably be incorporated in the integrated pest management (IPM) schedules. Tiwary1 and Sudershan Ganguly2 1 Department of Biotechnology. 2011 Current Topics in Biotechnology & Microbiology Chapter I BIOPESTICIDAL NEMATODES AND MOLECULAR TECHNIQUES INVOLVED FOR THEIR CHARACTERIZATION Jola Dubey1. 7 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.R.

KG. GERMANY .e. many plant pathogenic micro-organisms have developed resistance against known chemical pesticides [5. et al. Bacteria. fungi and nematodes or their biproducts in disease and pest control. In addition. 2011 Current Topics in Biotechnology & Microbiology INTRODUCTION In recent years. On the other hand. It has been estimated that hardly 0. accumulation in the food chain and extension of their influence to destroy both useful and harmful pests [1]. these are still being used despite their detrimental effects [2. DUDWELLER LANDSTR. investigation in the area of chemical control has been slowed down due to the rising cost of production and sales of the limited fungicides available in the market. It is now feasible to replace many of the highly toxic insecticides and fungicides by bio-pesticides which are safe and non hazardous to the environment. Fungi. H. leaving the remaining 99. Microbial control envisages the use of bacteria. Thus there is an all-round compulsion to go in for bio-rational alternative arsenals which can be ecofriendly and benign to environment. Environmental pollution and toxic hazards due to haphazard use of chemical pesticides have forced the scientists to look for safe and environmental–friendly alternative strategies. long degradation periods. such as the use of microorganisms including entomopathogenic nematodes (EPN) [8]. viruses and nematodes in disease and pest management during the past couple decades has led to the development of several microbial pesticides.3]. Bio-control agents are the best alternative available today. India’s vast potential of exporting agri-products will be successful only when we produce agri-products through minimum use of chemical pesticides or no pesticides. Basic research for over more than 40 years in biology and biochemistry has made it possible to envisage not only how new pesticides may be synthesized but also a completely new approach for the protection of plants using secondary plant products or microorganisms which may be toxic to a specific pest yet harmless to man. The term “entomopathogenic” is being widely used exclusively for steinernematid and heterorhabditid nematodes which kill the insect host quickly i. In the developing countries such as in India. viruses.7].1% of the agrochemicals used in crop protection reach the target pest.Dhingra. within a day or 2 after the 8 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.e. which the poor subsistent farmers are unable to afford.9% to enter the environment to cause hazards to non-target organisms including humans [4]. Research on the use of microorganisms i.6. a large number of synthetic pesticides including insecticides and fungicides have been banned in the western world because of their undesirable attributes such as high toxicity.

EPNs live inside the body of their host. have broad host range. from cultivated fields to deserts. and survivability of different nematode species are varied making them suitable in biological control programs [16]. the pathogenicity. beetles. as their name implies. can be easily applied using standard spray equipments. and mushroom. They infect many different types of soil insects. long term efficacy. EPNs are already being used commercially for managing insect pests of turf grass. and humans.13. India is yet to exploit this area of high biological control potential. compatible with many chemical pesticides.10. GERMANY .g. killing their host within a day or two. safe to the plant and animal health. and so they are designated as endoparasitic.Dhingra. et al. and the environment. active host seeking. These nematodes along with their symbiotic bacteria kill the host within a day or two and ultimately emerge from the insect cadaver in millions to infect the other insect host. 2011 Current Topics in Biotechnology & Microbiology invasion [9. It has also been established that EPNs are safe from the point of view of human health and environment and hence exempted from registration in USA and some other countries [18. They have very high potentialities as bioagents against the insect pests.19]. have a high reproductive potential. KG. H. can be cultured easily in vitro.pests. weevils on 9 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. host searching behaviour. are facultative parasites due to their capability to complete their life-cycle on the symbiotic bacteria without the insect host. EPNs have emerged as suitable biocontrol agents for eco-friendly pest management [12. These are soil-inhabiting. There are several species of EPNs used around the world against a variety of pests in niche markets (e. hydroponics. In some of the developed nations. DUDWELLER LANDSTR. have potential to recycle in environment. entomopathogenic nematodes (EPNs).14. commonly called roundworms. fungus gnats in nurseries.easy mass production. though highly pathogenic.. livestock. and flies. as well as adult crickets and grasshoppers [17]. like pathogens. lethal insect parasitoids that belong to the Phylum Nematoda. EPNs have been found in all inhabited continents and a range of ecologically diverse habitats. Although many other parasitic nematodes cause diseases in plants. However. The nematodes of the families Steinernematidae and Heterorhabditidae. fruit trees. they have chemoreceptors and are motile. including the larval forms of butterflies. mushrooms and other field crops [17]. they are highly virulent.11]. only infect insects and produce harmful effects on insect.15]. EPNs have tremendous biological control potential for managing a wide range of insect pests of crops as well as some household pests. However. due to their several attributes – like parasitoids / predators. moths.

et al. laboratory and field trials and commercialization of EPNs and their symbiotic bacteria resulting in over 2000 publications. host range. biology. DUDWELLER LANDSTR. peach borer moth in apples and carpenter worm in shade trees in China. cutworms. webworms. GERMANY . 2011 Current Topics in Biotechnology & Microbiology ornamentals.24]. EPN sale was only second to Bacillus thuringiensis at 80% [21]. and more comprehensively by Gaugler [15] and Grewal et al. The entomopathogenic activity of steinernematid and heterorhabditid species has been documented against a broad range of insect pests in a variety of habitats [13]. Considerable progress has also been made during the last 20 years on the subject dealing with taxonomy. and fig trees in USA) [20]. rabbits and monkeys) [26]. Some of the important EPN species belonging to Steinernema and Heterorhabditis with their original localities and sources of isolation are listed in the Table 1. They have been used inundatively in a number of high value cropping systems [25]. billbugs and mole crickets. reduction of pesticide residues in food. cranberries. When environmental benefits including safety for humans and other non-target organisms. ornamentals and blue berries. ecology. KG. production. This aspect was briefly reviewed by Friedman [17].19]. 10 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. scarabs of turf. application technologies. These nematodes have been produced in large scale in various countries for over 10 years and large numbers of production workers have been exposed without any adverse effects being recorded. The Environment Protection Agency (EPA) in the India. Kaya and Gaugler [13]. Recently. Under Heterorhabditidae. and increased biodiversity in managed ecosystems are taken into account. out of 13% bio-insecticide sale in industrialized countries. genetics. Akhurst [22]. increased activity of other natural enemies. there is only one genus – Heterorhabditis with only 18 known species. with 67 species in the former and only one species in the latter [28] have been characterized. Australia and many European countries has exempted EPNs from registration [18. termites in wooden articles and trees. [23. H. the advantages are numerous.Dhingra. there are two genera listed under Steinernematidae – Steinernema and Neosteinernema. USA. citrus and bananas. earthworms and other non-target organisms including plants have shown that the EPNs are harmless and of course nonpolluting [27]. strawberries. Various tests against mammals (mice. Till date.

feltiae (Filipjev 1934) Wouts et al. USA Central Province.1982 10. Kenya Germany Hamikita. 1994 S. Argentina Waslaco. 1994 9. intermedium (Poinar 1985) Mamiya 1988 12. abbasi Elawad et al. 1997 13. affine (Bovien 1937) Wouts et al. ritteri de Doucet & Doucet 1990 S. S. brevicaudis Liu 1994 4. USA Jeromesville. 21. S. NewZealand Sultanate of Oman Denmark Central Russia Strazilovo. S. kushidai Mamiya 1988 15. 1992 6. India Seaside. marelata Liu & Berry 1996 7. DUDWELLER LANDSTR. H. 1997 2. 1982 6. 22. GERMANY . China Western Cuba Denmark New Jersey. Florida. S. KG. India ORIGINAL SOURCE Graphognathus sp. Soil Soil Cydia pomonella Soil Soil Soil Agrotis feltiae Popillia japonica Soil Soil Cephaleia abietis Anomala cuprea Soil Soil Neocurtilla hexadactylla Soil Soil Soil Soil Soil Scapteriscus vicinus Soil 11 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 19. S. H. H.Dhingra.1982 3. 1987 8. neocurtillae Nguyen & Smart 1992 18. 1997 8. argentinensis Stock 1993 2. megidis Poinar et al. S. USA Auckland. Argentina Rivera. 1991 7. 1982 4. USA Puerto Rico Cordoba. South Australia Fujian Province. bacteriophora Poinar 1976 3. Guangdong. ceratophorum Jian et al. S. USA Oregon. riobrave Cabanillas et al. arenarium Wouts et al. USA Coimbatore. et al. S. 20. Uruguay IARI Farm. longicaudatum Shen & Wang 1991 16. thermophilum Ganguly & Singh. S. 1994 5. glaseri (Steiner 1929) Wouts et al. 1982 11. Oregon. H. H. S. carpocapsae (Weiser 1955)Wouts et al. S. 2011 Current Topics in Biotechnology & Microbiology Table 1: EPN species of Steinernema and Heterorhabditis with their original localities and sources of isolation NEMATODE SPECIES Heterorhabditis Poinar 1976 1. Texas. karii Waturu et al. S. H. S. Delhi. puertoricense Roman & Figueroa1994 S. S. rarum (de Doucet 1986) Mamiya 1988 S. USA South Carolina. 24. Yugoslavia Czechoslovakia China Jining Province. 23. H. cubanum Mracek et al. Heliothis punctigera Soil Soil Soil Soil Popillia japonica Heteronychus arator Soil Bibio sp. orgonense Liu & Berry 1996 S. indica Poinar et al. 1997 17. USA Cordoba. S. 1995 5. zealandica Poinar 1990 Steinernema Travassos 1927 1. caudatum Xu et al. S. scapterisci Nguyen & Smart 1990 S. bicornutum Tallosi et al. S. S. 2000 ORIGINAL LOCALITY Rafaela. H. krausei (Steiner 1923) Travassos 1927 14. hawaiiensis Gardener et al. H. monticolum Stock et al. Argentina Brecon. China Republic of Korea LaCrosse. China Hawaii.

et al. The cycle begins with an infective juvenile. H. and reproduce for several generations inside the cadaver. Although not closely related. After entering an insect. When food resources in the host become scarce. received from the internal host environment [30. KG. Steinernematid infective juveniles may become males or females.31. having been isolated from every inhabited continent from a wide range of ecologically diverse soil habitats including cultivated fields. and even ocean beaches. 2011 Current Topics in Biotechnology & Microbiology SYSTEMATIC POSITION OF EPN: Phylum Class Order Superfamily Family : Nematoda : Secernentea : Rhabditida : Rhabditoidea : Steinernematidae. 12 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 2). carrying with them an inoculum of mutualistic bacteria. forests. both share similar life histories (Poinar. The nematodes provide shelter to the bacteria.1. After about a week. entomopathogenic nematodes were recovered from 22% of sites sampled [29]. deserts.Dhingra. whereas heterorhabditids develop into self-fertilizing hermaphrodites with later generations producing two sexes. which. DUDWELLER LANDSTR. GERMANY .11] (Figs. Together. hundreds of thousands of infective juveniles emerge and leave in search of new hosts. 1993). Heterorhabditidae HABITAT Steinernematid and heterorhabditid nematodes are microscopic soil dwelling organisms. The bacteria of the two genera Xenorhabdus and Photorhabdus. along with steinerernematides and heterorhabditids. whose only function is to seek out and infect new hosts. respectively—cause host mortality within 48 hours. LIFE-CYCLE The life-cycles of the genera grouped under Heterorhabditidae and Steinernematidae are particularly well known. the adults produce new infective juveniles adapted to withstand the outside environment. grasslands. They are ubiquitous. phylogenetically. In New Jersey. infective juveniles release an associated mutualistic bacterium. the nematodes and bacteria feed on the liquefying host. kill the insect host and provide nutrients to the nematode. in return.

KG. whereas insects killed by heterorhabditids become red and the tissue assumes a gummy consistency (Fig. may be freeliving.. Infective juveniles of entomopathogenic nematodes range from 0. Disturbed nematodes move actively.g.5 mm in length and can be observed with a hand lens or microscope after separation from formulation materials. whereas nearly transparent nematodes are often active but possess low powers of infection. Infective juveniles are the only free-living and therefore environmentally tolerant stage. Figure1: Life cycle of Steinernema and Heterorhabditis [30] 13 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. gentle heat) to move before assessing viability. lack of movement is not always a sign of mortality. Steinernema carpocapsae. glaseri. nematodes may have to be stimulated (by e. Nematodes found within such cadavers tend to be free-living soil saprophages. however sedentary ambusher species (e. In short. lacking appendages. colourless.. Good quality nematodes tend to possess high lipid levels that provide a dense appearance. S. Low temperature or oxygen levels inhibit movement of even active cruiser species (e. unsegmented. A dim luminescence given off by insects freshly killed by heterorhabditids is a foolproof diagnostic for this genus (the symbiotic bacteria provide the luminescence). H.g. Heterorhabditis bacteriophora).Dhingra. predaceous or parasitic. GERMANY .5 to 1.g. S. Black cadavers with associated putrefaction indicate that the host was not killed by entomopathogenic species. scapterisci) in water soon revert to a characteristic "J"shaped resting position. DUDWELLER LANDSTR. probes. acetic acid. 3). 2011 Current Topics in Biotechnology & Microbiology APPEARANCE Nematodes are roundworms. Insects killed by most steinernematid nematodes become brownish-yellow. et al.

highly adapted. KG. The former group has been referred to as “ambusher”. The nematodes attack a far wider spectrum of insects in the laboratory where host contact is assured. S. and no ecological or behavioural barriers to infection exist [34]. S. DUDWELLER LANDSTR. responds strongly to longrange host chemical cues. carpocapsae and S. H. In laboratory tests. Some nematode species may search for hosts at or near the soil surface (e. bacteriophora and S. environmental conditions are optimal. whereas others are adapted to search deeper in the soil profile (e. et al. GERMANY . This rapid mortality permits the nematodes to exploit a range of hosts that spans nearly all insect orders. scapterisci). a spectrum of activity well beyond that of any other microbial control agent. glaseri). They have been used inundatively in a number of high value cropping systems [25].g.g.. H. The nematode-bacterium complex kills insects so rapidly that the nematodes do not form the intimate. which remains nearly sedentary while waiting for the mobile surface dwelling hosts [35]. The latter group has been referred to as “cruiser” which is highly mobile. carpocapsae alone infected more than 250 species of insects from over 75 families in 11 orders [33]..Dhingra. 2011 Current Topics in Biotechnology & Microbiology Figure 2: Enmasse production of nematode progeny from the insect cadaver HOST RANGE The entomopathogenic activity of steinernematid and heterorhabditid species has been documented against a broad range of insect pests in a variety of habitats [13]. host-parasite relationship characteristic of other insect nematode associations. and is therefore best adapted to find sedentary hosts [36] 14 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.

. indica. this list is certain to expand. EPNs are useful against a large number of insect pests. Tumkur. As field research progresses and improved insect-nematode matches are made. (B) Insect larvae killed by Heterorhabditis Because the symbiotic bacterium kills insects so quickly. When considered as a group of nearly 30 species. However. however. (Lepidoptera: Pyralidae) and formulation of these nematodes have been developed after three years of rigorous research by Multiplex Biotech Pvt. or corn rootworms. nematodes have yet to be found which are effective against several of the most important soil insects. to increase 15 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. including wireworms. the labour. et al. The in vivo process. thermophilum and S. IN VIVO PRODUCTION AND FORMULATION TECHNOLOGIES It is far too expensive to rear EPNs by in vitro media as they require separate media source for nematodes growth and their associated bacterium with suitable fermentor. 2011 Current Topics in Biotechnology & Microbiology A A B Figure 3: (A) Insect larvae killed by Steinernema. S. equipment. H. Consequently. with some species being quite narrow in host specificity. Field host range is considerably more restricted. fire ants. carpocapsae. Ltd. grape phylloxera. in India the first successful and commercial scale of mass production of EPN species. and material (insect diet) costs increase as a linear function of production capacity. many of which are listed in the Table 2. lacks any economy of scale. Regrettably. there is no intimate hostparasite relationship as is characteristic for other insect-parasitic nematodes. Karnataka. however. GERMANY . This is a pioneered organization commercializing these entomopathogenic nematodes reared in vivo. glaseri on larvae of greater wax moth. EPNs are lethal to an extraordinarily broad range of insect pests in the laboratory. S. India.Dhingra. DUDWELLER LANDSTR. H. Galleria mellonella L. KG.

flower. fungicides. et al. Ltd. relatively little is understood about their biology in natural and managed ecosystems [44]. so native nematodes are often baited using Galleria mellonella.g. The latest formulation developed by Multiplex Biotech Pvt. fertilizers. Some pesticides act synergistically with EPNs and therefore improve nematode efficacy in inundative applications [41. 39. APPLICATIONS Although the biological control industry has acknowledged EPNs since the 1980s.1 %). The company do have products of EPNs that are currently marketed satisfactorily in the name of ‘Soldier” recommended as soil application for soil dwelling insect pests and “Bouncer” as foliar application for leaf. because in a laboratory. The nematode shelf-life of 2 to 3 months was achieved at room temperature (27-28°C) with maximum of 80 % survival by increasing the nematodes metabolic rate by mixing silica powder (0. DUDWELLER LANDSTR.. contact with a host is assured and environmental conditions are ideal. surfactants (e.42]. Galleria mellonella are most commonly used host insect to mass produce these beneficial nematodes because of its rich nutrient source available in body and easy to multiply in economical semi-synthetic diet source containing wheat and corn flour based media. Infective juveniles are tolerant of short exposures (2-8 h) to most agrochemicals including herbicides. About 2. there are no 16 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. and more than half of the natural hosts for recognized Steinernema and Heterorhabditis species remain unknown [45]. horticulture and forest insect pests. Information is lacking because isolates of naturally infected hosts are rare. is comprised of hydro gel based semi solid cream with IJs suspension containing 1 million nematodes in a polythene pouch that can be readily mixed in a water spray tank without blocking the nozzles.Dhingra. fruit. pods. Nematode-host interactions are poorly understood. and soil amendments that may interact with nematodes. a lepidopteran that is highly susceptible to parasitic infection. EPNs are often applied to sites and ecosystems that routinely receive other inputs including chemical pesticides.5 billion infective juveniles (IJs) of EPN are recommended to treat one hectare where the insect pests are highly infested on any crop [37]. Laboratory studies showing wide host ranges for EPNs were often overestimates. KG. acaricides. GERMANY . wetting agents). stem and borer insect pests against agriculture. Fifty such pouches are recommended to spray in one acre against the highly infested crop. Nematodes are also compatible with most inorganic fertilizers when they applied inundatively [43]. 2011 Current Topics in Biotechnology & Microbiology their virulent and survival. and insecticides [38.. H. 40].

Comparing the life-histories of nematodes and target pests can often explain such failures [46]. bacteriophora. bacteriophora S. carpocapsae H. S. KG.46]. Webworm The lack of knowledge about nematode ecology has resulted in unanticipated failures to control pests in the field. bacteriophora S. DUDWELLER LANDSTR. S. feltiae H. scapterisci H. because nematodes are highly sensitive to UV light and desiccation [47]. carpocapsae S. GERMANY . bacteriophora S. Each nematode species has a unique array of characteristics. parasitic nematodes were found to be completely ineffective against blackflies and mosquitoes due to their inability to swim [47]. riobrave H. carpocapsae Mushrooms Ornamentals Sciarids Root weevils Wood borers Fungus gnats Turf Scarabs Mole crickets Billbugs Armyworm. Efforts to control foliage-feeding pests with EPNs were equally unsuccessful. the broad host range initially predicted by assay results has not always translated into insecticidal success. Cutworm. H. including different environmental tolerances. bacteriophora. H. H. carpocapsae S. dispersal tendencies. et al. Increased knowledge about the factors that influence EPN populations and the 17 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. For example. 2011 Current Topics in Biotechnology & Microbiology “ecological barriers” to infection [13. carpocapsae.Dhingra. carpocapsae S. and foraging behaviors [47]. Table 2: Current use of nematodes as biological insecticides COMMODITY Artichokes Berries Citrus Cranberries INSECT PEST Artichoke plume moth Root weevils Root weevils Root weevils Cranberry girdler NEMATODE SPECIES S. Therefore. riobrave. feltiae H. S. megidis S. bacteriophora.

g. Entomopathogenic nematodes should be applied at the first sign that a pest population is initializing to cause damage. Thus. Also.) but a large knowledge base has been developed to support their use. different species require different screen sizes. Accelerated implementation of nematodes into IPM (Integrated pest management) systems will require users to be more knowledgeable about how to use them effectively. et al. and are incompatible with several agricultural chemicals. nematodes are effective within a narrower temperature range than chemicals. mammalian toxicity. H. Nematode-based insecticides may be inactivated if stored in hot vehicles. it is crucial to apply them at the optimum environmental conditions needed for their better survival. nematodes are constrained by being living organisms that require specific conditions to be effective. resistance. 2011 Current Topics in Biotechnology & Microbiology impacts they have in their communities will likely increase their efficacy as biological control agents.. Entomopathogenic nematodes are remarkably versatile in being useful against many soil and cryptic insect pests in diverse cropping systems. soil is moist and direct sunlight is minimal. and are more impacted by suboptimal soil type. both before and after application because they need moist conditions to prevent desiccation and aid with movement to find hosts. the best results are obtained when the relative humidity is high. groundwater pollution. cannot be left in spray tanks for long periods. and irrigation frequency [48].Dhingra. it is best to irrigate the target site. All of these factors help to prevent the nematodes from drying out and increase their survival and virulence. Nematodes should be reapplied on 7 days intervals if damage continues. chemical insecticides are less constrained. ambient temperature is neither extremely hot or cold. Reapplying nematodes depends on the success of the first nematodes released. etc. thatch depth. KG. unused nematodes cannot be applied the following year. yet are clearly underutilized. Similarly. desiccation or ultraviolet light rapidly inactivates insecticidal nematodes. 18 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. DUDWELLER LANDSTR. moisture and soil type and percentage of living nematodes actually released during the first application. soil temperature is between 10 to 35°C. Therefore. GERMANY . Their survivorship and success are based on environmental condition. Certain species cannot be applied with highpressure application equipment. Like other biological control agents. In order to ensure maximum effectiveness. Chemicals also have problems (e.

an important consideration in commercial use of these microbes as bio. GERMANY .53]. bovieni with S. 2011 Current Topics in Biotechnology & Microbiology NEMATODE-BACTERIUM SYMBIOSIS These steinernematids and heterorhabditids have symbiotic relationships with the bacteria belonging to the genera Xenorhabdus and Photorhabdus. respectively.g. classified under the family Enterobacteraceae. Inhibition of the host’s antibacterial proteins. penetration into host’s haemocoel and. KG. indica with S.55] consequently inhibiting growth of other pathogens. To maintain monoxenic condition. X. X. poinarii with S. At this immuno-compromised state. which is required for development of the symbiotic 19 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.14. Breaking down the host tissues to serve as nutrient source for nematode. DUDWELLER LANDSTR. anaerobic rods. nematophilus with S.pesticides [12. bacterium synthesizes and secretes broad spectral antibiotics as well as narrow spectral bacteriocins [54. Excellent mutualism occurs between the nematode and the bacterium [49. 51].13]. On the other hand. These bacteria are known to exhibit peculiar behavior of phase transition [50.13. The bacterium needs the nematode for protection from the external environment. The bacterium replicates logarithmically in the insect rich haemolymph. the phase one produces better conditions for nematode reproduction. both phases are equally pathogenic to the insects. Serving as a food source and. then causes a suppression to the host immune capacity against both the symbiotic nematodes and the bacteria themselves by inhibiting phospholipase A of the insect host [52. as for e. The monoxenic state leads to lethal septicemia of the target insect. which are species specific. glaseri and Photorhabdus luminescens with Heterorhabditis bacteriophora. carpocapsae. Creating a suitable environment for nematode development and preventing the infection by secondary microorganisms. The nematode is dependent on the bacterium for Killing its host.Dhingra.15]. These bacterial symbionts are gram negative. thermophilum. feltiae. NEMATODE-BACTERIUM COMPLEX AND ITS MODE OF ACTION After entering through insect natural openings the EPNs deliver the symbiotic bacteria from their intestine to the haemocoel of the insect hosts [13]. H. 31. X. 13]. The nematode-killed insects are flaccid and do not have a putrid odor because the mutualistic bacteria produce antibiotics which prevent the growth of secondary microorganisms [31. X. Although. the infected insect can be susceptible to other saprophytic pathogens. et al.

et al. Presently. Japan. Some protocols for biosystematics of these nematodes have also been developed through multi-national COST (European cooperation in science and technology) Project involving working groups from 17 countries. there are more than hundred companies in the world. France. The mechanism of antibacterial activity has not been elucidated. China. Remarkable progress has been made on the techniques related to mass culturing.29]. RESEARCH SCENARIO The biological control potential of entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae is being exploited for managing the insect pests of crops as well as household pests in several parts of the world [62]. Argentina. GERMANY . guanosine-3 pyrophosphate (ppGpp) especially in indole compounds [60]. Canada and Europe. Soviet Union. but primarily speculated as inhibition of RNA and protein synthesis by an accumulation of the regulatory nucleotide. dithiolopyrrolones and xenocoumacins are the antibacterial compounds synthesized by Xenorhabdus spp [59]). mostly in USA. about 67 species of Steinernema and 18 species of Heterorhabditis have been described from different parts of the world [65.15]. and remarkable progress has also been made on the taxonomy and metabolites of the symbiotic bacteria associated with Steinernema and Heterorhabditis. New Zealand. including USA. biological (life-cycle and interbreeding tests). DUDWELLER LANDSTR. 2011 Current Topics in Biotechnology & Microbiology nematodes in the cadaver [56]. This antibiotic aspect of the entomopathogenic bacteria reflects the potential medical and agricultural importance of their metabolic products [61]. etc. biochemical and molecular tools (Base composition of rDNA) have been emphasized for the description and identification of species (67.Dhingra. ornamentals and some fruit crops [12. indoles [58].69]. Xenorhabdus and Photorhabdus [57]. The nematode-bacterium relationship is species-specific. formulating and marketing EPNs for managing insect pests of turf grass. Italy. Canada. There are now about 20 species of Xenorhabdus associated 20 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.66.64]. KG. wherein use of morphological. The bioefficacy of these nematodes as foliar spray as well as soil application against different insect pests have been demonstrated in at least in 44 trials in 12 countries. [15]. mushrooms. England. Various kinds of antibiotics against bacteria and fungi are synthesized and secreted during the bacterial cultures growth of two related entomopathogenic bacteria. H. formulations and application technologies of these nematodes [63. which are mass producing. Till now. Among them.

Some efforts have also been made to identify the biochemical and molecular basis of nematode-bacterium-insect relationships. S.74]. and other native strains [87-90]. MOLECULAR TECHNIQUES INVOLVED FOR CHARACTERIZATION Various biochemical techniques which have been used in the molecular taxonomy of Entomopathogenic nematodes are: PROTEIN ELECTROPHORESIS Species-specific isozymic patterns revealed through polyacrylamide slab gel electrophoresis have been found to be useful for differentiating the species of Meloidogyne [84. feltiae. and also exploiting these for developing insect resistant transgenic plants and biopesticides [45.57. carpocapsae 21 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. biotoxins and the genes involved. esterase and lactate dehydrogenase in different cultures and life stages of S. DUDWELLER LANDSTR. have been found to be more useful for detailed information about variation within and among nematode species than PCR-RFLP approaches (80-83].85].70. carpocapsae. feltiae. This biochemical technique was first applied by Herman and Jackson [86] wherein they examined the differences in alkaline phosphatase. the DNA sequences of internal transcribed spacer (ITS) region of ribosomal DNA. H. have been used in the descriptions of new species of Steinernema and deducing their phylogenetic relationships with other species [7779]. understanding their biology and evolution.Dhingra.73. 72]. Sha [87] observed qualitative differences in esterase patterns of S. et al. carpocapsae. The molecular approaches are very useful for differentiating the species. carpocapsae. Recently. glaseri and S. GERMANY . especially in the identification of S. quarantine and proprietory protection purposes [75]. later several workers studied isozymic profiles of different enzymes for resolving the taxonomic anomalies in steinernematids. KG. The polymerase chain reaction (PCR) in combination with restriction fragment length polymorphism (RFLP) of ITS region of ribosomal DNA. S. glaseri and a group of 3 isolates of S. identification of species and strains for registration. Molecular approaches have been applied in the taxonomy of not only entomopathogenic nematodes [76] but also their symbiotic bacteria [58]. 2011 Current Topics in Biotechnology & Microbiology with different species of Steinernema and 4 species of Photorhabdus with Heterorhabditis (10. subspecies or strains. glaseri and S. The common esterase patterns of 3 isolates of S.

DUDWELLER LANDSTR. non-specific esterase. acid phophatase. Ganguly and Pandey [89] and Ganguly et al. However. H. KG. GERMANY . thermophilum. [92] differentiated the Steinernema species on the basis of total protein patterns. Numerous genetic and systematic studies have focused on mtDNA. feltiae isolated by Stanuszek [91]. These isolates were analyzed for sequence variation. Its High rate of evolution permits comparisons of specific or subspecific taxa [97]. Similar results were obtained on the basis of morphological data as well as interbreeding tests. Kozodoi et al. Scientists also determined the complete sequence of the mitochondrial DNA of the entomopathogenic nematode Steinernema carpocapsae and analyzed 22 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The dissimilarity matrix and cluster analysis of these isolates based on 10 enzymes could categorize them into 3 –8 groups. phylogenetic relationships and molecular evolution [95]. 2011 Current Topics in Biotechnology & Microbiology clarified precisely the species status of S. Later on. Some ontogenetic variations in alkaline phosphatase were observed only in S. feltiae and 2 isolates of S. S.5 &6). Among the few mitochondrial genes investigated in other nematodes. Some differences were also noted between juveniles and adult stages within S. females and males). carpocapsae and S. anomali as the isozyme pattern varied in its different life-stages (IJs. glaseri. the taxonomic significance of total protein patterns. using both the enzymes (Fig 4. Divergence in mtDNA sequences may accumulate during a relatively short evolutionary time. Some more studies were done to obtain partial DNA sequences of ND4 gene from some EPN isolates collected from different regions of the world. a period in which traditional genetic and morphological markers might remain unchanged [96]. Akhurst [93] studied the isozyme patterns of 23 isolates of different species of Heterorhabditis. MITOCHONDRIAL DNA SEQUENCE ANALYSIS Mitochondrial DNA (mtDNA) is now widely used in studying speciation. non-specific esterase and alkaline phosphotase. there are currently not much published data on mtDNA sequences in Heterorhabditis species. alkaline phosphatase. [94] have demonstrated the utility of esterase. carpocapsae. Phylogenetic relationships among these isolates were inferred from the ND4 gene sequences and compared with the relationships inferred from the ITS1 region of the ribosomal DNA gene [99]. et al. the ND4 gene appeared to be a very useful marker for population genetics applications [98]. catalase and SOD isozymes of infective juveniles for the differentiation of some of the species of Steinernema. as evident from the species-specific enzyme phenotypes obtained for S. malate dehydrogenase have been also worked out at least to differentiate the morphologically similar species of Steinernema.Dhingra.

Almost the complete genome has been amplified in one fragment with long PCR and sequenced using a shotgun strategy (Montiel et al. thermophilum.Dhingra.. et al. DUDWELLER LANDSTR. c:Steinernema simkayai. carpocapsae. 23 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. c: S. b: S. riobrave (IARI-EPN-gj1). GERMANY . 2006) Figure 4: Esterase profiles of Infective Juveniles of Steinernema species. KG. H. glaseri. e: Steinernema sp. b: S. a: S. e: IARI-EPN-mg1. siamkayai. (strain IARI-EPN-mg1). glaseri. d: S. d: S. a: Steinernema thermophilum. 2011 Current Topics in Biotechnology & Microbiology its structure and composition as well as the secondary structures predicted for its tRNAs and rRNAs. f: IARI-EPN-gj1 Figure 5: Catalase isozymic profiles of infective juveniles of six Indian species of Steinernema. f: S. carpocapsae.

24 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 2011 Current Topics in Biotechnology & Microbiology Figure 6: Superoxide dismutase isozymic profiles of infective juveniles of six Indian species of Steinernema. S. a: S. e: Steinernema sp. IMMUNOLOGICAL TECHNIQUES Gel diffusion and immuno-electrophoresis techniques. d: S. siamkayai. using antisera raised against nematode homogenates injected in to rabbits. KG. glaseri. f: S. (strain IARI-EPN-mg1). DUDWELLER LANDSTR.Dhingra. c: S. In the absence of supporting morphological or cross breeding data. feltiae. carpocapsae. DNA sequence analysis was applied to Heterorhabditis isolates from North America by Curran and Webster [102]) and three genotypic groups based on RFLDs in repetitive DNA and rRNA gene. Steinernema spp. were identified. it was not possible to determine whether these groupings represented species or intraspecific forms. GERMANY . and Heterorhabditis isolates [101]. H. thermophilum. et al. riobrave (IARI-EPN-gj1). were capable of discriminating three isolates of Steinernema [100]. b: S. glaseri. DNA SEQUENCE ANALYSIS DNA sequence divergence between species of entomopathogenic nematodes was first studied by visualization of repetitive DNA restriction fragment length differences (RFLDs) in total genomic DNA of S.

a: S. H.chh1. HhaI. they recommended a combination of four restriction enzymes (Alu I. As per the protocol. Hinf I and Rsa I to differentiate some of the species of Steinernema and found AluI yielded polymorphism that could distinguish 8 out of 9 species (Figs. Kpn I. have been found to be more useful for detailed information about variation within and among nematode species than PCR-RFLP approaches [80. 8 & 9). KG. Sal I. PCR reactions should be performed using ITS primers of Vrain et al [104] and digested with the 17 restriction enzymes (AluI. the DNA sequences of internal transcribed spacer (ITS) region of ribosomal DNA. 2011 Current Topics in Biotechnology & Microbiology Hominick et al. DUDWELLER LANDSTR. [103] developed protocols and techniques under a multi-country project on entomopathogenic nematodes for the use of molecular data for identifying the species. Sau3A I. Hha I. Rsa I. Dde I. For screening the new isolates.105. then all the 17 enzyme profiles should be obtained.81. HinfI. Sau96 I and Xba I). glaseri b: IARI-EPN. c: IARI-EPNmg1 with 4 different restriction enzymes: AluI. et al. Hpa II. Hinf-I. BSt O I. 7. Pvu II. RsaI.83] Figure 7: RFLPs yielded by digestion of . Hae III. GERMANY .Dhingra. Recently. Pst I. 25 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Dubey et al. EcoR I. RFLPs should be obtained from total genomic DNA [77]) for knowing any intra population variations present in type sample. Dde I.82. Hha I. [83] used Alu I. Hinf I and Rsa I) and if the results do not tally with the database of the protocol. Hind III..

HhaI. DUDWELLER LANDSTR. HhaI Figure 9: RFLPs yielded by digestion of – a: S. KG. c: IARI-EPNdl2 with 4 different restriction enzymes: AluI. H. carpocapsae b: IARI-EPN -gj1. c: IARIEPN-gj2 with 4 different restriction enzymes: AluI. HinfI. et al. RsaI. b: IARI-EPN-dl1. GERMANY . thermophilum.Dhingra. 2011 Current Topics in Biotechnology & Microbiology Figure 8: RFLPs yielded by digestion of – a: S. HinfI. RsaI. 26 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.

sequencing of ITS region of rDNA and its alignment with the closely related species. Till to date. which are yet to be identified. and this will have far reaching impact on all round developments in research for exploiting these bioagents in integrated pest management. less time-consuming and less costly and therefore can be widely applied for identifying the samples obtained from large surveys. catalase and superoxide dismutase isozymic profiles from infective juveniles. Presentlly. The isozyme analysis technique is simpler. Conclusively.Dhingra. et al. there are 67 valid species of Steinernema. The accurate identification of the strains must be done before taking up any further research on them. DUDWELLER LANDSTR. integration of morphological data together with DNA analysis is the best approach for the biosystematics of entomopathogenic nematodes and for accurate identification of the species. Therefore. immuno-electrophoresis. and the number of researchers studying them has also increased considerably during the last one decade. enzyme analysis and DNA (genomic and mitochondrial) based molecular biological techniques. supplemented with morphological details. Numerous genetic and systematic studies have also focused on mtDNA. has been found to be useful in describing the species. GERMANY . The ribosomal genes flanking this region are highly conserved allowing the construction of primers that enable the PCR amplification of the highly variable ITS region between them. Morphological features of infective juveniles and males offer the best differentiating characters but the identification of these nematodes by these standard morphological criteria is rarely straightforward and for this reason scientists turned to gel diffusion. there are several strains isolated from different parts of the world. Besides. 2011 Current Topics in Biotechnology & Microbiology CONCLUSION The potential of entomopathogenic nematodes (steinernematids and heterorhabditids) for insect control has been now fully realized and well documented. one species of Neosteinernema and 18 species of Heterorhabditis. Polymerase chain reaction made it possible to identify nematodes using small sample size and now Internal transcribed spacer region (ITS) of ribosomal DNA repeat unit is preferably used for molecular taxonomy as well as identification and characterization. there is an urgent need to have strong biosystematics base in every country for the identification of entomopathogenic nematodes. Sequence variation in this region yields many RFLPs which can be used for identification. KG. proved useful for preliminary screening and differentiation of this strain from other species of the genus Steinernema. H. 27 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Combination of esterase.

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prebiotic substance may be added which stimulates growth of beneficial bacteria. GERMANY . MITS University. FASC .Dhingra. FASC. Keywords. Y shaped morphology in stools of children with diarrhea. INDIA 2 ACTREC. Extensive research is required for exploration and optimization of microorganism which can be exploited as probiotics to improve health. INDIA E. Sikar-332311. Historically. He suggested that the dependence of the intestinal microbes on the food enable them to adopt measures to modify the flora in our bodies and to replace the harmful microbes by beneficial microbes [1]. on the contrary. Assistant Professor of Biotechnology. he suggested that these bacteria could be administered to 37 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Sikar-332311. a French paediatrician. et al. abundant in healthy children [2]. was made by Eli Metchnikoff. Probiotics. These “bifid” bacteria were. Bifidobacter and certain other bacteria can be used as dietary supplement. DUDWELLER LANDSTR. Probiotics consisted of Lactobacillus. Navi Mumbai-410210 * Coressponding author: PratimaBajpai.mail:bajpai. Tata Memorial Centre. while working at the Pasteur Institute. H. In essence. probiotics may reduce level of pathogenic microorganism. KG. Lactobacillus and Bifidobacterium INTRODUCTION: The term “probiotic” refers to the bacteria which beneficial effects to humans and animals. Use of probiotics has been found to reduce risk of cancer and other serious ailments. administration of which improves microfloaral balance of gastrointestinal tract. Based on his observation and analysis. Department of Science. Rajasthan. Henry Tissier. 2011 Current Topics in Biotechnology & Microbiology Chapter II PROBIOTICS: POTENTIAL HEALTH TOOL *1 1 Pratima Bajpai.MITS University.pratima@rediffmail. noticed scanty number of bacteria characterized by a peculiar. Lakshmangarh. Kharghar. strengthen immune system. 2Anamika Pandey and 1Harish Dhingra Department of Science. the original observation of the positive effect exerted by some of the selected bacteria. Rajasthan. In addition. reduce level of toxic substances. assist in digestion of lactose and supply vitamins.com ABSTRACT: Probiotics are refered to beneficial microbes. Lakshmangarh. the Russian born Nobel Prize winner.

Replenishing the flora in our gut on a regular basis. even if the word "probiotic" was not coined until 1960. It is generally accepted that antibiotics deplete the friendly bacteria in our intestines which perform many important functions. KG. redefined the word as "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance" [4]. nonpathogenic microorganisms that may interact with gastrointestinal and vaginal microflora. to name substances produced by microorganisms which promoted the growth of other microorganisms [3]. In order to point out the microbial nature of probiotics. Investigations into different modes of administering probiotics may expand their applications in functional foods. when applied to animal or man. and poor digestion are also factors in the depletion of these vital factors. They provide many health benefits. beneficially affects the host by improving the properties of the indigenous flora”. et al. faulty diet. Gastric survival rates of probiotics are estimated at 20 to 40 percent. These bacteria are normal inhabitants of the large and small intestines. important and effective strategy to protect our overall health. Stress. but probably not the last definition is "live microorganisms. Although there are hundreds of different strains of bacteria that live in the digestive tract. which when consumed in adequate amounts. and eczema. Probiotics are live. respiratory allergies. 38 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. beneficial bacteria are generally placed in two categories: Lactobacillus acidophilus and Bifidobacterium bifidum. “a viable mono or mixed culture of bacteria which. A similar definition was proposed by Havenaar and Huis in 't Veld [5]. DUDWELLER LANDSTR. Clinical studies indicate that certain probiotics may be useful in treating some diarrheal disorders. Dietary sources of probiotics are usually found in dairy products such as Yogurt. confer a health effect on the host" [6]. is a simple. and particularly when undergoing a course of antibiotics. A more recent. GERMANY . The works of Metchnikoff and Tissier were the first to make scientific suggestions concerning the probiotic use of bacteria. as well as in controlling inflammation and reducing the risk of Candidal vaginitis and colon cancer. 2011 Current Topics in Biotechnology & Microbiology patients with diarrhea to help restore a healthy gut flora.Dhingra. which contains two groups of intestinal bacterial species of Lactobacilli and Bifidobacteria of probiotic bacteria. H. with the main obstacles to survival being gastric acidity and the action of bile salts. Probiotics are friendly and beneficial bacteria.

niacin. they may inhibit Candida albicans. et al. Acidophilus assists the body’s natural process of digestion. According to the National Center for Complementary and Alternative Medicine (NCCAM) and defined by the 39 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. In doing so. • Lowering levels of toxic by-products: Harmful bacteria can produce toxins. • Assist in digestion of lactose: Clinical studies have shown that L. such as indole. Therefore. These compounds include hydrogen peroxide. H. Bifidus aggressively attach themselves to the walls of the colon. VITAL ROLE OF PROBIOTICS • Probiotics reduce the levels of harmful bacteria such as E. a prebiotic aims at stimulating the growth of one or a limited number of the potentially health promoting indigenous microorganisms thus modulating the composition of the natural ecosystem. Acidophilus may inhibit pathogens by lowering the pH in the intestines. bacteria and the parasite Giardia lamblia. DUDWELLER LANDSTR. coli and Salmonella by producing metabolic end-products that inhibit or antagonize them. GERMANY . fungi and yeasts---that can be seen only under a microscope and that are often referred to as "healthy" or "good" bacteria. The production of organic acids effectively lowers intestinal pH to a level that is beneficial to good bacteria and destructive to pathogens.Dhingra. KG. compared to probiotics which introduce exogenous bacteria into the colonic micro flora. skatole. and methane because of their metabolic reaction to certain foods. and biotin. lactic and acetic acids. • • Protecting the immune system. Preventing establishment of harmful fungus and parasites: L. Acidophilus and B. pantothenic acid. • Synthesizing important B vitamins: Probiotics have been found to be beneficial in the synthesis of folic acid. particularly lactose and dietary carbohydrates. IDENTIFICATION OF PROBIOTICS Probiotics are microorganisms---such as bacteria. • Inhibiting levels of microbial pathogens: L. 2011 Current Topics in Biotechnology & Microbiology PREBIOTICS A prebiotics substance has been defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and or activity of limited number of bacteria in the colon. Reducing their numbers may lower toxin levels in the colon.

screening genetic traits offers considerable promise in attacking the almost insurmountable task of surveying for functional probiotic properties. confer a health benefit on the host.g. Moreover. as little evidence exists to support the long-term health benefits. bacteriocin levels). genetic modification of probiotic bacteria offers the added developmental potential to annex new beneficial activities (e. or building combinations of probiotic strains that can elicit multiple effects. including bacteriocins. KG. and organic acids • • Antagonistic toward pathogenic/cariogenic bacteria Resistant to bile 40 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. et al. There is a numerous of possible probiotic strains. the probiotic trend has swept the health and diet industries for their potential cleansing benefits. consumers may willingly accept and use genetically modified probiotics if there are substantial and tangible benefits provided over their traditional counterparts. immune boosting powers and nutritional value. Therefore. which. vaccine presentation) or improve the effectiveness of existing properties (e. 2011 Current Topics in Biotechnology & Microbiology World Health Organization. H. coupled with a highly diverse set of phenotypes and potential benefits. GENETIC MODIFICATION OF PROBIOTIC MICROORGANISMS The perceived desirable qualities of probiotics are many and it is highly unlikely that any one strain will harbor all the qualities or provide the multitude of proposed benefits. In this regard.g. DUDWELLER LANDSTR." The benefits of incorporating probiotics into one's diet have been widely speculated. because probiotics by definition. Consumers in developed countries will accept products manufactured or containing GMOs (genetically modified organisms). CHARACTERIZATION OF GOOD PROBIOTIC STRAINS Parameters that are employed for characterizing bacteria for being used in probiotics includes following steps or tests: • • Accurate taxonomic identification Production of antimicrobial substances. hydrogen peroxide. if they offer benefits to health and product quality that the consumer can clearly recognize [7].Dhingra. probiotics are "live microorganisms. offer health benefits. when administered in adequate amounts. GERMANY . Despite the lack of formal evidence.

concentration. DUDWELLER LANDSTR. 41 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. including the same or closely related species. inflammatory bowel diseases and allergic diseases. potentially resistant to bacteriocins. acid.Probiotics have been found as beneficial in the treatment of diarrhea. They improve the intestine’s immunological barrier functions. GERMANY . freezing. they offer considerable potential as probiotics because of their history of safe use and thegeneral body of evidence that supports their positive roles. and reduce generation of proinflammatory cytokines characteristic of local and systemic allergic inflammation [8]. and other antimicrobials produced by residing microflora • Lactobacilli and bifidobacteria constitute two extremely important groups of probiotic bacteria.Dhingra. The probiotic effects are attributable to the restoration to the normal of increased intestinal permeability and an unbalanced gut micro ecology. Effects of probiotics in treatment of diarrhea: The use of probiotic microorganisms for the prevention or therapy of gastrointestinal disorders is an obvious measure and perhaps the most usual application of probiotics because most health effects attributed to them are related directly or indirectly (i. KG.e. storage. et al. 2011 Current Topics in Biotechnology & Microbiology • • Immunostimulatory Amenable to production processing: adequate growth. alleviate the intestinal inflammatory response. • Genetic analysis and manipulation of these bacteria will be paramount to understanding their probiotic roles and maximising their performance in vitro and in vivo. mediated by the immune system) to the gastrointestinal tract. ROLE OF PROBIOTICS IN VARIOUS DISEASES Health effects of probiotics in pediatrics: In probiotic foods. H.. recovery. As members of the normal microflora of the gastrointestinal tract of humans. and distribution • Able to compete with the normal microflora. dehydration. cultures of beneficial live microorganism’s characteristic of the healthy human gut microbiota are administered in order to provide a safe microbial stimulus.

and maturation and optimal development of the immune system after birth depend on the development and composition of the indigenous microflora and vice versa. et al. epithelial and mucosal barrier function. H. Alteration of the intestinal microbiota Probiotic bacteria can antagonize pathogenic bacteria by reducing luminal pH. GERMANY . They exert their effects on numerous cell types involved in the innate and adaptive immune responses. The gut or its associated lymphoid system. GALT [9] is the largest immunologically competent organ in the body.Dhingra. redox potential. 2011 Current Topics in Biotechnology & Microbiology The mechanisms and the efficacy of a probiotic effect often depend on interactions with the specific microflora of the host or immunocompetent cells of the intestinal mucosa. B cells. One of the mechanisms by which the gut flora resists colonization by pathogenic bacteria is by the production of a physiologically restrictive environment. T cells. probiotics can influence mucosal cell– cell interactions and cellular “stability” by the enhancement of intestinal barrier function through modulation of cytoskeletal and tight junctional protein phosphorylation [13] Probiotics are already widely used to prepare fermented dairy products that are becoming popular in Europe and Japan. MECHANISM OF PROBIOTICS Probiotic bacteria have different influences on the host. DUDWELLER LANDSTR. this pH reduction was associated with increased animal survival [12] Augmentation of barrier function In this mechanism probiotics increase mucous production and enhance the barrier integrity. inhibiting bacterial adherence and translocation.In addition to the inhibition of growth of “conventional” organisms or potential pathogens. KG. such as epithelial cells. E. monocytes/macrophages. coli. or Vibrio cholerae) [10] to modulate (temporarily) the intestinal microflora and to have immunostimulatory or -regulatory properties. Shigella. and hydrogen sulfide production. and NK cells [11]. Many strains of probiotic microorganisms have been shown to inhibit growth and metabolic activity as well as the adhesion to intestinal cells of enteropathogenic bacteria (Salmonella. and the mucosal immune system. including T cells with regulatory properties. Probiotic bacteria decrease the luminal pH. Different organisms can influence the intestinal luminal environment. or producing antibacterial substances and defensins. dendritic cells. Some of the commercially available probiotic bacteria are listed in 42 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. with respect to pH.

rhamnosus L. 2011 Current Topics in Biotechnology & Microbiology table 1. bifidum B. Probiotic bacteria affect the various cells which are the key members of innate and adaptive `immune responses as like: 1.Dhingra. lactis L. johnsonii L. paracasei L. thus enhancing their effects in the large bowel. KG. thermophilusz 43 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. H. salivarius Bifidobacterium species B. breve B. DUDWELLER LANDSTR. Effects on lymphocytes (B lymphocytes. plantarum L. Effects on epithelial cells 2. Immunomodulation: This concept was demonstrated by Lammers et al. et al. 15]. on 2002 and Otte and Podolsky on 2004 [14. T cell redistribution) Table 1: commercially available human probiotic microorganisms Lactobacillus species L. casei L. T cells. acidophilus L. These 2 effects may be mechanistically related. Combining probiotics with prebiotics could improve the survival of the bacteria crossing the upper part of the gastrointestinal tract. NK cells. Effects on monocytes/macrophage 4. Effects on dendritic cells 3. GERMANY . reuteri L. lactis B. fermentum L. longum Streptococcus species S. These products favorably influence digestive functions and colonic flora. The most promising health benefits are the prevention of diarrhea and enhancement of the immune system. gasseri L.

Vol 1. Hinrichs. Vidales. & Lu.F. & Schaafsma. Eizaguirre.J. 9. R. Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB. Tritement des infections intestinales par la methode de translormation de la flore bacterienne de l’intestin. Fuller. Hudault. V. F. & Berg D. Urkia. P. Asensio. S. 2.Barriers to application of genetically modified lactic acid bacteria. & Servin... Coconnier. 44 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 747-748 4. R. E..Dhingra.. C R Social Biology. Kirjavainen.Growth promoting factors produced by micro-organisms. (2002) Probiotic supplementation reduces the risk of bacterial translocation in experimental short bowel syndrome. Guarner. 41.L. Zubillaga.J.(1965) Probiotics.. A.. S. M. P. H. Lilly.B. Journal of Pediatric Surgery.). 1046–52. Bernet-Camard. M. Science 147. Kallimäki. I. W. GERMANY . Metchnikoff. the next therapeutic targets for inflammatory bowel disease. (1998) F. H. Zubillaga. International Immunopharmacology 7:409–416. 5.. 39. Antimicrobial Agents Chemotherapy. Rautava. J.. Amsterdam.H. & Stillwell. D. DUDWELLER LANDSTR. 365-378.: Probiotics ((1992)) a general view. et al. International Journal of Food Microbiology. Z.M. (1997). Current Opinion in Allergy and Clinical Immunology. Garcia-Arenzana.. I. Havenaar.: Wood. 60: 359-361.B. Tissier.J. A.: Probiotics in man and animals ((1989)) Journal of Applied Bacteriology 66. C.. 237–238. 8. 7. Antonie van Leeuwenhoek 70: 299-316. M. M. KG. 2011 Current Topics in Biotechnology & Microbiology REFERENCES 1. Lactic acid as inhibiting intestinal putrefaction In: The prolongation of life: optimistic studies. & Salminen.C. (2007). Isolauri. 161-183. 3. C.699–702 10. Probiotics. Elsevier Applied Science Publishers. (1996).M. 2: 263-271. London. Verrips. & Aldazabal. & Huis in’t Veld.T. Zhang. E. D. (1907).J. 11.. Lievin.H. S. J. H. Heinemann.H. R. 37. P. G.. (1906). 6.J. (2002) Role of probiotics in food hypersensitivity. In: Lactic acid bacteria in health and disease (Ed.G. N.

et al. 15. Gionchetti. 116:301–309. 1182–1186. DUDWELLER LANDSTR. 2011 Current Topics in Biotechnology & Microbiology 12. Effect of probiotic strains on interleukin 8 production by HT29/19A cells. KG.. 14. (1999). American Journal of Physiology.M. D. Lammers. (1999) Impact on the composition of the faecal flora by a new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Alimentary Pharmacology Therapy 13:1103–1108. Gastroenterology. U. A. & Rizzello. H. Gastrointestinal and Liver Physiology. Barmeyer. GERMANY . 286. Helwig. G613–G626. C. K. (2004) Functional modulation of enterocytes by Grampositive and Gram-negative microorganisms. M. & Podolsky. Schmitz. E.. Americal Journal of Gastroenterology.Dhingra.M. Otte. 13. Venturi. F...K. H. J. & Fromm. 97. P. & Swennen. 45 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis. (2002).

slow and costly non automated tissue assembly processes are some of the limitations of traditional tissue engineering. Bone TE. to the micro. incapability of precisely controlling pore size. Liver TE and Urinary bladder TE. Cartilage TE. Atul Kumar Singh1.N. vascularization of thick tissue constructs.and 46 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. such as organ transplants.Mithila University.Department of Biotechnology. pore geometry. proliferate.Email:jaykarjha@yahoo.Mithila University. et al. Jaykar Jha4* 1 Department of Biotechnology. Darbhanga. Khan4. It allows cells to attach. The various types of polymers used for scaffold preparation can be categorized under two broad groups: either natural or synthetic materials.com. GERMANY . migrate and differentiate. Indian Institute of Technology. Ministry of Science &Technology. grow. However. Bihar-*Corresponding Author:Dr Jaykar Jha. G. Sharad S Lodhi2.Dhingra. Sudhanshu Singh3. On the whole.Darbhanga-846608. Gwalior 2 3 Department of Biotechnology. Dental TC. TE uses living cells and their extracellular components with polymer based biomaterial scaffolds to develop biological tissues for human body repair. Chandrabhan Seniya1. Madhav Institute of Technology & Science. TE appears to be the new frontier of medicine for its impact on regenerative and reconstructive procedures in humans.L. Skin TE. 2011 Current Topics in Biotechnology & Microbiology Chapter III TISSUE ENGINEERING AND ITS THERAPEUTIC APPLICATION. Each phase in TE must be understood in an integrated manner from the polymer material properties. Bihar. Extensive research is going on in the areas of Cornea TE. Scaffold serves numerous functions critical for the success of tissue regeneration. Cardiovascular TE.J. Low level of precision in cell placement. organ printing technology and use of bioreactors provide new insights for TE. India. spatial distribution of pores and construction of internal channels within the scaffold and extremely laborious. cell sheet engineering. DUDWELLER LANDSTR. KG. ABSTRACT: Tissue Engineering (TE) is an interdisciplinary field that applies the principles of engineering and life sciences to develop a fully functional human organ in vitro to cope up with the problem of organ scarcity. H.Professor & Head. L. New Delhi Department of Chemical Engineering.N. New Delhi 4 Department of Biotechnology.

Regenerative medicine is also a promising field in this case but this technique is most effective in the early stage of a disease. The problem in case of artificial mechanical organ is the very poor integration between the organ and native tissue and also they deteriorate with time in vivo and loose their function. there is a still need for organ replacement. or improve tissue function or a whole organ”. At terminal stages or for injured tissue. Scaffold: Structure and Function Scaffold serves numerous functions critical for the success of tissue regeneration. such as organ transplants. to the cell. 2. Tissue engineering has also been defined as “understanding the principles of tissue growth. proliferate.Dhingra. Scaffolds also enable the efficient transport of nutrients. 2011 Current Topics in Biotechnology & Microbiology macro-architecture of scaffold. 47 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The main problem of organ transplantation from donor (human/animal) is the – immune response problem in patient’s body and any disease can be transferred from the donor to the patient. KG. maintain. INTRODUCTION Tissue Engineering (TE) is “an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore. grow. growth factors. neotissue formation and remodeling. 1. DUDWELLER LANDSTR. in this case tissue engineered organ is the right answer to overcome the problem of organ shortage. tissue engineering and regenerative medicine. to the tissue-engineered transplant and finally to the host tissue. Tissue engineering uses living cells and their extracellular components with polymer based biomaterial scaffolds to develop biological tissues for human body repair. Currently the number of donors available is far less than the requirement of organs for transplantation. It provides the space for vascularization. The ultimate goal of tissue engineering is to develop a fully functional human organ in vitro to cope up with the problem of organ scarcity. H. and applying this to produce functional replacement tissue for clinical use. migrate and differentiate. GERMANY . Several approaches are currently being used to overcome the donor scarcity like– artificial mechanical organs. blood vessels and removal of waste materials. tissue engineering research has made significant progress towards the design of tissue constructs to repair or replace lost morphology and functions in diseased or damaged organs.” During the last decade. xenotransplantation (using animal organs). et al. It allows cells to attach. So.

GERMANY . ability to provide the bimolecular signals to the cells and. DUDWELLER LANDSTR. Polymers used in Tissue Engineering: The various scaffold materials investigated to date can be categorized under two broad groups: either natural or synthetic materials. silk fibroin. deliver inductive molecules or cells to the repair site and provide cues to control the structure and function of newly formed tissue. KG. 3. The interconnectivity of pores determines the transport of nutrients and waste and thus influences the success of tissue engineering. proliferation. heparin. such as poly(a-hydroxy ester)s. chitosan. et al. porosity. interconnected pores with proper pore size to support cell infiltration and vascularization. but for this to happen the pores need to be highly interconnected. ability to mimic in part the structure and biological function of the extracellular matrix [18]. migration.1 Characteristics of Scaffolds: The scaffold used in tissue engineering should at least have some important features which include biocompatibility to avoid unwanted host tissue responses to the implant.Dhingra. support for the formation of extra cellular matrix (ECM) by promoting cellular functions. The reproducibility of scaffolds is also very important as it determines the dimensional stability of the scaffold and consistency of the tissue formation. collagen. poly(glycolic acid) 48 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. permeability/porosity ratio is an indicator of the percolative efficiency per unit porous volume of a scaffold. poly(lactide) (PLA). excellent surface chemistry to allow attachment. 2. These aspects are vital as they provide optimal spatial and nutritional conditions for the cells. and other naturally occurring biodegradable polymers of sugar units. Generally. fibrin. acquisition of the sufficient mechanical properties to provide better environment for cells. The best natural scaffold into which cells naturally migrate during spontaneous wound repair is the fibrin clot. H. polysaccharides such as alginate. or fibrin fibres. fibres with fibrin coating. an increase in porosity leads to increase in the permeability. pore connectivity and reproducibility of pores. pore size distribution. hyaluronic acid derivatives. a controlled biodegradability to aid the formation of new tissue. Synthetic resorbable polymers. 2011 Current Topics in Biotechnology & Microbiology The structural aspect of scaffolds includes pore size. Examples of commonly used polymers include proteins such as albumin. and differentiation of the cells. adequate mechanical properties to maintain the structure and function immediately after implantation and during remodeling of the implants. Therefore.

3. poly[β(1→4)-2-amino-2-deoxy-Dglucopyranose] . Deacetylation of chitin by alkali generates chitosan. degradable polycyanoacrylates and degradable polyurethanes have been developed. insects. glycosaminoglycan (GAG) and collagen than those in monolayer culture on petri-dish [10]. fungi. H. which is present in crustacean.. A typical preparation of chitosan fibers can be done by wet spinning method [12]. GERMANY .D-glucopyranose]. yeasts. This anti-adhesive nature of alginate can have dramatic effect for tissue regeneration process. Karl Meyer described a procedure for 49 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. DUDWELLER LANDSTR. PLA. Chitin.3 glycosidic bonds.1 Alginate: Alginic acid is 1. polyanhydrides. There is no specific interaction known between mammalian cells and the alginate polysaccharide. as for cartilage tissue engineering chondrocytes encapsulated within alginate found to proliferate less but produce higher levels of cartilage specific proteins. The alginate is usually derived from seaweed source. KG. linked together by alternating β-1. 3. biodegradability and acceleratory effect on wound healing [11].S. polyphosphazens.3 Hyaluronic acid: Hyaluronan is a naturally occurring linear polysaccharide.2 Chitosan: Chitin and chitosan attracted attention of tissue engineers mainly due to its cheap availability. Hyaluronan consists of basic disaccharide units of D-guluronic acid and D-N-acetylglucosamine.g. polyorthoesters. 3. Furthermore. copolyesters of the lactic acid and glycolic acid.4-linked (homopolymeric or heteropolymeric) copolymer of β-Dmannuronic acid and α-L-guluronic acid. 2011 Current Topics in Biotechnology & Microbiology (PGA). polycarbonates.e. In 1934. polyamino acids. polyortho esters. biocompatibility. polyanhydrides. polyacetals. poly[ β(1→4)-2-acetoamido-2-deoxy. poly(lactide-co-glycolide) (PLGA). so proteins are not readily adsorbed due to electrostatic repulsion and thus alginate hydrogel and/or fibrous matrix provide somewhat anti-adhesive surface [9]. PGA and their copolymers have a long history of use as synthetic biodegradable materials and are approved by U. alginate carries a negative charge balance. et al.4 and β-1. polyamides.Dhingra. is one of the most abundant natural polysaccharide in the world. Food and Drug Administration (FDA) for some specific clinical uses.

3. To mimic the high proportion of collagen present in most native tissues. KG. with a diameter of 500 nm. the ability to engineer the materials with specific and impressive mechanical properties. Silk fibrous scaffolds compression resistant. and challenges the mechanical properties of synthetic high performance fibers. has the highest strength of any natural fiber. and a diverse range of surface chemistries for modification or decoration suggests that silk has the potential to be used as tissue engineering (TE) scaffold. Silk structures have been studied as a model for the study of structure-function relationships of fibrous proteins. acting as a mechanical support and creating a microenvironment to which the cells can respond.Dhingra. and also to act as a lubricant. At physiological pH all carboxy. Such a scaffold has the potential to promote cell growth and to restore key functions to damaged tissues and organs. Nephilia clavipes. and to stabilise the structures by electrostatic interactions [13]. Thus. which consists of up to 90% type I collagen among other matrix proteins [15-17]. 2011 Current Topics in Biotechnology & Microbiology isolating a novel glycosaminoglycan from the vitreous humor of bovine eyes. GERMANY . DUDWELLER LANDSTR. and hence the polysaccharide should be called hyaluronate. collagen scaffolds are widely used in tissue engineering [14].4 Silk fibroin: Recently silk is emerging as the most highlighted candidate for designing of biomaterials and tissue-engineering scaffolds with a wide range of mechanical properties. biocompatibility. Collagens – as a source of natural polymers – are in particular interesting as scaffolds for bone tissue engineering as they reflect the major source of proteins in bone extracellular matrix. while in solution the chain behaves as an extended random coil. These properties enable hyaluronan to regulate water balance. 50 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The molecular weight could be in the range of several millions. groups are dissociated. osmotic pressure and flow resistance. and named it hyaluronic acid. et al. Constructing a matrix or scaffold that simulates the ECM environment is therefore desirable and a widely used strategy in tissue engineering.5 Collagen: The extracellular matrix (ECM) is the natural scaffold for the cells. to interact with proteins. The dragline silk from the orb weaving spider. are stable at physiological temperatures and. 3. are insoluble in aqueous and organic solvents. H. The physico-chemical characterisation of hyaluronan was carried out during 1950s and 1960s.

Dhingra. modifications with hydrophilic functional groups or polymers are necessary to improve wettability for tear protein and mucin interactions and to improve glucose permeability for cellular health. To induce bone in-growth into the scaffold.8 Polydimethylsiloxane (PDMS): Polydimethylsiloxane (PDMS) belongs to a group of polymeric organosilicon compounds which are commonly referred to as silicones. meaning that at long flow times (or high temperatures). it acts like a viscous liquid. 3. 4. non-toxic and non-flammable. DUDWELLER LANDSTR. Traditional tissue engineering methods have generally focused on one of two 51 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. contact lens and in other applications. 2011 Current Topics in Biotechnology & Microbiology 3. GERMANY .e. However. can be included [18]. It is occasionally called dimethicone. However at short flow times (or low temperatures) it acts like an elastic solid. for use as an artificial cornea.7 Poly-caprolactone (PCL): The repeating molecular structure of PCL homopolymer consists of five nonpolar methylene groups and a single relatively polar ester group. 3. excellent oxygen permeability and transparency [19-21]. At temperatures below 32 °C PIPAAm molecules are highly hydrated. so the PIPAAm grafted surfaces are hydrophilic. i. The presence of hydrolytically unstable aliphatic-ester linkage causes the polymer to be biodegradable. high mechanical strength. H. similar to rubber. et al. a bioactive coating of the pore surface or additional bioactive fibres. calcium phosphate glass fibres.9 Poly (N-isopropylacrylamide): Poly (N-isopropylacrylamide) (PIPAAm) exhibits thermo-responsive hydrophobicity changes in aqueous solutions. At temperatures above 32 °C the surfaces suddenly change to hydrophobic due to extensive dehydration of PIPAAm molecules [22-27].1 Cornea Tissue Engineering: Tissue engineering has great potential to appease the growing demand of corneal transplantation. similar to honey. and is generally considered to be inert. PDMS is viscoelastic. KG. PDMS is optically clear. Applications of tissue engineering: 4. Poly (e-caprolactone) (PCL) fibres are produced by wet spinning from solutions in acetone. This property of PIPAAm is well exploited in cornea tissue engineering. PDMS has been widely used as a biomaterial in ophthalmic and other applications due to its good compatibility.

DUDWELLER LANDSTR. Allografts. Several attempts failed due to presence of synthetic scaffold material. [30] Bone grafts can be categorized into three types: autografts. reversible surface property alterations with slight temperature changes. including dramatic. strip the tissue of osteoinductive factors necessary for stimulating bone 52 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. endothelial cell sheets and with randomly oriented cells that affects transparency. which impairs transparency and causes strong inflammatory responses upon biodegradation. and it is hard to form the required surface curvature. et al.2 Bone Tissue Engineering Although bone is a dynamic and well-vascularized tissue with innate healing and remodeling capacities up to 10% of the bony fractures are complicated by non-union. and have attached these polymers to various surfaces. commonly the iliac crest. Moreover. 4. Tissue engineered cornea is generated by temperature-responsive polymers of N(isopropylacrylamide).Dhingra. such as demineralization. following which production of corneal tissue seems to be an achievable target. no dependence on donor and most of all transparent. it is important to have a tissue engineered properly oriented complete corneal construct which suffers no rejection. However. In fact. This new surface modification technology demonstrates several intelligent functions. Scientists are still facing some problems regarding tissue engineered (TE) cornea. KG. causes a nonspecific inflammatory response [28-29]. However. bone grafts harvested from another donor (mainly from cadavers). processing techniques. Cell Sheet Engineering is a promising approach to treat the cornea. One of the major problems is that these TC corneas have not enough tensile strength to allow surgical manipulation and fixation. Autografts are harvested from a secondary site from the patient’s own body. epithelial cell sheets. So for proper transplantation. they introduce the possibility of immune rejection and pathogen transmission. current strategies for developing tissue engineered corneal construct has few hurdles to cross and hence until now no successful bioengineered keratoplasty strategy has been clinically implemented. allografts. which require additional treatment with bone grafts in order to achieve defect union and healing. GERMANY . 2011 Current Topics in Biotechnology & Microbiology strategies: either the injection of isolated cell suspensions or the use of biodegradable scaffolds to support tissue formation. H.e. It has been observed that the implantation of nearly all polymer materials. are an alternative with enhanced flexibility of graft size and shapes [31-33]. bone grafts have become the second most transplanted tissue in the world after blood. and synthetic grafts. Till now the designed cell sheets are of single layer i. even if non-toxic.

H. have been hampered by their poor speed of healing and inability to remodel in tandem with the natural healing process [30]. et al. In vitro reconstructed skin relies on the hypothesis that tissue can.5 Cardiovascular Tissue Engineering: Cardiovascular disease remains a major cause of morbidity and mortality worldwide. accounting for 29% of all deaths globally. resulting in impeded healing times. During the several preceding decades. tissue engineering and new biopharmaceutical as well as recombinant genetic therapies. 2011 Current Topics in Biotechnology & Microbiology repair. provide a promising step towards a new therapy for reforming enamel but further study is needed on how to combine the newly-generated enamel with the original dental dentin or enamel in the tooth [35-37]. resulting 16. As EOE cells disappear in adult teeth after tooth eruption we also need to discover new cell sources to advance this technology in the clinical setting [38-40]. Sumita et al. as compared to autografts [34]. GERMANY . which can be fashioned to different shapes and sizes and are nonimmunogenic. DUDWELLER LANDSTR. 4. 4.Dhingra. The major modality of death due to cardiovascular disease remains acute myocardial infarctions (MI) and sudden cardiac death with more than one third of first time MI not making it to the hospital. enamel organ epithelial (EOE) cells is a significant advance towards enamel replacement and therefore attempts have been made to develop a strategy to generate enamel based on subcultured EOE cells using tissue engineering technology. Cadaver skin has offered an alternate pathway to skin replacement for many years. in principle. Autografting is a widely used method of treating organ loss. there have been breathtaking achievements in medical science in the field of in vitro cell cultivation. KG. 4. be cultured in vitro. with or without living cells [41]. but thus far.3 Dental tissue engineering Developing a technique to manipulate follicles.7 million deaths each year. cadaver skin and in vitro reconstructed skin. we are witnessing the evolution of 53 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. there are three methods of skin repair and replacements are autografting. The skin has been the first tissue-engineered organ from the lab bench to the patient [41]. Cardiovascular tissue engineering is an emerging field with an enormous potential for revolutionizing the next generation of heart failure therapies. Alternatives to cadaveric skin are represented by native biological substitutes.4 Skin Tissue Engineering: Basically. Synthetic grafts are made from metals or ceramics.

4. plasma proteins).g. or foam. Differentiated hepatocytes are difficult to culture in vitro because they do not proliferate well and quickly lose viability. This has been the focus of intense research in the cardiovascular tissue regeneration field over the past decade [42-45]. of clotting factors. The reason for this discrepancy lies in the challenging environment the implant will be exposed to in human body. This inert biocompatible material will then implanted with differentiated cells to form appropriate functional tissue to be transplanted in-vivo. DUDWELLER LANDSTR. high cost. need for immunosuppression to control allograft rejection (which can cause long-term damage to both the liver and kidneys) are highlighting about the need to develop tissue engineered liver. sufficient mechanical properties of the implant. and prevention of cell dedifferentiation are required. strength. Function of Liver is challenging enough to simulate in vitro. metabolism (of endogenous and exogenous substances) and elimination (bile secretion) of specific substances over prolonged time periods. 2011 Current Topics in Biotechnology & Microbiology therapies from pharmaceuticals and device treatments to the era of biological therapies with cell and engineered tissue do mechanisms Cardiovascular tissue engineering is a materials-based approach and involves preformed three-dimensional (3D) scaffolds in the form of mesh. The success of tissue engineering of other organs such as bone. resiliency and shock absorption. et al. represents at first sight a relatively simple example of tissue engineering but still no fully successful tissue engineered cartilage product is commercially available. and skin has been comparably easier to achieve because they are not as highly metabolic and do not 54 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. It is amazing that only few millimetres thick (500 µm up to 7 mm) articular cartilage. as an avascular tissue consisting of only one cell type (chondrocytes).7 Liver Tissue Engineering: Orthotopic liver transplantation is currently the only treatment option available for the people suffering from end-stage liver diseases. Accordingly.Dhingra. Significant progress has been made by using fibrous scaffolds for tissue engineering of articular cartilage. for example the enormous mechanical forces acting on knee articular cartilage. patch. integration of the implant into the host tissue.6 Cartilage Tissue Engineering: Cartilage is a remarkably complex bio-composite material which exhibits outstanding stiffness. H. But technical complexity. GERMANY . cartilage. KG. which includes synthesis (e. 4. Once damaged. human cartilage has a poor regenerative ability.

poor nutrition leading to cell death and consecutive tissue fibrosis [48-49].e.Dhingra. the clinical application of engineered tissues has been hampered by slow vascularization. H. including an adequate surface for stable attachment of urothelial cells. several studies have confirmed feasibility of bladder reconstruction using engineered segments which were formed using biomaterials seeded with autologous cells in vitro [47]. pore geometry. An intrinsic problem with vascularization of thick tissue constructs. Recently. serve as a barrier between luminal contents and the body cavity and support the formation of unidirectional muscle tissue in defined layers and allow for rapid innervations and vascularisation [50-51]. scaffolds produced by solvent-casting particulate-leaching cannot guarantee interconnection of pores because this is dependent on whether the adjacent salt particles are in contact. 4. To date. using autologous cells for implantation might offer a solution to this problem. For successful bladder reconstruction revascularization of the construct is essential to support short and long term survival. to restore physiological bladder function reinnervation is indispensable. 4. But the complex physiology of liver tissue demands preestablish a vasculature bed and seed the cells around the network. give adequate biomechanical support to harbor a high density of smooth muscle cells on the exterior surface without inducing premature collapse of the hollow organ. Incapable of precisely controlling pore size. Furthermore.8 Urinary bladder Tissue Engineering Tissue engineering. et al. slow and costly non automated tissue assembly process. GERMANY . DUDWELLER LANDSTR. 2. Furthermore. 3. especially when engineering multicellular constructs. Limitations of traditional Tissue Engineering The main limitations of the traditional tissue engineered solid scaffold approach are the following: 1. Low level of precision in cell placement. i. 2011 Current Topics in Biotechnology & Microbiology require an extensive vasculature.. skin layers are formed during evaporation and agglomeration of salt particles makes controlling the pore size difficult. For example. In respect to bladder TE the ideal scaffold material should prrovide structural support for distinct cell layers. 5. spatial distribution of pores and construction of internal channels within the scaffold. difficult to obtain 3D precision positioning of several cell types. Extremely laborious. KG. 55 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.

Cellular structure plays a major role in determining cellular activity. Furthermore. cell colonisation at the scaffold periphery is consuming. GERMANY . or acting as an effective barrier to the diffusion of oxygen and nutrients into the interior of the scaffold. this approach has resulted in the in vitro growth of tissues with cross-sections of less than 500 µm from the external surface. signal transduction. Cells are then seeded and expected to grow into the scaffold. H. KG. like chloroform and methylene chloride. 6. 56 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Non-woven fibre meshes have poor mechanical integrity. Thus. The presence of residual organic solvent is the most significant problem facing these techniques due to the risks of toxicity and carcinogenicity it poses to cells. to dissolve synthetic polymers at some stage in the process. Thus cells are only able to survive close to the surface. But this cellular metabolism gets disturbed in 2D cell culture because the cells isolated directly from higher organisms frequently alter metabolism and alter their gene expression patterns. most other 3D tissues require a high oxygen and nutrient concentration. However. DUDWELLER LANDSTR. et al. For gas foaming. the adaptation of cells in case of 2D cell culture requires a lot of significant adjustment of the surviving cell population not only to changes in oxygen.Dhingra. The cellular membrane structure. Conventional fabrication techniques produce scaffolds that are foam structures. neural information transmission and countless other biological processes. 7. 2011 Current Topics in Biotechnology & Microbiology Moreover. the extracellular matrix and basement membrane influences cellular metabolism. nutrients and extracellular matrix interactions. In case of traditional scaffold based tissue engineering the cultured cells lack key signaling and hormonal agents supplied in the in vivo situation by the circulatory system. conventional scaffold fabrication techniques use organic solvents. further limiting the mass transfer to the interior of the scaffold. the high rates of nutrient and oxygen transfer at the surface of the scaffold promote the mineralization of the scaffold surface. The human body supplies its tissues with adequate concentrations of oxygen and nutrients via blood vessels. This is probably due to the diffusion constraints of the foam. but also to alter waste disposal. determination of cell fate. for bone tissue engineering. The pioneering cells cannot migrate deep into the scaffold because of the lack of nutrients and oxygen and insufficient removal of waste products. only thin scaffold cross-sections can be produced due to difficulty in removing salt particles deep in the matrix. it has been reported that only 10-30% of the pores gets interconnected. Excluding gas foaming and melt molding. However. Molecular gradients play a vital role in biological differentiation. via the protein–protein interactions. 5. organ development.

biodegradable polymer scaffolds have been seeded with cells to be used to fabricate threedimensional tissue-like grafts. However. However. without any enzymes or 57 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. which enables reversible cell adhesion to and detachment from the dish surface by controllable hydrophobicity of the surface. cells are believed to proliferate and migrate to replace the polymer scaffold.g. the injected cells are expected to remain around the damaged host tissues for the maintenance and recovery of native functions. To overcome these problems. The monolayer cell sheet can be transplanted to host tissues without using biodegradable scaffolds and sutures. This allows for a non-invasive harvest of cultured cells as an intact monolayer cell sheet including deposited extra cellular matrices. The monolayer cell sheet can be collected simply by reducing culture temperature lower than 32°C for less than 30 minutes. Thick tissue constructs and patterned cell sheets using two or more kinds of cell source are also developed by means of layered cell sheets in vitro. the injected cells cannot be retained around the target tissue. H. Therefore. Advances in Tissue Engineering: 6.1 Cell Sheet Engineering: Cell sheet technology enables novel approaches to tissue engineering without the use of biodegradable scaffolds. GERMANY .Dhingra. This is due to the limits on passive diffusion. an increase in spreading). DUDWELLER LANDSTR.. Cell sheet technology consists of a temperature-responsive culture dish. Strong inflammatory responses are often observed upon biodegradation of the scaffolds. Growing cells can significantly alter production of their own extracellular matrix protein and often undergo morphological changes (e. With the injection of single cell suspensions. that is. et al. Macrophages and neutrophils with collagenase and elastase activities migrate to the implant site during the early wound healing response [52]. KG. Because of this the receptors on cell surface might not be in correct orientation and clustering and thus this would affect communication between cells. 2011 Current Topics in Biotechnology & Microbiology 8. cells in the center of the constructs become necrotic though the cells on the periphery are unimpaired. a new method to avoid the use of biodegradable scaffolds is strongly expected for the further development of tissue engineering. causing difficulties to control the size. 6. Upon polymer degradation. insufficient delivery of oxygen and nutrients and removal of metabolic waste. in most cases. shape and location of the injected cells. the places previously occupied by the polymer scaffolds are filled with large amount of extracellular matrix (ECM). For larger constructs.

6. 2011 Current Topics in Biotechnology & Microbiology chelating agents. chemical. The answer to this has been solved by the same emerging interdisciplinary field of tissue engineering that has integrated developmental biology in a biomimetic approach. Advantages of cell sheet engineering compared with more conventional approaches: • Cell sheets harvested from thermoresponsive substrates adhere to the host tissues without the need for sutures [54]. GERMANY . reflecting the varying cell densities of the different native tissues [58]. Scaffolds may elicit adverse host responses and interfere with direct cell–cell interaction. fabrication techniques for production of scaffold-free engineered 58 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. • Control over the spatial distribution of cells within three-dimensional stratified tissues can be achieved by layering cell sheets created from different cell types [57]. This biomimetic approach intends to provide the biological. final tissue-like constructs have greater cell densities and less ECM. which can complicate or delay integration of the tissue implant [52]. • Tissue engineering strategies require the scaffold material to be surgically placed between cultured cells and the injury site. KG. • Cell sheet engineering offers better control over cell seeding. • Cell sheet engineering avoids the use of scaffold materials. Thus. et al.Dhingra. that is. which can have potential for inflammatory or foreign body reactions or other complications arising from the by-products of scaffold biodegradation [55].2 Organ Printing Technology: It has always been a question to be answered that how a tissue engineer can closely imitate the developmental morphogenesis for capturing the above. Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. and mechanical clues to guide the gradual cell differentiation taking place and also the assembly into a 3D tissue construct with a real-time insight. we can transplant cell sheets to host tissues without using biodegradable scaffolds [53]. as well as assembly and alignment of cell-produced ECM. when cells are seeded at a cell concentration similar to that observed on a confluent monolayer. • Thermoresponsive substrates avoid the use of deleterious enzymes that typically are used to remove cell monolayers from conventional culture dishes [56]. DUDWELLER LANDSTR. By this technology. H.

The main rationale behind this approach is a need to maintain. it can be defined as computer aided. hours to days). Thus. drug discovery and drug toxicity. et al. which we implement through a rapid prototyping bioprinting method for scaffold-free small diameter vascular reconstruction. atleast initially. The main practical outcomes of this technology are industrial scalable robotic biofabrication of complex human tissues and organs.and local-environment where the functional properties of cells can be observed and manipulated that is not possible in 2D culturing. It has become apparent that 3D cell culture offers a more realistic micro. Tissue engineering is based on fabrication of porous solid biodegradable scaffolds with sequential cell seeding in bioreactors. DUDWELLER LANDSTR. in a more narrow sense. and complex in vitro models of human diseases. the shape and mechanical properties of the tissue engineered construct and to provide a substrate for cell attachment and signals for cell differentiation and tissue development. The ultimate goal of this technology is to fabricate 3D vascularised functional living human organs suitable for clinical implantation. GERMANY . 2011 Current Topics in Biotechnology & Microbiology tissue constructs have recently emerged. Biomimetic-writing or organ printing.Dhingra. Here we report on a fully biological self-assembly approach. H. Most bone TE methods involve seeding of acellular constructs or insertion of acellular implants with the expectation of 59 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. layer by layer deposition of biologically relevant materials. which is also known as the biomedical application of rapid prototyping is defined as additive layer by layer biomanufacturing. Thus. organ printing or biomimetic writing or robot fabrication offers an interesting alternative to solid scaffold based tissue engineering. The basis for this technique is to produce usable scaffolds in a short time scale (i. Now the question arises – how can this biomimetic approach be utilized in a beneficial way to serve the above purpose? This is done by using the principle of Biomimetic-writing. It is an emerging transforming technology that has the potential of surpassing traditional scaffold based tissue engineering.e. Solid freeform fabrication (SFF) and 3D printing are two of the more popular rapid prototyping techniques that are capable of generating multi-material and multi-cellular anatomical constructs. Rapid prototyping has many different variations. KG. automated tissue based in vitro assays for clinical diagnostics. It is the ultimate traditional or classic way of making scaffolds but this standard or method itself has lot of limitations that makes a person to think of an alternative for constructing or making scaffolds.

NJ. as termed by Klebe involved alternating deposition of layers of cells and materials to generate 2D and 3D tissues. An excellent example of this is by Cohen et al. Cell printing efforts by Chang et al. More recently several groups have demonstrated simultaneous co-deposition of cells and materials. GERMANY . DUDWELLER LANDSTR. such as the meniscus. 3D printing brushite implants and a cranial segment using tricalcium phosphate (TCP) and tetracalcium phosphate respectively. Shek et al. The composite structure exhibited region specific mechanical properties and integration between the two biomaterials making it suitable for implantation. USA) produced osteochondral composites using TCP combined with either PLG or PLA for the chondral surface. two different grades of alginate) and in different structurally sound shapes including a disc. 2011 Current Topics in Biotechnology & Microbiology cellular ingrowth in vivo. used localized gene therapy to increase and localize cellular and tissue ingrowth using an SFF polypropylene fumarate/TCP composite that provided a stable matrix that could be matched to specific patient defect geometry. The micro-organs had vascular networks serving as pharmacokinetic models and were able to replicate consistent prints with 250 m resolution. Rapid prototyping has also been used in the fabrication of 3D hepatic tissues with complex internal microstructure. and altered scaffold porosity. Inc.Dhingra. Therics. 60 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. crescent and meniscus based on CT data with printing resolution of 270 m. et al. chemically bonded growth factors. in conjunction with Therics. Klebe established this technique using a variety of different cell types from different species and bound them to substrates using fibronectin that was deposited via Hewlett Packard graphics plotter of ink jet printer. heart valve and liver.e. Constructs were generated using both multi-cell and multi-material as means to improve nutrient transport. via SFF using alginate and chondrocytes. have evaluated cell viability of HepG2 cells based on dispensing pressure and nozzle diameter with calcium cross-linked alginate and combined these SFF techniques with lithography methods to generate 3D micro-organs. Some successful studies include the use of porous coral in the shape of a distal phalanx seeded with periosteal cells for thumb reconstruction. H. The work established the ability to print cell seeded alginate using different materials (i. Inc. KG. For more heterogeneous tissues. also has a number of other TCP based therapeutic products that are currently undergoing clinical studies. (Princeton. Achieving structures that have the necessary cell distributions and biomechanical properties is a major challenge. Cytoscribing. control over spatial and temporal differences in cell type/morphology and mechanical properties is necessary. SFF techniques are able to produce patient specific scaffolds that can be modified to increase and guide cellular in growth through variation of surface roughness. Work by Sherwood et al.

ballistic particles manufacturing and others. et al. as well as development of novel deposition systems specifically designed for biologically relevant materials. Cells respond to mechanical stimulation and bioreactors can be used to apply mechanical stimulation to cells. This can encourage cells to produce extracellular matrix (ECM) in a shorter 61 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.Dhingra. 3D printing. 2011 Power Switch No. Y Axis Power Plug Air compressor Figure 1: Schematic Diagram of Direct Write Assembly Rapid prototying is already well established and includes many technology variants such as stereolithigraphy. Bioreactors are devices in which biological or biochemical processes develop under a closely monitored and tightly controlled environment. selective laser sintering. GERMANY .3 Bioreactors in Tissue Engineering: A tissue engineering bioreactor can be defined as a device that uses mechanical means to influence biological processes [59]. KG. fused deposition modelling. H. DUDWELLER LANDSTR. The main challenge of this technology is the adaptation of existing systems for specific biological materials when it is technically possible. Bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. 6.1 Power Switch No.2 Current Topics in Biotechnology & Microbiology Feed back switcher Z Axis Y axis X Axis AC/ DC 220V/ A 48V Ethernet Switching regulator Pressure Valve Controller S yringe Z Axis Adapter Ethernet X.

in comparisons between ECM protein levels of equine articular chondrocytes cultured on polyglycolic acid scaffolds after 5 weeks in culture. A heterogeneous cell distribution is a major obstacle to developing any threedimensional tissue or organ in vitro. GERMANY . H. Another important application of bioreactors is in cellular differentiation. problems arise when culturing cells on these scaffolds. resulting in core degradation of tissue engineered constructs. after long periods in culture. Defects requiring tissue engineering solutions are typically many millimeters in size [63]. Static culture conditions result in scaffolds with few cells in the centre of the construct. or cell death in the centre of the scaffold [63]. A six-fold increase in equilibrium aggregate modulus (an intrinsic property of cartilage which is a measure of stiffness) was found after 28 days of culture in a compression bioreactor compared to free swelling controls [61]. Bioreactors can provide biochemical and physical regulatory signals that guide differentiation [62]. Mechanical stimulation can be used to encourage stem cells down a particular path and hence provide the cell phenotype required. et al. constructs cultured under hydrostatic pressure showed significant improvements over constructs cultured in static medium [60]. It is hypothesized that this is due to limited cell penetration during seeding. There is great potential for using mesenchymal stem cells and other multipotent cells to generate different cell types and bioreactors can play an important role in this process. As well as providing mechanical stimulation.Dhingra. The only mechanism by which nutrients and waste can move when a scaffold is in static culture is by diffusion. the move towards clinical trials has been slow and progress to date in engineering significant quantities of functional tissue in vitro for implantation in humans in vivo has been somewhat disappointing. For this reason. It has been shown that despite homogeneous cell seeding. cell migration to the scaffold periphery during culture. This is of major concern in the field of tissue engineering. For example. Scaffolds in such a size range are easily fabricated. DUDWELLER LANDSTR. bioreactors can also be used to improve cellular spatial distribution. for a number of tissue types. 62 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. A benefit of ECM production is the increase in mechanical stiffness that it provides to the construct. however. 2011 Current Topics in Biotechnology & Microbiology time period and in a more homogeneous manner than would be the case with static culture. diffusion of nutrients to the centre of the construct becomes more difficult. more cells are found on the periphery of constructs [64] leading to peripheral encapsulation which hinders nutrient and waste exchange from the centre. and is a major obstacle in the formation of a viable tissue in vitro. KG. As the size of the scaffold increases.

In addition. However. Equilibrium is needed between highly porous scaffolds that allow rapid tissue ingrowths and minimize diffusion limitations and less porous materials that retain both construct shape and the ability to bear mechanical loads in a complex biochemical and mechanical environment. 4. 63 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. deliver inductive molecules or cells to the repair site. In fact. Maintain the desired concentration of gases. 3. its ultimate goal is to develop powerful new therapies.Dhingra. GERMANY . Matrix /polymer hybrid scaffold with improved biomechanical characteristics may be advantageous for many tissue engineering aspects such as heart valve tissue engineering. Provide a spatially uniform cell distribution. differentiation and migration by up regulating and down regulating the synthesis of protein and growth factors. Scaffolds also provide clues to control the structure and function of newly formed tissue. Expose the construct to physical stimuli and/or 5. providing “cell-to-tissue replacement parts” of the human body it can ultimately afford the irremediable shortage of transplantable organs. and nutrients in culture medium. for structural and functional disorders that have been proven to be difficult or impossible to tackle successfully with the current approaches of interventional medicine. CONCLUSION: Scaffolds are used to provide sites for cells attachment. Recently decellularized extracellular matrix has been suggested as a scaffold for heart valve tissue engineering or direct implantation. In general. H. bioreactors are designed to perform at least one of the following five functions to 1. tissue engineering appears to be the new frontier of medicine for its impact on regenerative and reconstructive procedures in humans. 7. bioreactors can be used in tissue engineering applications to overcome problems associated with traditional static culture conditions. et al. cell removal damages the physical and biochemical properties of the valve leaflet structure. Provide information about the formation of 3D tissue. They provide mechanical support. improve cellular distribution and accelerate construct maturation [65] whilst applying biophysical signals to constructs to improve tissue formation in vitro prior to in vivo implantation. Facilitate mass transport to the tissue. DUDWELLER LANDSTR. proliferation. KG. On the whole. namely “biological substitutes”. 2011 Current Topics in Biotechnology & Microbiology Thus. 2.

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Darbhanga-846608. 2011 Current Topics in Biotechnology & Microbiology Chapter IV RNA INTERFERENCE: MOLECULAR MECHANISM AND THERAPEUTIC APPLICATIONS G. Birla Institute of Technology & Science. It is triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of singlestranded target RNAs homologous in response to dsRNA. India. Bihar.N. Pilani. Department of Biotechnology. India 3 Dept. Atul K. Singh2.Khan1. siRNAs are approximately twentyne nucleotides in length. INTRODUCTION: RNA interference (RNAi) is a phenomenon leading to post-transcriptional gene silencing (PTGS) after endogenous production or artificial introduction into a cell of small interfering double strand RNA (siRNA) with sequences complementary to the targeted gene [1]. Bihar-. which are produced from long dsRNA by enzymatic cleavage in the cell. H. KG. Mithila University. L. ABSTRACTS: RNA interference (RNAi) is a conserved cellular defence mechanism for controlling the expression of foreign genes in most eukaryotes including humans. siRNAs hold great potential as gene-specific therapeutic agents. GERMANY . India *Corresponding Author: Dr Jaykar Jha. L. Jaykar Jha1* 1 2 Department of Biotechnology. and have a base-paired structure characterized by two nucleotide 3'-overhangs. Chemically synthesized siRNAs have become powerful reagents for genome-wide analysis of mammalian gene function in cultured somatic cells. Phone: +919431627901 Email:jaykarjha@yahoo. India Department of Biotechnology. of Biosciences. Gwalior. RNAi prevents the translation of the target protein by selective sequence specific degradation of its encoded messenger RNA (mRNA). The mediators of mRNA degradation are small interfering RNA duplexes (siRNAs).J. et al. .com. Madhav Institute of Technology and Science. Darbhanga.Dhingra.N. Chandrabhan Seniya2. RNAi is an RNA-dependent gene silencing process that is 70 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Professor & Head. DUDWELLER LANDSTR.Mithila University. Prabhat N Jha3.

4]. it is involved in the response to exogenous pathogenic and endogenous parasitic nucleic acids [3. such as antisense oligonucleotides (ODNs). Posttranscriptional gene silencing occurs when the guide strand base pairs with a complementary sequence of a mRNA molecule and induces cleavage by argonaute. DUDWELLER LANDSTR. The natural function of RNAi refers to the mechanism involved in cellular defence against viruses. where they interact with the catalytic RISC component argonaute. genomic containment of retrotransposons. Two types of small RNA molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. namely the passenger strand and the guide strand. et al. ribozymes. siRNAs have apparently become the most powerful and widely used gene silencing reagents for manipulating gene activity. viral-induced gene silencing) [11]. This process is known to spread systemically throughout the organism despite initially limited molar concentrations of siRNA. H. GERMANY . or upon infection with recombinant RNA viruses that carry the target gene (VIGS. as well as in the basic cellular functions. creating knockout phenotypes. 2011 Current Topics in Biotechnology & Microbiology controlled by the RNA-induced silencing complex (RISC) and is initiated by short doublestranded RNA molecules in a cell's cytoplasm. but also as an effectively potent strategy for gene silencing-based therapeutics [9. It is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription (TGS) or by activating a sequence Specific RNA degradation process [PTGS/RNA interference (RNAi)] [2]. either in transformants that can produce the required hairpin RNAs. where endogenous RNAi controls different 71 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer. the catalytic component of the RISC complex.Dhingra. RNAi can specifically silence individual genes. and post-transcriptional regulation of gene expression. The gene silencing molecules (RNAi) is amplified and the concentration of RNAi increases by many fold. As compared with other gene silencing reagents. The siRNA is unwound into two ssRNA.10]. The effects of both RNAi and PTGS are potent and systemic in nature. and DNAzymes. RNAi has been widely used not only as an extremely powerful approach for reverse functional genomics [8]. such as gene regulation and heterochromatin formation [5–7].13]. and the guide strand is incorporated into the RNA-induced silencing complex (RISC). KG. The integrity of the RNAi pathway is essential in mammals [12. Primarily. The amplification of the RNAi makes it potent even if the concentration is quite low at initiation stage. The passenger strand is degraded. Currently. which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of about 20 nucleotides called siRNA.

Knowing the biological processes in which RNAi participates would allow us to anticipate and monitor the appearance of unexpected adverse effects derived from the application of these therapeutic molecules. certain invariant features are observed. (iii) the degradative machinery requires a set of proteins which are similar in structure and function across most organisms [16]. have converged on a universal paradigm of gene regulation. However. H. instead of increasing anthocyanin levels.Dhingra. [19] who observed that the introduction of sense or antisense RNA to par-1 RNA resulted in degradation of the par-1 message in Caenorhabditis elegans. The levels of endogenous as well as introduced CHS were 50-fold lower than in wild-type petunias. quelling in fungi. DUDWELLER LANDSTR. Understanding these functions is of critical importance when exploiting RNAi for different purposes. antisense was one of the most attractive means of eliminating gene expression. In most of these processes. In 1992. the transgenic model showed apparent silencing of both the endogenous and exogenous CHS genes. GERMANY . including the formation of small interfering RNA (siRNA) and the organism specific systemic transmission of silencing from its site of initiation.15]. et al. andvirus-induced gene silencing in plants and animals. which led them to hypothesize that the introduced transgene was “cosuppressing” the endogenous CHS gene. The common components of the paradigm are: (i) the inducer is the dsRNA. RNA silencing was first documented in animals by Guo et al. BRIEF HISTORY OF RNA INTERFERENCE: Napoli and Jorgensen were the first to report an RNAi type of phenomenon in 1990 [17].. KG. as exogenous manipulation could undesirably interfere with the function of endogenous RNAi. Antisense was thought to function by hybridization with endogenous mRNAs resulting in double stranded RNA (dsRNA). At that time. which either inhibited translation or was targeted for 72 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. IMPORTANT FEATURES OF RNA SILENCING: RNA silencing has been coined variously as PTGS in plants. (ii) the target RNA is degraded in a homology dependent fashion. RNAi in animals. noting that introduction of homologous RNA sequences caused “quelling” of the endogenous gene. 2011 Current Topics in Biotechnology & Microbiology developmental and physiological processes and has been implicated in the development of cancer and other diseases [14. The discovery of RNAi was the unexpected result of attempts to make the colour of petunia blooms more purple by over expressing the transgene chalcone synthetase (CHS)). Romano and Macino reported a similar phenomenon in Neurospora crassa [18].

Due to the versatile application of RNAi in diverse systems. Guo et al. elegans by direct injection of antisense siRNA (par1 gene) and a similar inhibitory effect on par-1 gene function was elicited by the injection of sense siRNA [19]. H. GERMANY . Systemic silencing. Some 73 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. and the plant Arabidopis thaliana to search for mutants defective in quelling.Dhingra. Silencing can be introduced in different developmental stages. the nematode Caenorhabditis elegans. Genetic screens were carried out in the fungus Neurospora crassa. cancer. DUDWELLER LANDSTR. KG. reported that double-strand RNA was at least tenfold potent as a silensing trigger than were antisense or sense RNA alone [20]. In 1998. Avoids problems with abnormalities caused by a knocked out gene in early stages Silencing effects passed through generations. High degree of specific gene silencing with less effort.. SALIENT FEATURES OF RNA INTERFERENCE The mechanism by which RNAi inhibits the conversion of mRNA into protein is a potent and robust system which protect the organism from genetic contamination and bears the following features. RNA interference. 24]. have provided insight into the RNAi pathway. produced the loss-of-function or gene-knockout phenotype in the offspring of the injected C. Highly potent and effective. Fire et al. It has been established that siRNAs are the key mediators of RNAi [21] and the cloning of the RNAse III-like enzyme Dicer [22]. 2011 Current Topics in Biotechnology & Microbiology destruction by cellular ribonucleases. COMPONENTS OF RNAi: Both genetic and biochemical approaches have been undertaken to understand the basis of silencing. the alga Chlamydomonas reinhardtii. et al. RNAi has also shown exceptional promise as a genome-wide screening tool [25-27]. and dominantly inherited disorders [23. or PTGS. • • • • • • • Double stranded RNA rather than single-stranded antisense RNA is the interfering agent. RNAi renders promising result in therapy specially in infectious disease.. different groups around the globe are trying to explore the molecular mechanism of its action. Analyses of these mutants led to the identification of host-encoded proteins involved in gene silencing and also revealed that a number of essential enzymes or factors are common to these processes.

Bernstein et al. while others serve as effectors. 2011 Current Topics in Biotechnology & Microbiology of the components identified serve as initiators. amplifiers. siRNAs were subsequently discovered in Drosophila tissue culture cells in which RNAi was induced by introducing 500-nucleotide long exogenous dsRNA [34]. Some Dicer proteins. some recombinant Dicers have also been examined in vitro.Dhingra. Dicer homologues from many different sources have been identified. who first demonstrated the enzyme's dsRNA "dicing" activity [28]. Dicer: Dicer was given its name by Emily Bernstein. [31] identified an RNase III like enzyme in Drosophila extract which was shown to have the ability to produce fragments of 22 nucleotides. DUDWELLER LANDSTR. similar to the size produced during RNAi. siRNAs were detected first in plants undergoing either cosuppression or virus-induced gene silencing and were not detectable in control plants that were not silenced. about 20-25 nucleotides long usually with a two-base overhang on the 3' end. two independent groups. and phylogenetic analysis of the known Dicer-like proteins indicates a common ancestry of these proteins [32]. also in Drosophila embryos that were injected with dsRNA [36].. Dicer contains two RNase III domains and one PAZ domain. overturned the long-held belief that helicases played a major role in unwinding ds siRNAs by demonstrating that Ago2 is responsible for cleaving the non incorporated (passenger) strand of the siRNA duplex. RNase III family members are among the few nucleases that show specificity for dsRNAs [29] and cleave them with 3’ overhangs of 2 to 3 nucleotides and 5’ -phosphate and 3’-hydroxyl termini [30]. allowing the other strand to be 74 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. siRNAs are formed and accumulate as double-stranded RNA molecules. the distance between these two regions of the molecule is determined by the length and angle of the connector helix and determines the length of the siRNAs it produces. Guide RNAs and RNA-Induced Silencing Complex: At the end of 2005. et al. H. Cleavage by Dicer is thought to be catalyzed by its tandem RNase III domains. who identified the product of RNA degradation as a small RNA species (siRNA) of 25 nucleotides of both sense and antisense polarity. in Drosophila embryo extracts that were carrying out RNAi in vitro [35] and. including the one from D. contain an ATP-binding motif along with the DEAD box RNA helicase domain. Dicer is an endoribonuclease in the RNase III family that cleaves double-stranded RNA (dsRNA) into short double-stranded RNA fragments called small interfering RNA (siRNA). and Matranga et al. Gregory et al. KG. and/or transmitters of the gene silencing process. GERMANY . melanogaster. Small interfering RNA (siRNA): The key insight in the process of PTGS was provided from the experiments of Baulcombe and Hamilton [33].

Transgenic and virus-infected plants show an accumulation of aberrant transgenic and viral RNAs. Winston et al. a group led by Jayasena addressed this question by comparing the thermodynamic properties of functional and nonfunctional siRNAs [39].43] which probably reflects the requirement for energy driven unwinding of siRNA . How the RISC/Ago2 determines which strand of the doublestranded siRNA to cleave and which strand to incorporate into the RISC complex were a mystery. The siRNA duplex containing ribonucleoprotein particles (RNP’s) are subsequently rearranged into the RNA induced silencing complex. elegans (HC57). GERMANY . One thing these effector complexes have in common is the presence of an Argonaute protein. elegans and plants. Transmembrane Protein (Channel or Receptor): The systemic spread of gene silencing from one tissue to another has been well established in C. but also at later phases [40. Recent evidence has suggested that Dicer may not only play a role in the initiator phases of the RNAi pathway. [48] constructed and used a special transgenic strain of C. This observation suggests that additional cofactors are needed for RISC activity. 41] The assembly of RISC is ATP dependent [42. providing a basis for entry into RISC. They identified a systemic RNA interference deficient (sid) locus required to transmit the effects of gene silencing between cells with green fluorescent protein 75 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. KG. 2011 Current Topics in Biotechnology & Microbiology incorporated into RISC [37. melanogaster cell lysates. This has led to a proposed mechanism in which RNA-dependent RNA polymerases (RdRPs) play a role in both triggering and amplifying the silencing effect. The functional RNP’s contain only single stranded RNA. To investigate the mechanism of systemic RNAi. 38]. H.Dhingra. Evidence that the passenger strand of the siRNA serves as the first target of RISC is derived from detection of the predicted 9-nucleotide 5’ cleavage and 12nucleotide 3’ cleavage products. Their results indicated that the 5’ antisense region of the functional siRNAs were less thermodynamically stable than the 5’ sense regions. The RdRP enzymes might recognize these aberrant RNAs as templates and synthesize antisense RNAs to form dsRNAs that are finally the targets for sequence-specific RNA degradation [44-47]. In 2003. purified Ago2 cannot use double-stranded siRNAs as a substrate for target mRNA cleavage. Although Ago2 is the enzyme responsible for cleaving the passenger strand siRNA as well as target mRNA. et al. DUDWELLER LANDSTR. RNA-Dependent RNA polymerase: The effect of RNAi is both potent and systemic in nature. The estimated apparent molecular mass ranges from between 130 to 160 kDa in the case of high salt purified RISC from human cells to 500 kDa or up to the 80S range in the case of RISC isolated from D.

perhaps functioning as a receptor. these mutants also failed to transmit the effect of RNAi to the progeny.Dhingra. is loaded onto a specific Argonaute protein and assembled into the active RISC. KG. In the execution phase. are small RNA strand unwinding and preferential strand selection. siRNAs are then incorporated into large ribonucleoprotein complexes. No homologue of sid1 was detected in D. which may be consistent with the apparent lack of systemic RNAi in the organism [49. in D. MECHANISM OF RNA INTERFERENCE RNA interference takes place predominantly within the cytoplasm of the cell and is triggered by the introduction of a double stranded oligonucleotide into the cell cytoplasm. the presence of SID homologues in humans and mice might hint at the systemic characteristics of RNAi in mammals. it was suggested that it might act as a channel for the import or export of a systemic RNAi signal or might be necessary for endocytosis of the systemic RNAi signal. which must dissociate into ‘competent’ single strands in order to function as guides for RISCs.RISC). providing a basis for entry into RISC. 50]. DCR-2 does not simply transfer siRNAs to a distinct RISC but. 2011 Current Topics in Biotechnology & Microbiology (GFP) as a marker protein. Of the 106 sid mutants belonging to three complementation groups (sid1. Their results indicated that the 5’ antisense region of the functional siRNAs were less thermodynamically stable than the 5’ sense regions. et al. melanogaster. [52]. sid2. However. Interestingly. DUDWELLER LANDSTR. In the gene silencing pathways initiated by dsRNA precursors (small RNA duplex). the guide strand. H. and sid3). The key factors in converting the RISC from its precursor form (the preRISC). is destroyed. The SID1 polypeptide is predicted to be a 776-amino-acid membrane protein consisting of a signal peptide and 11 putative transmembrane domains. melanogaster. For example. indicating that the role of DCR-2 76 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. the passenger strand. The sid1 mutants had no readily detectable mutant phenotype other than failure to show systemic RNAi. instead. other strand. GERMANY . they isolated and characterized sid1 mutants. These effector complex interfere with gene expression by using the small RNA strand to identify their complementary mRNA and the same is cleaved and degraded [51]. The initiation phase involves processing of dsRNA into small interfering RNA molecule (siRNA). Based on the structure of SID1. The mechanism can be divided into two distinct phases: an initiation and an execution phase. forms part of the RISC together with the siRNAs. which contains the small RNA duplex. to its mature form (the holo. For each small RNA duplex. which contains the guide strand. Dicer-mediated cleavage yields small dsRNA intermediates.

Although the conversion of long dsRNA into many small siRNAs results in some degree of amplification. single-stranded miRNAs are found associated with AGO2 in humans. the unwinding of the siRNA duplex and the loading of a single strand into the RISC are facilitated by the slicing of the unincorporated (passenger) strand by AGO2. it is 77 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. premiRNAs are known to bind to a preformed trimeric complex of AGO2. The particular strand of the siRNA duplex that is loaded onto AGO2 seems to be determined by the orientation of the DCR-2–R2D2 heterodimer on the siRNA duplex [55]. The loading of siRNA duplexes onto AGO2 is facilitated by the RISC loading complex.60]. This process by which a pre-RISC is converted to a holoRISC can also occur by a slicer-independent mechanism. in humans. This complex can cleave target RNAs using pre-miRNA and can distinguish miRNA from miRNA* in the absence of ATP hydrolysis [59. Similarly. KG. The heterodimer probably recruits AGO2 through an interaction between DCR-2 and AGO2. DUDWELLER LANDSTR. 54]. Amplification Of siRNAs: One of the many intriguing features of RNA interference is the apparently catalytic nature of the phenomenon. Cleavage in the middle of the passenger strand. a cleavage-independent (bypass) mechanism for RISC assembly must exist. Previous models have proposed that the transition from a double-stranded silencing trigger to a single-stranded one is mediated by an unidentified ATP-dependent RNA helicase. A few molecules of dsRNA are sufficient to degrade a continuously transcribed target mRNA for a long period of time. 2011 Current Topics in Biotechnology & Microbiology extends beyond the initiation phase. whereas DCR-2 is recruited to the less stable end. However. suggesting that DICER1-mediated cleavage and sensing of thermodynamic stability occur in series in the AGO2–DICER1–TRBP complex. despite the expectation that mismatches in the unwound pre-miRNA should block the passenger-strand cleavage activity of AGO2. as though the passenger strand were an mRNA target. AGO3 and AGO4) lack slicer activity but are nonetheless loaded with singlestranded guide siRNAs [61-63]. GERMANY . Thus. DICER1 and DICER1’s dsRBDcontaining partner.Dhingra. These data support a model in which siRNAs are initially loaded as duplexes onto an AGO2-containing pre-RISC. would be expected to reduce the annealing temperature and the free energy of duplex formation. R2D2 is thought to sense the thermodynamic stability of the siRNA duplexes and bind to the more stable end of the siRNA. a process that does not require ATP [56–58]. H. By contrast. et al. Three of the four Argonaute proteins in humans (AGO1. TRBP [59]. R2D2 [53. RNA helicase A has been identified as a candidate for unwinding the duplex in this process [64]. which in turn facilitates the separation of the siRNA strands. which contains DCR-2 and its dsRBD-containing partner.

Dhingra. Lipardi et al. Both single stranded RNAs (equivalent to target mRNA) and dsRNAs served as templates for copying by RdRP. [66] provided convincing biochemical and genetic evidence that RdRP indeed plays a critical role in amplifying RNAi effects. New full-length dsRNAs were formed rapidly and cleaved. Figure1: Different steps in Gene Silencing: 78 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. elegans. DUDWELLER LANDSTR. Since mutations in genes encoding RNA-dependent RNA polymerase (RdRP) affect RNAi. [65]. Recent studies by Lipardi et al. showed the generation of full-length cognate dsRNAs from labeled siRNAs at early time points. [65] and Sijen et al. permitting their spread throughout plants and between generations in C. GERMANY . 2011 Current Topics in Biotechnology & Microbiology not sufficient to bring about such continuous mRNA degradation. They also showed a strict requirement for the 3’ hydroxyl group and 5’ phosphate group on siRNAs for primer extension in the RdRPmediated reaction [65]. while investigating the dsRNA-dependent degradation of target mRNA in a Drosophila embryo cell extract system. KG. H. et al. it was proposed that this type of polymerase might replicate siRNAs as epigenetic agents.

Finally. In an RNAi reaction. H. GERMANY . was converted into a 100 kDa complex upon being activated by ATP. Additionally. The size and constitution of the precursor as well as the activated RISC might vary depending on the choice of system [68]. It is widely believed that this nuclease is probably different from Dicer. as mentioned earlier. This activated complex cleaved the substrate. judging from the substrate requirements and the nature of the end products. and the complex cuts this mRNA approximately in the middle of the duplex region. [66] further revealed the role of RdRP activity in RNAi. A part of cleaved fragments of mRNA at the end of step 2 might also be converted to the duplex forms by the RdRP-like activity. This kind of RNAi induced by secondary siRNAs was named transitive RNAi. The antisense siRNAs in the activated RISC pair with cognate mRNAs.Dhingra. the cleaved mRNAs are perhaps degraded by exoribonucleases [70]. KG. Zamore et al [69] demonstrated that a 250 kDa precursor RISC. DUDWELLER LANDSTR. Amplification of siRNAs might occur at various stages of the RNAi reaction and has been documented in plants. They named these new siRNAs secondary siRNAs. elegans gene with sequence homology to RdRP. N. a C. the double-stranded siRNAs bind an RNAi-specific protein complex to form a RISC. The mRNA cleaving RNA-protein complexes have also been referred to as siRNP (small interfering ribonucleoprotein particles). With a primary trigger dsRNA specific for the lacZ region of the target mRNA that encoded a GFP-LacZ fusion protein. These authors demonstrated the requirement for the rrf1 gene. found in Drosophila embryo extract. melanogaster [68]. a conformational rearrangement or a change in the composition of an siRNP ahead of the cleavage of target mRNA is postulated. C. Degradation of mRNA: In the effector step of RNAi. This complex undergoes activation in the presence of ATP so that the antisense component of the unwound siRNA becomes exposed and allows the RISC to perform the downstream RNAi reaction. 2011 Current Topics in Biotechnology & Microbiology Sijen et al. crassa. these authors demonstrated the degradation of a separate GFP mRNA target. it is surprising that the fly genome does not code for RdRP. These forms might have 79 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. in the generation of secondary siRNAs and transitive RNAi [66]. Since the target cleavage site has been mapped to 11 or 12 nucleotides downstream of 5’ end of the guide siRNA. numerous experiments also suggest that RdRP is not required for RNAi in D. et al. Though the RdRP activity is present in Drosophila embryo extract.elegans. they observed the formation of new siRNA species corresponding to target mRNAs but different from trigger dsRNAs. A few independent studies demonstrated the importance of the RISC complex in this part of RNAi reactions. and Dictyostelium discoideum but not in flies and mammals [67].

a single point mutation in the tumour suppressor p53 was effectively targeted but not the wild-type product using siRNAs [74]. dsRNA 80 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Cancer: Cancer cells exhibit two main abnormalities. it is likely that amplification of the RNAi reaction takes place at both step 1 and step 2 of RNAi. Uncontrolled growth and resistance to cell death (apoptosis) [72].The goals for RNAi approaches for cancer therapy are therefore to modulate the expression of apoptotic protein/cell cycle protein in the cancer cells thereby stopping tumour growth and killing the cancer cells. a retroviral vector was used to specifically and stably inhibit expression of the oncogenic K-RASV12 allele in human tumor cells [73]. RNAi technology can be directed against cancer by specifically modulating the expression of oncogenes. All these models were summarized by Schwarz et al. Ideally this would be accomplished with no or tolerable side effects. Similarly. et al. A. the RNAi is used to target a gene specifically involved in the growth or survival of the cancer cell. For example. but instead assemble along the length of the target RNA and are then ligated together by an RNA ligase to generate cRNA. we will focus on four different types of diseases that are very common and for which RNAi approaches are currently being tested in preclinical studies. [71]. Advantages of RNAi methods for the growth suppression and killing of cancer cells have been shown in different studies. The ability of siRNAs to silence specific oncogenic variants while sparing the wild-type products of genes has been demonstrated in human pancreatic carcinoma [73]. In another model. Although there are candidate gene targets for many different diseases. In addition to blocking the expression of normal genes that are required for cancer cell growth and survival. The cRNA and target RNA hybrid would then be diced by the DCR protein. H. 2011 Current Topics in Biotechnology & Microbiology siRNA-like functions and eventually enter the pool of the amplification reaction. Depletion of K-RASV12 resulted in loss of anchorage-independent growth and tumorigenicity. GERMANY . it has been proposed that siRNAs do not act as primers for the RdRP-like enzymes. KG. RNAi can be used to target specific cancer-causing mutations. Thus. For example. To selectively eliminate cancer cells without damaging normal cells. Viral vectors have also been used to express siRNAs and inhibit cancer cell growth and tumorogenicity. promoting apoptosis and regulating cell cycle.Dhingra. DUDWELLER LANDSTR. Most of the steps involved in the mechanism of RNAi have been illustrated schematically in Fig 1. THERAPEUTIC APPLICATIONS OF RNA INTERFERENCE RNAi is an excellent therapeutic tool for disease in which selective knockout of a gene would slow or completely halt the disease process in the affected cells.

vif [80]. Fas siRNA treatment abrogated hepatocyte necrosis and inflammatory infiltration and markedly reduced serum concentrations of transaminases demonstrating a clear hepatoprotective effect of the siRNA therapy. Tumor cells require a rich supply of blood and achieve this by stimulating the process of angiogenesis. Intravenous injection of Fas siRNA specifically reduced Fas protein levels in the livers of mice during 10-day period. resulting in the inhibition of HIV replication in numerous human cell lines and in primary cells 81 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. holds promise for the treatment of human patients. gag [83. Despite the success of RNAi-mediated inhibition of HIV-encoded RNAs in cell culture. 82]. Transfection of human cells with siRNAs against different genes in the poliovirus genome resulted in resistance of the cells to infection with poliovirus [78]. it may therefore be possible to inhibit tumor growth by targeting the vascular endothelial cells involved in angiogenesis. RNAi-mediated downregulation of the cellular cofactors required for HIV infection is an attractive alternative or complementary approach. DUDWELLER LANDSTR. B. 2011 Current Topics in Biotechnology & Microbiology was employed to target the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement [75]. the HIV receptor CD4 [83] and the co-receptors CXCR4 and CCR5 [86] have been successfully downregulated by RNAi. The next step in the development of RNAi technology for cancer therapy will be to establish methods for targeting tumor cells in vivo. nef [80] and reverse transcriptase [82]. Another approach might be to target genes that promote angiogenesis. 84]. Infectious Diseases: The ability of RNAi to inhibit the replication and cellular uptake of viruses and other infectious agents has been clearly demonstrated in cell culture studies and therefore. it was shown that depletion of the crk adaptor protein using RNAi inhibited the migration of cultured vascular endothelial cells [77]. targeting the virus directly represents a substantial challenge for clinical applications because the high viral mutation rate will lead to mutants that can escape being targeted [85]. As evidence. tat [81. env [84]. Cellular cofactors such as NF-B [82]. [79]. et al. H. Leukemic cells without BCR/ABL rearrangement were not killed by MBCR/ABL-dsRNA. The ability of siRNAs targeting the gene encoding the death receptor Fas to protect mice from liver failure and fibrosis in two models of autoimmune hepatitis was tested by Song et al. GERMANY .Dhingra. KG. rev [ 81]. Synthetic siRNAs and expressed siRNAs have been used to target several early and late HIV-encoded RNAs in cell lines and in primary haematopoietic cells including the TAR element [80]. Several other studies have demonstrated efficacy of liposome-mediated or viral vector-mediated transfection of cancer cells in suppressing their growth and/or inducing their death [76].

2011 Current Topics in Biotechnology & Microbiology including T lymphocytes and haematopoietic -stem-cell-derived macrophages [80. dopamine-producing neurons in the substantia nigra that control body movements in amyotrophic lateral sclerosis. 84. Huntington’s disease and Amyotrophic lateral sclerosis (ALS) are examples of relatively common age related neurodegenerative disorders that are increasing as average life expectancy increases. Specific genetic mutations are responsible for a small percentage of cases of Alzheimer’s and Parkinson’s disease and Amyotrophic lateral sclerosis [94]. Cardiovascular and Cerebrovascular Diseases: Cardiovascular disease is the leading cause of death in many industrialized countries. The production of cell adhesion molecules can be selectively suppressed in cultured cells [93]. Advances in targeted delivery of RNAiinducing molecules has raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. In addition. whereas all cases of Huntington’s disease result from mutations (polyglutamine expansions) in the Huntington protein [95]. DUDWELLER LANDSTR. et al. 92]. Each disorder is characterized by the dysfunction and death of specific populations of neurons: hippocampal and cortical neurons involved in learning and memory processes in Alzheimer’s disease. A key step in the process of atherosclerosis is the up-regulation of cell adhesion molecules in vascular endothelial cells. H. KG. which play an essential role in the recruitment of macrophages to the site of endothelial damage. Neurodegenerative Disorders: Recent rapid progress in the application of RNAi offers new approaches to drug target identification and validation. Studies of patients and of animal and cell culture models of each disease have revealed shared biochemical cascades that result in neuronal death.Dhingra. many other cells die more slowly by apoptosis. apoptosis of foam cells and vascular smooth muscle cells occurs [90]. The severe irritability that occurs in heart or brain cells during a myocardial infarction or stroke results in the death of cardiac tissue rapidly by necrosis. 81. It may be possible to use RNAi technology to intervene in the process of atherosclerosis or to reduce the damage to heart tissue and brain cells that patients suffer following a myocardial infarction or stroke. local production of inflammatory cytokines and the recruitment of macrophages to the site forming foam cells. Parkinson’s disease. Atherosclerosis involves damage to vascular endothelial cells. It most commonly results from the progressive occlusion of arteries in a process called Atherosclerosis. 8689]. data from animal studies suggest that such cardiac myocytes and brain neurons that die by apoptosis can be saved [91. GERMANY . 82 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. which can ultimately culminate in a myocardial infarction or stroke. D. C. Alzheimer’s disease.

2011 Current Topics in Biotechnology & Microbiology Those cascades include increased oxidative stress. CHALLENGES IN RNA INTERFERENCE • Intravascular degradation: Naked siRNA is unstable in circulation owing to serum RNase A-type nucleases and rapid renal clearance. the backbone or the bases of oligoribonucleotides for stabilization [101. Although nanoparticles by definition range in size from 1 nm to 1. KG. H. to selectively silence a transcript associated with an important group of time genetic diseases by RNAi. Some investigators have turned towards the chemical modification of the sugars. at least in cell culture. Thus. the hydrophobic cell membranes create a challenge for the intracellular delivery of negatively charged polymers. Therefore. many variables must be negotiated.000 nm in size. whereas the second strategy targets downstream events in the neurodegenerative cascade. spleen. a protein in the TNF receptor family that has been implicated in neuronal apoptosis in certain settings [96]. However. lung and bone marrow. In one study. DUDWELLER LANDSTR. et al. • Tissue penetrance and intracellular delivery: For safe and effective delivery of RNAi to the mRNA target of interest. leading to degradation and a short half-life [100]. One strategy is to block the disease-specific events that are believed to initiate the neurodegenerative process. there have been two different strategies for preventative and therapeutic interventions in neurodegenerative disorders. extracellular factors in the microenvironment are less well studied 83 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.[103] • The fate of RNAi in biological fluids: Although intracellular barriers to RNAi delivery are being explored in depth. it has become clear that intravenously injected particles > 100 nm in diameter are likely to be trapped by the reticuloendothelial system (RES) in the liver. it was shown that cultured neurons can be depleted of the p75 neurotrophin receptor. now it is possible. dysregulation of cellular calcium Parkinson’s disease and spinal cord motor neurons in homeostasis and apoptosis [91]. leading to degradation by activated monocytes and macrophages. Recent studies have shown that cultured neurons can be efficiently transfected with siRNAs and that the targeted genes are effectively silenced. GERMANY .Dhingra. Caplen and colleagues [61] performed studies aimed at determining whether RNAi could be used to target the pathogenic process in inherited neurodegenerative disorders caused by polyglutamine expansions.102]. Proapoptotic members of the Bcl-2 family [97] and caspases [98] have been effectively targeted and neuronal death have been prevented using RNAi methods.

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2011 Current Topics in Biotechnology & Microbiology Chapter V BIOTECHNOLOGICAL INTERVENTIONS TO COMBAT ABIOTIC STRESS IN PLANTS Rajesh Mehrotra1. KG. and grain yield of most crops [1. Louisiana 70803. Pilani. biomass production and accumulation. DUDWELLER LANDSTR. Nevertheless. nuclear factors and stress responsive genes. Purva Bhalothia1. in recent times. Keywords: Stress. 2 Louisiana state university. H. Rajasthan. Bhavya Ravi1 and Sandhya Mehrotra1 1 Department of Biological sciences. Abiotic stress has been shown to negatively influence survival. This chapter focuses on the various types of stress conditions such as drought. chilling INTRODUCTION Abiotic factors such as water availability and temperature extremes have had a major influence on the evolution of plant systems. Uthra Balasubramaniyan1. salinity. salinity and temperature extremes and their molecular manifestations with an emphasis on signaling cascades involving phosphatases. drastic environmental disturbances have affected agricultural productivity to a large extent. E-mail: rajmeh25@hotmail. Baton Rouge. protein engineering.2. The role of cis elements present in promoters and the design of synthetic promoters for achieving inducible stress-specific expression profiles have also been explored.com ABSTRACT: Molecular programming of plants enables them to withstand diverse environmental stress conditions by dynamic alterations in physiology and cellular composition. India Pin-333031. GERMANY . kinases. Birla Institute of Technology & Sciences (BITS). The perception of varying environmental conditions and the processes of adaptation for survival under these conditions represent a complex interplay between various nuclear factors and signaling molecules. et al. Corresponding Author: Rajesh Mehrotra.Dhingra. An insight into the mechanisms involved in such adaptations will help not only in engineering stress resistance in agronomically important crop varieties but also in understanding patterns of plant development and distribution in response to the environment. Engineering of stress tolerance therefore demands a comprehensive knowledge of genes and regulatory components activated in response to different 93 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.2].

which is the basic unit of chromatin structure [3]. These have interesting implications both in terms of stress tolerance and understanding of plant biochemical and developmental processes. Histone methyltransferases on the other hand transfer methyl groups to histones and can either activate or further repress transcription depending on the amino acid that is methylated and other covalent modifications in the vicinity. eukaryotic genes are present as chromatin which is a complex of DNA and histone proteins. research has focused on the characterization of abiotic stress related genes. trans-acting elements and the membranous and intercellular proteins involved in signal transduction pathways is indispensable in the events that finally result in transforming the genetic information on DNA into protein products. require a change in this chromatin structure. Many recent studies have suggested that genes involved in abiotic stress tolerance are regulated at the level of transcription and hence a brief understanding of transcription regulation becomes essential at this stage. TRANSCRIPTIONAL REGULATION: AN OVERVIEW DNA. 94 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. cannot result in protein synthesis. thus loosening the histones and making DNA available for transcription. Histone acetyltransferase (HAT) and deacetylase mediate the addition of negatively charged acetyl groups to the basic residues of histone tail. DUDWELLER LANDSTR. H. transcription factors and regulatory sequences present in plant promoters. This represents the default condition of an inactive gene. in isolation. H3 and H4) in two left-handed super helical turns and the assembly is referred to as a nucleosome.Dhingra. A stretch of approximately 160 base pairs of DNA wraps around the central kernel of the histone octamer (H2A. Even within the nucleus. This reduces the electrostatic interaction between the histones and DNA. Since abiotic stress responses are multigenic in nature. 2011 Current Topics in Biotechnology & Microbiology kinds of stress. The nucleosomes are attached by short stretches of linker DNA and are wound into tight solenoids stabilized by histone H1 which assembles the nucleosomes into tight arrays thereby preventing the transcription factors and RNA polymerase from gaining access to the genes [4]. GERMANY . KG. et al. The protein machinery comprising the basal transcription factors. H2B. Gene activation and regulation therefore. [4] In a very elegant study Wada et al have shown association between up regulation of stress responsive genes and hypomethylation of genomic DNA in tobacco plants [6]. The two major classes of chromatin modifying complexes that have been characterized are histone acetyltransferases and methyltransferases and ATP dependent chromatin remodeling complexes [5].

The most common consensus sequences found in promoters are the CCAAT box (known as the CAT box). GC box and the TATA box (consensus TATAAA sequence). they have been classified into two main groups. They are usually located upstream of the transcription start site where the RNA polymerase binds. Trans-acting factors are proteins that bind to the cis-acting sequences like promoters. eukaryotic RNA polymerase II which is responsible for transcribing mRNAs cannot directly bind to the DNA sequence of a promoter.Dhingra. TFIIA. Through their DNA binding domain. A third class of ATP-dependent complexes contains a Snf2-like ATPase which also displays deacetylase activity. Multiple families of transcription factors have 95 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. et al. An enhancer that is important for the expression and regulation of AtHKT1gene involved in salt tolerance has been reported [8]. Based on the identity of this subunit. or within the intron sequence. 2011 Current Topics in Biotechnology & Microbiology ATP dependant chromatin remodeling complexes use the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA. It requires other protein factors called basal transcription factors such as TFIID. They contain an ATPase subunit that belongs to the SNF2 superfamily of proteins. H. TFIIE. Enhancers are modular in the sense that a gene can have several enhancer elements turning it on in different cells. Coupling element 1 and coupling element 3 have been shown to be involved in dehydration stress. TFIIB. Cisregulatory elements are small nucleotide sequences usually arranged in a unique fashion in a promoter regulatory region. Enhancers are cis-regulatory elements which act on promoters situated on the same chromosome and are situated upstream or downstream relative to the gene. TFIIF and TFIIH which bind to the promoter in a defined order and interact with RNA polymerase II to efficiently transcribe the mRNA. thus regulating the expression of gene located in front of them. KG. they bind to the cis-acting sequences and through their trans-activating domain interact with proteins bound with RNA polymerase to activate or suppress the transcription of a gene. DUDWELLER LANDSTR. In addition to the open reading frame (ORF) sequences coding for proteins. For instance. These elements have an ACGT core motif to which bzip class of proteins bind. specific DNA sequences called cis regulatory elements are crucial for the regulation of transcription. enhancers and silencers and interact with one another to activate or repress transcription of a gene. GERMANY . the SWI2/SNF2 group and the imitation SWI (ISWI) group [7]. The size of cis-regulatory elements varies from 3 to 25 nucleotides and they interact specifically with the transcription factors.

exposure of leaf and root tissues of Agrostis stolonifera to high temperatures showed significant changes in membrane lipid composition with high levels of palmitic acid and reduced amounts of linolenic acids [11]. 96 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Temperature stress resistance in plants can be mainly categorized into: 1. KG. can generally withstand higher temperatures because they keep their stomata closed during the day and as a result do not lose water by transpiration [10]. proteins like Hox and Pax belong to the homeodomain family and are involved in turning on several developmental genes. Other families include the zinc finger proteins. H. et al. Inositol triphosphate (IP3) and reactive oxygen species (ROS). DUDWELLER LANDSTR. For instance. These include ligand binding to specific cell surface receptors.Dhingra. GERMANY . These factors may be expressed in a spatial or temporal stage inside the cell or upon activation by modifications like phosphorylation or binding of specific ligands to the enhancer sites for the initiation of transcription. Heat tolerance 2. phosphorylation and activation of nuclear transcription factors. helixloop-helix family and basic leucine zipper [4]. For example. phosphatases and nuclear transcription factors [9]. Generally plant systems are maintained 5-8 degrees lower than the surroundings through transpirational cooling. The signaling machinery is complex and involves secondary messengers (Ca2+. sensory kinases. CAM plants. The activation of specific transcription factors in cells is achieved through specific signal transduction pathways. 2011 Current Topics in Biotechnology & Microbiology been characterized and grouped based on structural similarities and their mode of action. ENVIRONMENTAL STRESS CONDITIONS TEMPERATURE STRESS The temperature maintained in plant systems relies heavily on the ambient environment. Change in the lipid composition of membranes thus has severe effects on the action of membrane-associated enzymes and electron carrier molecules which are involved in photosynthesis and respiration. Chilling tolerance and 3. Freezing tolerance Heat tolerance: Elevated temperature levels have disastrous consequences on cellular organization due to the denaturation of proteins and alterations in membrane fluidity. in particular.

A specific transcription factor. Chilling tolerance: Chilling temperatures generally lead to inhibition of photosynthesis. Chilling – sensitive plants have been found to contain a high proportion of saturated fatty acids in their membrane phospholipids which tend to crystallize at lower temperatures. but they are found to be mainly expressed during certain developmental stages [13]. glycine betaine. γ-4amino butyric acid (GABA).Dhingra. Plants exposed to high temperatures also begin to accumulate osmotic compounds to reduce the water potential of cells. particularly the chloroplast and mitochondrial membranes. Certain compounds such as sucrose. The physiological changes include development of leaf hairs and waxes. GERMANY . change in orientation of the leaf and the development of small leaves so as to minimize the boundary layer resistance for heat transfer to the environment. flowering and seed dormancy are also found to be overproduced in thermo tolerant plants [12]. ice crystals begin to form outside cells resulting in dehydration of cells. et al. raffinose and sorbitol also help in freezing tolerance by acting as cryoprotectants. This implies an impaired membrane function. Plant proteins called anti-freeze proteins (AFPs) help in preventing ice crystal formation by binding to ice crystal surfaces and inhibiting their growth [14]. Their function is still not clear. H. the Hsp90 family. acts on the transcription of the HSP mRNA. Molecular responses to heat stress involve the production of heat shock proteins (HSPs). mitochondria. KG. chloroplast and endoplasmic reticulum. which act as molecular chaperones and assist in proper folding of cellular proteins. Low molecular weight HSPs (15-30 kDa) are found abundantly in plants. 97 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. degradation of cellular proteins and lower respiration rates. This lowers the temperature at which phase transition of membrane lipids occur. the Hsp100 (Clp) family. plants have evolved several adaptations to heat stress. DUDWELLER LANDSTR. Plant hormones such as abscisic acid and ethylene which have roles in normal developmental processes such as seed germination. HSF. mannitol. membrane lipids of chilling resistant plants have been found to possess a high proportion of unsaturated fatty acids and desaturase enzymes [10]. 2011 Current Topics in Biotechnology & Microbiology Therefore. Five different classes of HSPs have been found in plants which have been found to be localized to cytosol. When plants are exposed to long periods of freezing temperature. the chaperonins (GroEL and Hsp60) and the small Hsp (sHsp) family. The trimeric form of this factor accumulates during heat stress and upon phosphorylation activates the production of HSP mRNAs [10]. These include proline. In contrast. These include the Hsp70 (DnaK) family.

As the temperature drops below 0 degrees. ICE (Inducer of CBF expression) [21]. has been cloned by using a polyclonal antibody [18]. photosynthesis. et al. The interaction between AFPs and ice crystals has been studied. signaling pathways which are membrane dependent. CBF2. cDNA encoding an antifreeze protein of 64kDa from the bittersweet nightshade. KG. The difference between melting and freezing temperature is defined as thermal hysteresis and it forms the basis for the quantitative assay of AFPs [17]. 2011 Current Topics in Biotechnology & Microbiology Freezing tolerance: Freezing tolerance in plants generally refers to prevention of cellular dehydration. DUDWELLER LANDSTR. This is thought to be the reason behind evolution of different AFPs. CBF/DREB1-type transcription factors bind to CRT/DRE elements (C-repeat/dehydration-responsive.Dhingra. H. apoplastic ice formation begins to occur [15]. ABA-independent sequence elements) in gene promoter sequences. Genes coding for antifreeze proteins have been cloned by several researchers. Several cryoprotective proteins such as antifreeze proteins are produced by plants against freezing stress [16]. GERMANY . dulcamara. This is accompanied by the formation of extracellular ice crystals. This severely impairs membrane-associated processes such as ion transport. AFPs are thought to minimize freezing damage by inhibiting recrystallisation and minimizing the size of crystals. This freezing is further enhanced by ice nucleators which trigger ice formation. and CBF3). They result in an overall decrease in the freezing temperature of the plant. metabolite synthesis. Studies have revealed that CBF1/DREB1 is induced by cold stress whereas DREB2 is induced by dehydration stress [20]. The expression of CBF/DREB1 is controlled by a transcription factor. Chilling-resistant plants can be produced by acclimation to cold conditions and exogenous application of ABA. Cold-stress induced genes are activated by transcription factors such as C-repeat binding factors (CBF1. winter S. Different AFPs interact with different sides of ice crystals and inhibit growth. Plants employ both colligative and non-colligative mechanisms to combat freezing-dehydration. It is thought that AFPs interact with the relatively flat surfaces of ice crystals through van der waal’s interactions and hydrogen bonds [19]. Membrane phospholipids: Functions and possible engineering mechanisms: Membrane lipids form the backbone for several cellular functions and are the most susceptible to 98 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. This protein was found to contain a zinc finger motif and a specific DNA binding ability. Intracellular ice formation is highly deleterious to the cell as the plasma membrane is ruptured. Apart from inducing freezing tolerance. AFPs have been mainly isolated from the apoplastic space where they interact with ice crystals and modify their growth.

It was observed in such mutants that the eukaryotic pathway predominated and compensated for the shortcomings in the prokaryotic pathway thus maintaining a constant lipid composition [23] . KG. As they are not tightly packed. DUDWELLER LANDSTR. Several desaturases have been characterized by mutational studies of A. phosphatidylethanolamine (PE) and phosphatidylinositol (PI) [22]. fab1 mutants of A. GERMANY .Dhingra. et al. The lipid components of plastid and cytoplasmic organelle membranes follow separate biosynthesis pathways. 2011 Current Topics in Biotechnology & Microbiology environmental stress conditions. While the chloroplast membrane is mainly composed of monogalactosyl diacylglycerol (MGD). Additional polyunsaturation is achieved by the action of membrane associated desaturases. All the major glycerolipids present in membranes are initially synthesized with 16:0 and 18:0 acyl groups [22]. Studies with fad3-2fad7-2 fad8 mutants have shown that trienoic fatty acids in membrane lipids may have important functions in enhancing the tolerance of the photosynthetic machinery to temperature extremes [24]. Both these mutants show enhanced levels of saturated fatty acids compared to wild type. H. thaliana have reduced levels of the enzyme ketoacyl synthase II which is responsible for the elongation of 16:0 fatty acids to 18:0 acyl entities. This is in contrast to phosphatidic acid produced in the extrachloroplast membranes where 18 carbon chains are found at the sn-2 position and 16:0 fatty acids at the sn-1 position predominantly. Thus it has been observed that every membrane has a specific lipid composition and these lipids in turn have defined fatty acid compositions. For instance. phosphatidic acid produced in the plastids always contains 16:0 at sn-2 position and 18:1 at sn-1 position. the endoplasmic reticular membrane and other extrachloroplast membranes are mainly composed of phosphatidylcholine (PC). For instance. fad6 mutants contain reduced levels of 18:3 and 16:3 FAs and elevated levels of 18:1 and 16:1 precursors due to the deficiency of chloroplast desaturase. They have been studied for roles in membrane stability in relation to cold tolerance. Polyunsaturated fatty acids are very important when considering low temperature membrane behavior. digalactosyl diacylglycerol (DGD) and sulfoquinovosyl diacylglycerol. thaliana [23]. The engineering of membrane lipids therefore offers exciting possibilities for enhancing stress tolerance. This is shown in studies involving act1 mutants which are deficient in chloroplast acyl-ACP sn-glycerol-3-P acyl transferase. Fab2 mutants which have a mutated 18:0 ACP desaturase isozyme have also been studied. Efforts have also been made to alter the level of polyunsaturated fatty acids (PUFA). they don’t crystallize easily at low temperatures. The importance of PUFAs for endowing 99 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The different pathways for lipid biosynthesis go hand in hand for maintaining the defined lipid composition of membranes.

The level of saturated and unsaturated fatty acids is very important. Several studies have shown that the thylakoid membrane is extremely sensitive to temperature changes. K+. They are mainly regulated by Ca2+. and diacylglycerol kinase. Since PLDs play a major role in membrane lipid hydrolysis.Dhingra. Salt stress and drought stress have overlapping consequences as salt concentration and water deficit are closely linked. The model plant A. PLD activated during low temperature acclimation induced the production of salicyclic acid and HSP73 which are important mediators of thermo-tolerance [28]. Excessive quantities of Na+ cause membrane disintegration. DUDWELLER LANDSTR. Regulating the levels of PLD in plants would therefore aid immensely in preserving the integrity of cell membranes during conditions of stress. thaliana has been found to contain 12 distinct PLD families. Studies with low temperature acclimatised grape berries (Vitis vinifera) showed that when exposed to high temperatures. They mainly contain MGD. Phosphatidic acid (PA) is an important secondary messenger which is produced with the help of phospholipase D (PLD) and phospholipase C (PLC). H. Phospholipase D: Threatening membrane integrity: Phospholipase D cleaves membrane phospholipids such as phosphatidylcholine. DGD. The major ions involved in salinity stress are Na+. acyl hydrolases and lipoxygenases. The main domains that are present are phox homology domain. High salinity causes an increase in 100 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. These mutants show poor phenotypic characteristics at low temperatures which underlines the importance of PUFAs [25]. SL and PG as component lipids. 2011 Current Topics in Biotechnology & Microbiology low temperature tolerance is further strengthened by studies with fab2 mutants that are deficient in ER 18:1 desaturases. it might be overexpressed during conditions of stress. It can be further broken down to soluble molecules by phosphatases. pleckstrin homology domain and C2 domain. toxicity to metabolic pathways and a reduction in photosynthesis. H+ and Ca2+. This is reflected in studies with mutants of Chlamydomonas reinhardtii with reduced levels of unsaturated fatty acids which show high heat tolerance with enhanced activity of PSII [26]. PIP2 and alpha subunit of G-proteins [29]. et al. KG. SALINITY STRESS High salinity leads to osmotic stress in plants and is largely dependent on the amount of evaporation and precipitation. GERMANY . their regulation is very important. phosphatidylinositol and phosphatidylethanolamine (PE) to produce phosphatidic acid [27]. Since this enzyme is involved in disruption of membrane integrity.

This effect is compensated by the accumulation of metabolites called as compatible solutes [32]. Arginine decarboxylase (ADC). GERMANY . SOS3 and SOS2 are thought to down regulate AtHKT1 activity under salt stress. proline and ectoine are also accumulated. high salinity and potassium deficiency while ADC1 is responsible to cold stress. Salt stress almost invariably leads to cellular dehydration. ABA produced during abiotic stresses also induces genes involved in polyamine synthesis. These include sugars and sugar alcohols such as sucrose. SOS2/CIPK24 encodes a serine/threonine protein kinase and contains an auto inhibitory domain. H. Salt stress mechanisms mainly focus on efflux of Na+ across the plasma membrane and sequestration into intracellular compartments. It has been found that Athkt1 mutants also have deficiency in SOS3 function. trehalose. SAMDC1 and SAMDC2 have been found to be induced by cold [33].Dhingra. SOS3 interacts with SOS2 through this auto inhibitory domain and activates it in a calcium-dependent manner. raffinose and mannitol. SOS3 is a myristoylated calcium binding protein that can change its conformation in response to calcium ion concentration. The presence of such compatible solutes increases the water potential of cells and thereby prevents water loss. Mutants in Arabidopsis with reduced ADC activity. In Arabidopsis. Osmolytes such as glycine-betaine. The transport of solutes such as Na+ and K+ across the plasma membrane is aided by the H+-ATPases that generate the proton motive force required for these processes. 101 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. For instance. Polyamines: Polyamines are aliphatic nitrogen compounds that are positively charged at physiological pH. AtHKT1 codes for a histidine kinase transporter which mediates Na+ entry into the root cells of Arabidopsis [30]. KG. 2011 Current Topics in Biotechnology & Microbiology cytosolic Ca2+ levels. The genes involved in salt stress belong to the SOS (Salt Overly Sensitive) family. spe-1 and spe-2 are found to have reduced salt tolerance [34]. Many Arabidopsis mutants have been studied to determine their function in salinity stress. agmatine iminohydrolase and N-carbamoyl putrescine amidohydrolase and S-adenosine methionine decarboxylase (SAMDC) are the major enzymes involved in the biosynthesis of polyamines. et al. DUDWELLER LANDSTR. SOS2 has been found to interact with vacuolar Na+/H+ antiporter and H+/Ca2+ antiporter [31]. wounding. fructose. ADC2 has been found to be induced in response to drought. Overexpression of genes involved in the biosynthesis of osmolytes has been investigated as a possible mechanism for salt stress induction.

In response to drought stress. The accumulated ABA causes a depolarization of the guard cell membrane which results in reduced stomatal conductance. H. DUDWELLER LANDSTR. the synthesis of LEA (Late Embryogenesis Abundant)/dehydrin-type genes and proteinases is up regulated. dehydration response element (DRE) and DRE binding factors involved in ABA-independent regulation of gene expression during osmotic stress [20. Several major regulatory elements that are active in response to abiotic stress have been identified in Arabidopsis. Dehydration-responsive element binding protein 1(DREB1)/C-repeat binding factor (CBF) and DREB2 function in an ABA-independent gene expression. showed that dehydration response gene RD22BP1.In addition to these major pathways. which have been shown to be present in the promoters of stress responsive genes [35]. But this response may affect the rate of photosynthesis with the limited availability of CO2. and ATMYB2. hydrogen peroxide and hydroxyl radicals are produced. whereas the ABA-responsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) functions in an ABA-dependent gene expression . transport of ABA to the shoots is enhanced. 39]. 2011 Current Topics in Biotechnology & Microbiology DROUGHT STRESS Drought is considered as one of the major abiotic stresses in the world. MYC and MYB related proteins act as transcriptional activators of 102 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Due to a decrease in pH of the xylem sap during water deficit. ABRE motif is somewhat similar to the G-box that functions in regulating plant gene expression in response to light conditions and wounding. The electron transport chain is affected and reactive oxygen species such as superoxide. ATAF1. Water stress affects the integrity of the lipid bilayer and leads to increased salt concentration inside cells thereby creating osmotic imbalance. 38. other regulatory factors including the NAC (NAM. On the other hand. KG.Dhingra. Hydroactive closure of stomata which is metabolically dependent on ion fluxes is regulated by ABA. Most of the plants respond to drought stress by closing their stomata to avoid transpirational water loss. The promoter of rd29A has been found to contain both ABRE as well as DRE/CRT elements. et al. ABA-mediated regulation of gene expression involves cis ABA responsive (ABRE) and coupling (CE) elements. 37. required for ABA responsiveness. Proteins (ABFs) that bind to ABRE elements containing a basic leucine zipper (bZIP) motif activate ABA stress responsive genes [36].2 and CUC) and MYB/MYC (myeloblastosis/ myelocytomatosis) factors are involved in abiotic stress-responsive gene expression [20]. GERMANY .

ABA promotes the efflux of potassium ions from the guard cells which reduces the turgor pressure of the stomata and causes stomata closure and inhibits its opening during stress conditions. DUDWELLER LANDSTR. [44] AREB/ABF bZIP proteins are found to require post-translational modifications for their activation. bZIP subfamily members such as AREB1/ABF2. Overexpression of AtHD2C. 2011 Current Topics in Biotechnology & Microbiology rd22 (dehydration response) in response to drought and ABA. or the combination of an ABRE with a coupling element (CE). [45] 103 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Due to its capability of inducing desiccation tolerance in developing seeds. it is known to have functions in water stress tolerance. H. KG. Enhanced levels of ABA are often found in plants subjected to stress. which is repressed by ABA results in a lower transpiration rate in comparison to the wild type plants. AREB2/ABF4 and ABF3 have been found to be induced in the case of dehydration and ABA treatment in vegetative tissues. [10] At the molecular level. respectively. which are crucial for plant responsiveness to ABA and negatively regulate ABA signaling. Conditions of water scarcity lead to a rise in the pH of the xylem sap which promotes the transport of ABA through the root xylem into the shoot.Dhingra. ERF7 (Ethylene responsive factor 7) from Arabidopsis thaliana is found to interact with atsin3 (arabidopsis thaliana sin3) and hda19 (histone deacetylation 19) to repress transcription of genes. This is performed by SnRK kinase family members in the presence of ABA. GERMANY . Chromatin remodeling has also been found to be responsible for stomatal closure in plants. [42] RECENT ADVANCES IN ABIOTIC STRESS TOLERANCE ABA is an important plant hormone which has roles in inhibition of growth and stomatal opening. et al. PyACGTGG/TC). [30] The promoters of ABA-inducible genes contain multiple copies of cis elements known as ABA-responsive elements (ABREs. particularly under conditions of stress. 41] isolated and characterized arabidopsis ABI1 and ABI2 homologous (ABA insensitive 1 and 2) loci. It is also involved in the regulation of seed maturation and dormancy. Leung et al. [40. this is achieved by chromatin remodeling through changes in histone acetylation. Transgenic Arabidopsis plants have been created which express aterf7 and are sensitive to ABA in guard cells leading to an increase in transpirational water loss [43].

ABI2. ABI1 is expressed in seeds and guard cells and found in both cytosol and nucleus. AHG1/AHG3 (ABAhypersensitive germination I) have been developed which act as negative regulators of ABA. 50]. The expression of PP2Cs varies in different tissues. DUDWELLER LANDSTR. The protein serine/threonine phosphatases are further classified into the PPP (protein phosphatase P family) and PPM (protein phosphatase magnesium divalent ion dependent family) gene families. et al. The whole mechanism is reversed in the absence of ABA. These receptors are known to inhibit the activity of phosphatases especially protein phosphatase 2C (PP2C) [51]. form hydrogen bonds with a conserved tryptophan residue in PP2C and result in inhibition of its phosphorylating activity [46]. PP2Cs are monomeric enzymes and the largest protein phosphatase family in plants known. [49. 2011 Current Topics in Biotechnology & Microbiology Regulation of ABA mediated stress responses: ABA signaling involves the following essential components [46]: 1) PYR1/PYL/RCAR1 receptors. PP2A. whereas AHG1 and AHG3/AtPP2CA are expressed mostly in seeds and localized to the nucleus. The PPP family comprises PP1. For instance. [46] This evidence was supported by developing double or triple PP2C knockout mutants which shows more hypersensitivity to ABA [49. 3) Protein kinases (SnRK2) and.Dhingra. H. was found to encode protein phosphatase 2C (PP2C). PPM family includes PP2Cs and these have not been found to share any sequence homology with PPPs [47]. The receptor serine residues (PYL1 S112 and PYL2 S89) that are exposed upon ABA binding. HAB1 (homology to ABI1) HAB2. As soon as ABA binds one of the loops containing β-pleated sheet closes like a gate bringing all the amino acids present in the cavity in close vicinity to the bounded ABA [52]. ABA interacts with the lysine residues of the target receptor molecule through a carboxylic acid residue. The first ABA-insensitive mutant that was developed. 2) Protein phosphatases (PP2C). 4) Downstream targets (ABRE) Phosphatases as negative regulators: Protein phosphatases are broadly classified into two major classes: protein tyrosine phosphatases and protein serine/threonine phosphatases. Several ABA-insensitive mutants such as ABI1. PP2C interacts with the 104 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 76 members have been found in Arabidopsis thaliana [48]. and PP2B which share sequence homology in their catalytic domains and are sensitive to specific inhibitors like okadaic acid. ABI1. KG. ABA interacts with the water filled space of the receptor cavity. 46] During stress ABA directly interacts with its target receptors PYR/PYL/RCAR (pyrabactin resistance 1/pyrabactin resistance like/regulatory component of aba receptor 1). GERMANY .

3 (SRK2I) from Arabidopsis thaliana. cannot bind to the ABAresponsive element. SAPK 8. SRK2E/SnRK2. Internal phosphorylation by SnRK2 occurs at Ser175 residue in its kinase activation loop. 2011 Current Topics in Biotechnology & Microbiology PYR1/PYL/RCAR1 receptor thereby inhibiting the auto phosphorylation of the kinases. GERMANY . Kinases as Positive Regulators: Kinases mainly act by phosphorylating transcription factors which regulate stress-responsive genes. PKABA1 from wheat and SAPK8.3 are strongly activated by ABA. PP2C inactivates SnRK2 by dephosphorylation when ABA is not present or plants are not exposed to stress. SAPK10 from rice. The inactive kinases are unable to phosphorylate AREB which.Dhingra. and phosphorylates myelin basic protein (MBP) and histone protein and it is found to be induced under conditions of dehydration [53]. et al. subclass III. It has been determined that three kinases within the Arabidopsis SnRK2 family. the ABA-bound PYR/PYL/RCAR receptors interact with PP2C and inhibit its phosphatase activity. The negative regulation of SnRK2 by PP2C is eliminated. Kinases lead to auto phosphorylation of a family of SnRKs (sucrose non-fermenting -related protein kinase). SRK2E has been found to function primarily in regulation of the stomatal responses. These include SNF1-related protein kinase 2 SRK2D/SnRK2.6/OST1 (SRK2E) and SRK2I/SnRK2. 53]. In the case of tobacco NtOSAK kinase was found to be activated by osmotic stress [46.2 (SRK2D). SRK2D/SnRK2. This shows that PP2C is involved in the regulation of the ABA-responsive activation of SnRK2. SAPK 9 and SAPK 10 kinases were found to be activated by ABA. KG. It has been reported that PP2C directly dephosphorylates sub class III SnRK2 kinases. mature leaves and vessels and present in high quantities in reproductive organs. It has been observed that the SnRK2 phosphorylation which happens in the presence of ABA is absent in abi1-1 mutants. Among the ten SnRk2 kinases characterized in rice. Therefore. A maize ZmSPK1 has been reported to be expressed in roots. SAPK9. Therefore stress inducible gene expression is hindered. SnRK2 (OSRK1) is capable of auto phosphorylation. [46] Several kinases have been characterized in different plants. SRK2E/OST1/SnRK2. DUDWELLER LANDSTR.2. in turn. The same sites are dephosphorylated in the absence of the ABA. AnRK2 phosphorylates serine /threonine residues at R-X-X-S/T sites in the conserved regions of its 105 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. H. while SRK2D & SRK21 are involved in seed germination and root growth. It has been reported in soyabean that the SPK1 and SPK2 kinases are stimulated in response to hyperosmotic stress while the activity of SPK3 and SPK4 kinases increases in response to dehydration and high salinity.6 and SRK2I/SnRK2. Once ABA level rises because of adverse environmental conditions or developmental cues.

This happens in a cyclic manner where PYR/PYL/RCAR negatively regulatesPP2C. Helicases: The partial unwinding of the double helical DNA is crucial for life-defining processes such as replication. The ABA-dependent phosphorylation of AREB/ABF transcription factors is required for the complete activation of stress-inducible promoter cassettes. DUDWELLER LANDSTR. Strategies for the construction of synthetic promoters depend on the cis-elements located upstream of the core or basal promoter. salinity and other stress conditions. Highly conserved domains are mainly involved in ATP-hydrolysis and unwinding of the nucleic acids. and PP2C negatively regulates SnRK2. H. engineering of helicases has been explored as a possible mechanism for stress tolerance. bZIP transcription factors have been characterized in different crop varieties such as TaAbF from wheat [54] and TRAB1 from rice [55].Dhingra. Microarray analysis has been performed to 106 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. the helicase family is also called DEAD-box protein family [56]. As helicases are indispensable in basic cellular processes governing phenotypic expression and physiological adaptation. DEAH or DEAX). PDH45 is thought to exert its role in a coordinated fashion alongwith DNA topoisomerase I which is involved in relieving the tension caused by partial unwinding of DNA [32]. This is achieved by DNA helicases which are proteins capable of disrupting the hydrogen bonds between nucleic acid duplexes. In plants. This unwinding of the duplex DNA is NTP/dNTP hydrolysis dependent and all the RNA and DNA helicases have inherent NTPase activity. recombination and transcription. 2011 Current Topics in Biotechnology & Microbiology target site in the transcription factor AREB/ABF. Genes encoding DEAD-box DNA helicases such as PDH45. KG. AREB is known to bind to the ABA-responsive element ( PyACGTGG/TC). as many as 469 cis-regulatory elements have been documented in relation to transcriptional regulations according to Plant cis-acting regulatory DNA elements (PLACE) database. GERMANY . Helicases have been classified into 3 super families: SF1. PDH47 and LOS4 have been expressed and found to be induced in response to cold. a conserved cis-element found upstream of the ABA-inducible gene. SF2 and SF3. Synthetic promoters are created by combining natural or artificially synthesized core-promoters with multiple cis-regulatory elements either derived from plants or other sources. PROMOTER ENGINEERING The diverse molecular patterns in a cell are a result of a complex interplay between cisand trans-acting factors. Due to the presence of the sequence motif II (DEAD. et al.

H. in cases where the gene expression needs to be tailored to a specific organ or a specific time. especially for the stress-induced genes. thaliana under the control of CaMV 35S (constitutive) and rd29a (stress induced) promoters [61].. KG. However. such constitutive promoters may not be a suitable choice. Two-component gene switches can also be constructed. The possibility of inducing gene regulatory units by chemical compounds which do no have any side-effects on normal plant developmental processes such as growth and cell division have directed research strategies towards chemically inducible promoters. i. This is because the constitutive expression of some stress induced genes may have serious deleterious effects on the plant growth and developmental processes. Such promoters are called bidirectional promoters. A surplus of synthetic TF binding sites can cause depletion of endogenous TFs and thereby lead to transcriptional inactivation of other essential housekeeping genes [59].Dhingra. The use of the strong CaMV 35S promoter to drive the expression of DREB1A also resulted in severe growth retardation under normal growing conditions. But the number of such regulatory motifs has to be controlled in order to prevent the monopolization of transcriptions factors that may regulate endogenous gene expression. More than 40 genes were identified as the DREB1A downstream genes [58]. they are turned on all the time and throughout the plant life cycle.e. expression of DREB1A under the control of rd29a gene promoter caused minimal negative effects on plant growth. For instance. tetracycline. two novel seed specific promoters 107 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. GERMANY . dexamethasone and ethanol [62]. the DREB1A cDNA has been overexpressed in A. et al. An artificial bidirectional promoter for high level expression of transgenes which can carry two genes at a time in different orientations has been designed [60]. With a prior knowledge of the type of cis elements influencing specific genes. For instance. Full length cDNA micro array and Gene Chip array were employed for exploring downstream transgenes overexpressing DREB1A. The chemicals commonly used for the induction of transgenic plants are benzothiadiazole. 2011 Current Topics in Biotechnology & Microbiology identify target stress inducible genes of DREB1A [57]. The most widely used promoters in generating transgenic plants are constitutively expressed. two core promoters can be placed in the opposite orientation with the common cis-regulatory elements placed in between. while providing even greater tolerance compared to CaMV 35S promoter. A gene sequence coding for a transcription factor forms the initial component followed by a regulatory module comprising the core promoter and multiple cis motifs upstream of it. DUDWELLER LANDSTR. In contrast.

et al. salt and freezing stress tolerance. 2011 Current Topics in Biotechnology & Microbiology namely HaFAD2-1 and HaAP10. promoter was induced by abscisic acid but not salicylic acid [74]. The differential induction is expected to 108 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. drought or salinity stresses when promoters responding to these are fully characterized and then cloned in suitable plant transformation vector systems. KG. SEF binding sites and GCN4 motif [63]. 66. Heat shock promoters. orientation. The over expression of DREB1A was found to improve drought and low temperature stress tolerance in tobacco [72]. Two cis-motifs ACGT and GT which are involved in the regulation of pathogen defense have been studied. salt stress or exposure to ABA [66]. Most of the stress promoters contain an array of stress specific cis-acting elements that are recognized by specific transcription factors [64. DUDWELLER LANDSTR. 69]. bZIP and MYB.Dhingra. 68. Transcriptional activation of stress-induced genes has been possible in transgenic plants over expressing one or more transcription factors that recognize promoter regulatory elements of these genes. 65. Two copies of ACGT when separated by 5 nucleotides allowed promoter activation by salicylic acid and when separated by 25 nucleotides. 71]. copy number and spacing. Two families. rd29 (induced by osmotic stress). These promoters contained seed specific motifs like AACA motif. and adh (induced by anaerobic stress) gene promoters have been subject of intensive research. GERMANY . ACGT element. 67. The levels of rd29 mRNA changes differentially in response to dehydration. It will become feasible to employ stress. EBoxes. These results highlight the importance of stress inducible rd29A promoter and the DREB1A gene in improving drought. The factors pertaining to cis-elements taken into consideration are position. The basic findings on stress promoters have led to a major shift in the paradigm for genetically engineering stress tolerant crops in recent years. are involved in ABA signaling and its gene activation.induced promoters for raising transgenics against low or high temperatures. Many ABA inducible genes share the (C/T) ACGTGGC consensus. H. cis-acting ABA-responsive element (ABRE) in their promoter regions [70. low temperature. have been isolated and functionally characterized from sunflower. These motifs placed 50 nucleotides or 100 nucleotides upstream to TATA box were studied [73]. It was interpreted that a single ACGT motif enhanced promoter expression when placed 100 nucleotides upstream to TATA box while two ACGT motifs separated by 5 nucleotides enhanced promoter expression when placed 50 nucleotides upstream to minimal promoter.

S. Current Science. et al. where the effect of eight cis-acting motifs on transcription from the basal promoter (Pmec) was studied by placing these upstream of the TATA-box at the -38 position as in plant genes. information about additional signaling components is required to make sense of the complete picture. plant engineering will require manipulation of complex metabolic or regulatory pathways involving multiple genes.Dhingra. Therefore. R. a better approach would be to control stress responsive genes themselves through cis-element engineering instead of overexpressing components produced in response to stress. This implicates essential roles of membrane lipid composition. Various cis-acting DNA motifs could function as an activator by itself as well as a synergizing activator in the presence of other neighboring homologous as well as heterologous motifs. However. Therefore. Khanna. KG. 2011 Current Topics in Biotechnology & Microbiology involve the recruitment of different bZIP transcription factors [75] and this change in spacing between two copies of a given motif can alter the signal pathway to which a promoter responds. REFERENCES 1. Current research methodologies include manipulation of genes encoding several stress-responsive components to mitigate the effects of stress. A common denominator among different types of stress responses seems to be the loss of membrane integrity. a comprehensive knowledge about various cis-elements and their corresponding effect on stress-responsive genes will pave way for designing synthetic promoter cassettes for achieving enhanced levels of stress-specific resistance. Signal transduction pathways involved in cellular responses to myriad stress conditions seem to be tightly interlinked. Therefore. engineering of membrane lipid composition and activity of membrane-associated enzymes such as PLD also offers exciting possibilities for enhancing stress tolerance. Signaling pathways involved in stress responses have been characterized through mutant studies. (1998). [76]. CONCLUSION Poor plant growth and productivity under adverse environmental stress conditions has triggered research to devise novel strategies for the induction of stress resistance. As abiotic stress resistance is polygenic. A. H. DUDWELLER LANDSTR. 109 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Simultaneous transfer of several genes will be the likely next step to achieve practical levels of plant stress tolerance. & Sinha. GERMANY . thaliana. Prospects of success of biotechnological approaches for improving tolerance to drought stress in crop plants. C. mostly of the model plant. 25-34. 74. This synergistic effect is well illustrated in the work of Sawant et al. K.

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The later section of the chapter discusses various biological applications of MINPs viz. healthcare and consumer goods and therefore building great expectations not only in the academic community but also among investors. detection and diagnostics. Department of Biological Sciences Birla Institute of Technology & Science. Pilani-333 031 * Corresponding author: Jitendra Panwar E-mail: drjitendrapanwar@yahoo. in separations of biomolecules and cells. jpanwar@bits-pilani. environment.co. Further. hyperthermia. 2011 Current Topics in Biotechnology & Microbiology Chapter VI SYNTHESIS AND APPLICATION OF MAGNETIC NANOPARTICLES: A BIOLOGICAL PERSPECTIVE Arpit Bhargava. Owing to the limitations and drawbacks of physiochemical methods of MINP synthesis. fungi and plants. DUDWELLER LANDSTR. This chapter deals with most recent advances in the biogenic synthesis and biological applications of magnetic iron nanoparticles [MINPs]. imaging. H. Magnetotactic bacteria [MTB] represent a fascinating example of biologically controlled mineralization forming MINPs in the form of magnetosomes. considerable interest has been generated in biogenic synthesis methods as the later being ecofriendly and cost effective. the chapter evaluates the advantages of biosynthesized MINPs over commercially available ones and finally concludes with the possible directions of future research in this field.in ABSTRACT: The fast growing field of nanotechnology presents great potential to influence diverse areas like energy. cell manipulation and tissue engineering and in agriculture.in. KG. nucleic acid delivery and transfer. et al. 117 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. drug and therapeutic delivery. agriculture. Progress in the biological methods can be realized with the number of reports coming in the past decade on MINP synthesis using bacteria. governments and industries.ac. GERMANY . Navin Jain and Jitendra Panwar* Centre for Biotechnology.Dhingra.

The chapter finally concludes with a brief discussion and propose hypothesis for future trends of research in this field. Nanoparticles are of great scientific interest as they bridge the gap between bulk material and atomic or molecular structures. This chapter explores new insights in the synthesis and applications of MINPs in biological perspectives. specific advantages and importance of magnetosomes and biosynthesized MINPs have been discussed. a class of nanostructured material possess amenable properties making them important in view of biological applications.Dhingra. drug and gene delivery [13. GERMANY . sol-gel synthesis. The physicochemical methods include microemulsion. cell tagging and tracking [12]. The nanometric size interacts efficiently with biological entities like cell [10-100 m]. 118 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Over the years the increasing inflow of investment in the field of nanotechnology proves its potential [7. Presence of nanoparticles is well known in many natural processes. flow injection synthesis. Magnetic iron nanoparticles (MINPs). hyperthermic effect [11]. electrochemical synthesis. 8]. KG. In the later part of the chapter. pyrolysis and chemical co-precipitation method [17]. from magnetotactic bacteria that sense the earth’s magnetic field using “nanomagnets”. protein [5-50nm] and gene [10-100nm] thus leading to exciting opportunities in the field of MRI imaging [10]. BIOSYNTHESIS OF MINPs Significant application of MINPs at commercial platform makes their synthesis protocols a valued subject for research and innovation. et al. 4]. Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. 16] as well as a prospective material to be applied in agriculture. energy generation [3. Physicochemical as well as biological methods are employed for the synthesis of MINPs. clean water technology [2]. hydrothermal reactions. 14] and detection of probes [15. viruses [20-450nm]. information storage [5] and biomedical applications [6]. Recent developments in the fabrication and application of nanostructured materials has delivered solution to various problems like climate change and pollution control [1]. H. DUDWELLER LANDSTR. 2011 Current Topics in Biotechnology & Microbiology INTRODUCTION Nanotechnology serves as a prospective tool for overcoming critical issues faced by society. to the facilitated transport of radionuclides in groundwater [9].

The green synthesis involves selection of solvent medium. which is probably due to their adaptation to complex chemical gradients typically encountered in stratified sediments. which are nanosized. sizes and arrangements [Figure 1. Despite of their high abundance and ubiquitous occurrence in marine and fresh water habitats. MTB are the oldest reported microbial factories for MINPs fabrication. thus precluding their application [17. complexity of these processes owing to colloidal nature of nanoscale iron particles is a major constraint which limits their applications. environmentally benign reducing agent and non-toxic substances for the stability of nanoparticles. GERMANY . most MTB are difficult to isolate and cultivate invitro. alternate methods for nanoparticle synthesis deserves merit. KG. In general. 2011 Current Topics in Biotechnology & Microbiology Although the physicochemical methods are fast. 20]. Magnetosome crystals have species-specific morphologies. Only a limited number of MTB strains have been isolated in pure culture [28]. DUDWELLER LANDSTR. 23] and their formation is genetically regulated by a complex set of genes situated mainly at magnetosome island in MTB genome [24. it involves the formation of intracellular magnetic structure. 18]. The major problem is the difficulties in controlling the size and shape of nanoparticles. et al. The group is highly diverse in terms of phylogeny. Magnetosomes are involved in magnetotaxis and direct the bacterial cells to swim towards the growth favouring microoxic zones of the chemically stratified natural water [26]. H. the particles are 119 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The landmark discovery of magnetotactic bacteria [MTB] by Richard Blakemore made the path for enormous research in finding biological agents capable of syntheszing MINPs [22].Dhingra. Almost all the reported MTB are microaerophiles which can form multiple magnetosomes within them through an oxygen sensitive process and are motile by means of flagella. polydispersity. 25]. 29]. efficient with considerable ease in upgrading to industrial scale production. Considering the ill-effects of physicochemical methods. The increased awareness towards the development of clean. membrane enclosed crystals of magnetic iron minerals composed of magnetite [Fe3O4] or greigite [Fe3S4]. the magnetosomes. Other issues surrounding these methods are difficulties in controlling experimental conditions. morphology and physiology [27]. reliable and eco-friendly alternate for synthesis of MINPs has encouraged the researchers to look into newer “Greener” methods [19. out of which species of Magnetosiprillum are extensively studied for elucidation of mechanism for magnetosome formation [Figure 2. tedious downstream processing with generation of toxic by-products and yield of particle in nonpolar organic solvent. As a naturally occurring phenomenon. which must be evaluated based on green chemistry perspective [21].

were challenged with mixture of potassium ferricyanide/ferrocyanide at room temperature thus producing magnetite nanoparticles extracellularly. with single domain magnetism [30]. maintenance of reductive conditions and mineralization of iron either by oxidation or by partial reduction and dehydration of ferrihydrite intermediate to magnetite [23]. 120 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. recent years have observed reports of many other aerobic bacterial species synthesizing iron nanoparticles. magnetosomal iron transport and. Bacteria like acid tolerant Leptospirillum ferriphilum [33] and iron oxidising Leptothrix ochracea [34] were found to produce magnetosome like mineral nanoparticles. the nanoparticles produced by Actinobacter under aerobic condition were found to have a protein coat [36]. et al. GERMANY . KG. Surrounding the mineral core is the magnetosome membrane which is a lipid bilayer membrane with composition considerably similar to cytoplasmic membrane [31]. Bharde et al. where Fusarium oxysporum and Verticillium sp.Dhingra. It originates from the cytoplasmic membrane by invagination. [37]. control the crystallization and intracellular arrangement of MINPs [32]. 2011 Current Topics in Biotechnology & Microbiology usually arranged along the axis showing single or multiple chains with particle size ranging between 35 and 120 nm. Both of these bacterial species belongs to the class of Chemolithotrophs which utilizes iron as an energy source. 36] have successfully obtained iron oxide (magnetite and maghemite) and iron sulphide (gregite) nanoparticles from an aerobic bacterium. Somewhat similar to magnetosomes membrane. represents a distinct subcellular compartment and has a unique biochemical composition. Apart from the group of MTB which synthesize MINPs in microaerophillic environment. H. Roughly 20 magnetosome-specific proteins function in the vesicle formation. It has been hypothesized that these membrane proteins may play functional roles like accumulation of supersaturating iron concentration. The group also explored the possibility of using mixture of ferric and ferrous chloride but the particles formed were irregular in shape and had poor crystal structure. [35. DUDWELLER LANDSTR. The only account of fungi producing iron nanoparticle is by Bharde et al. an Actinobacter species using aqueous mixture of potassium ferricyanide/ferrocyanide.

2011 Current Topics in Biotechnology & Microbiology Figure 1: TEM images of magnetotactic bacteria. low cost. sun dried leaves. 121 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. GERMANY . et al. platelets and nanorods) have been synthesized using tea polyphenols [40]. DUDWELLER LANDSTR. iron nanoparticles were synthesized at room temperature using aqueous sorghum bran extract as both the reducing and capping agent [42]. Herrera-Becerra et al. (a) Magnetospirillum magneticum strain AMB-1 and (b) Desulfovibrio magneticus strain RS-1. amorphous iron nanoparticles of various size and morphologies (spherical. Reproduced from [23] with permission from American chemical Society. soyabean sprouts were used for synthesis of superparamagnetic Fe3O4 nanoparticles [41].Dhingra. rapid. H. and fruits can be used and many have proved to be successful [38]. In a separate study. eco-friendly and a single step method. [39] successfully demonstrated a green chemistry method for the production of MINPs with size less than 5 nm by exposing a homogeneous suspension of powdered milled alfalfa and FeNH4(SO4)2. Recently. KG. Various plant parts viz.12H2O solution. In a very recent examination. Phytosynthesis of nanoparticles is a recent. leaf extract. BacMPs of (c) the AMB-1 strain and (d) the RS-1 strain.

GERMANY . OM. Protein names in black indicate factors discovered in previous studies in AMB-1. inner membrane. KG. Black octagons represent growing or mature. USA BIOLOGICAL APPLICATIONS OF MINPs Nanotechnology is beginning to allow scientists. DUDWELLER LANDSTR. proteins whose potential functions were defined in the present study.Dhingra. and the factors known to play a role in each of these steps are indicated on the right side. Asterisks indicate proteins characterized in MSR-1. H. 2011 Current Topics in Biotechnology & Microbiology Figure 2: Model for step-wise assembly of magnetosomes in Magnetospirillum magneticum AMB-1. Steps leading to magnetosome formation are indicated on the left side of the model. outer membrane. Interdisciplinary approach combining the field of nanotechnology. Red symbols indicate inner membrane proteins. engineers and physicians to work at the cellular and molecular levels to produce major advances in the life sciences and health care. in orange. purple dots indicate magnetosomeassociated proteins. 122 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. IM. material sciences and biology enable us to fully comprehend the potential uses of nanoparticles in biomedical applications. yellow lines represent the MamK cytoskeletal filaments. et al. euhedral magnetite crystals. Reproduced from [29] with permission from Proceedings of the National Academy of Sciences.

In general. MINPs coated with high affinity ligand are targeted towards specific biological entity to which these modified MINPs attaches and thus can be eluted out by using magnetic separator. In the indirect approach which can also be said as sandwiched separation. followed by the introduction of MINPs coated with high affinity ligand against the antibody. and (ii) separating out these tagged entities via a fluid-based magnetic separation device [46]. an antibody specific to the molecule need to be separated is introduced first. This phenomenon arises because these nanoparticles consist of a single crystal domain. and thus exhibit a net magnetic moment directed along an anisotrophy axis. In the direct or targeted separation. This part of the chapter deals with various biomedical applications of MINPs. separation can be performed by one of the two ways. KG. MINPs with diameter less than 20 nm are said to be superparamagnetic and can have potential biomedical applications. Magnetic separation is based on the simple principle of magnetic attraction of biocompatible MINPs towards a magnet [45] and likely to be a cost effective alternative technique.Dhingra. the magnetic separation using biocompatible nanoparticles is a two-step process. Although variety of techniques are available for the separation process but all suffer from issues like cost. et al. 2011 Current Topics in Biotechnology & Microbiology MINPs offer attractive possibilities in biomedicine as they can be manipulated and transported to a particular site by using external magnetic field gradient. Magnetic separation technique has been successfully employed in various applications like cell sorting. [i] direct or targeted separation and [ii] indirect separation. In general. GERMANY . A series of modification have been performed on the magnetic separation technique but the core principle remains the same. separation and purification of biologically active compound. H. DUDWELLER LANDSTR. involving (i) the tagging or labelling of the desired biological entity with magnetic material. protein purification and nucleic acid extraction. eco-friendliness and complexity [44]. SEPARATION OF BIOLOGICAL MOLECULES AND CELLS Isolation. biomolecules and cells raise the cost of downstream processing in many industries. This is in contrast to the multi-domain ferromagnetic particles which aggregate after exposure to an external magnetic field [43]. 123 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Strategies can be made either to separate the desired molecule or cell from the mixture using magnetic separation or removing the contaminants and undesired one [45]. as they do not retain any magnetism after removal of external magnetic field.

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Conventionally proteins are being separated using chromatography, ultracentrifugation and membrane filtration technique, where each method requires tedious pre-processing of sample to achieve high efficiency and purity. The same task can be done using MINPs coated with protein specific ligand like streptavidin, protein A or antibody, which can easily and quickly separate out target protein from crude cell extract without tedious frills [47]. Kim et al. [48] reported an efficient method for purification of native polyhistidine-tagged proteins using modified MINPs. In this experiment they synthesized superparamagnetic iron oxide [SPIO] nanoparticles coated with bis-nitrilotriacetic acid [NTA] chelate using catechol anchor which when loaded with Ni [II], binded to polyhistidine fusion proteins in their native, folded conformations. In an another study, SPIO nanoparticles were coated with hydrophilic resins prepared by radical polymerization of magnetite [Fe3O4], styrene, divinyl benzene [DVB] and glycidyl methacrylate–iminodiacetic acid [GMA–IDA] in ethanol/water medium to improve purification of His-tagged proteins [49]. Wei et al. [50] devised a novel strategy based on MINPs coated with zirconium phosphonate for the enrichment of phosphopeptides used for mass spectroscopy. Under optimized experimental conditions, 1 × 10-9 M phosphopeptides in 50 L tryptic digest of β-casein could be enriched and identified successfully using the adopted strategy. Nucleic acid separation had been a time consuming and laborious process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products and unsuitable for automation and up-scaling. Although availability of nucleic acid extraction kit has diluted the complexity but it involve increased cost and still the process lacks rapid extraction. Use of MINPs explored the possibility to use them to isolate and purify nucleic acids. During last few years, specifically functionalised magnetic particles have been developed that allow quick and efficient purification directly after extraction from crude cell extracts, eliminating repeated steps of centrifugation and incubation. In addition, the new approach provided an easy automation of entire process and isolation of nucleic acids from larger sample volumes [51]. SPIO nanoparticles [Fe3O4] coated with a multivalent cationic agent, polyethylenimine [PEI] were employed to simplify the purification of plasmid DNA from bacterial cells [52]. Approximately 35 µg of high-purity plasmid DNA was isolated using magnetic nanobeads within 10 minutes from 3 ml of overnight bacterial culture [53]. Yet in another study, Park and Chang [54] developed a method for high throughput human DNA 124

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purification with the amino-functionalized silica coated MINPs having average particle size of 9 nm and coating thickness of 10–40 nm. In advancement to this method, isolation of messenger RNA (mRNA) from mammalian cells and extraction of the supercoiled form of plasmid DNA from agarose gel was achieved using Carboxyl-coated MINPs [55]. These MINPs were attached with 5′-NH2-tagged oligo-[dT]25 primer and were used to isolate mRNA from breast cancer cells. The ability to separate living cells is an essential aspect of research in cell biology. The need of cell separation arises during some advanced medical procedures and certain cell replacement therapies [56]. Magnetic cell separation methods are among some of the most efficient methods for bulk cell separation [57]. The background principle exploits the concept of immunogenic interaction between antigen and specific antibody or ligand and receptor which in this case is coated or decorated on MINPs. Magnetic cell separation typically employs the use of antibodies against specific cell surface epitopes to tag cells of interest with magnetic particle conjugates [47]. Under the effect of applied magnetic field, the magnetically tagged cells tend to have higher magnetophoretic mobility enhancing their separation [57]. Inglis et al. [58] have developed a continuous-flow microfluidic device that enables cell by cell separation in which cells were selectively tagged with MINPs. One of the most critical contributions of this technique has been in ART [Assisted Reproductive Technique], where MACS [magnetic activated cell sorting] uses annexin V-conjugated SPIO nanoparticles to separate non-apoptotic spermatozoa based on expression of phosphatidylserine [59]. Qiu et al. [60] used the MACS strategy with anti-CD14 and anti-CD16 antibody beads to deplete macrophages and neutrophils from sputum and to enrich the content of bronchial epithelial cells from 1.1% in the starting population to 40%.

The rapid detection of biomolecules and specific cells in biological samples is of paramount concern in the field of medicine. Biosensing strategies based on MINPs have received considerable attention because they offer unique advantages over the other detection techniques. Biological samples exhibit virtually no magnetic background, and thus highly sensitive measurements can be performed in turbid or otherwise visually obscured samples without further processing [61]. The superb structural and functional properties of MINPs have made them a promising candidate to be used in nanoscale biosensors for detection of biomolecules, cells and 125

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other biological entities [62]. Although the detection/ diagnostic or sensing principle may vary in different type of applications but the biological principle is more or less common relating to interaction between target molecule or cell and its specific ligand which in many cases may be monoclonal antibodies. A sensor generally consists of two components: a recognition element for target binding and a transduction element for signalling the binding event [63]. Majorly, three sensory principles are used in development of various biosensors using MINPs [64]. The first one, called magnetic relaxation switches suggests that aggregated and dispersed states of MINPs have different transverse spin-spin relaxation times [values of T2]. When used as targeted contrast agents, surface-modified nanoparticles bind specific molecules producing local inhomogenieties in the applied magnetic field in tissues where molecular targets are present. These inhomogenieties result in decreases in the T2 relaxation time (or increases in 1/T2) which in turn lead to changes in the contrast of magnetic resonance images [65]. The second type of biosensors, called magnetic particle relaxation-based sensors, exploits the change in relaxation of magnetic moments within magnetic particles. In theory, the effective relaxation rate depends on the Brownian relaxation rate and the Neel relaxation rate. Faster relaxation time between these two, governs the effective relaxation process. Target induced aggregation can decrease the rates of the Brownian or Neel relaxations and this is the basis of assays for molecular targets [17]. The third principle, called magnetoresistive sensors, involves the binding of magnetic nanoparticles to a sensor surface. The magnetic fields of the particles alter the magnetic fields of the sensor which result in electrical current changes within the sensor [66]. Two mechanisms have been suggested for binding of magnetic nanoparticles to the sensor surface [67]: [i] direct labeling and [ii] indirect labelling (a sandwich type binding) which has been principally discussed in the previous section of this chapter. Even though there can be different type of detector systems and biosensors particular for enormous range of biological units present, it is beyond reach to discuss the specific application of all the available systems. Mass spectrometry specifically MALDI-TOF MS is one of the standard method for the study of biomolecules. However, this approach is not suitable for detection of small molecules due to the interference from matrix in the low molecular weight region of the mass spectrum. To overcome this limitation, Lin et al. [15] have developed nanoprobe-based affinity mass spectrometry [NBAMS] in combination with direct protein identification by MALDI-TOF MS. It 126

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eliminates conventional time-consuming sample purification and enrichment processes and also removes the interference caused by the background matrix as in NBAMS, the surface of MINP acts as a matrix. One of the methods for detection and quantification of DNA has been proposed by Kouassi and Irudayaraj [68], in which DNA can be labelled with appropriate fluorophores decorated on MINPs. Another variant for detection of DNA using MINP is electrochemical detection, which usually involves immobilization of a single-stranded probe nucleic acid to a solid substrate and the production of an electrochemical signal using a redox couple when the desired target hybridizes to the probe. The advantage of this method lie in the fact that since the electrochemical redox couple does not bind to the DNA, the nucleic acid retains its natural state and can be recycled [69]. MINPs are helpful in detection of viral particles as in case of supramolecular assemblies that take advantage of changes in the nanoparticle’s optical or magnetic properties upon viralinduced assembly. Such structures act as magnetic relaxation switches by causing extensive spinspin relaxation time changes of surrounding water molecules and thus can be used as potential nanosensors for rapid and sensitive detection of clinically relevant viruses. Perez et al. [70] have developed method for detection of Herpes simplex virus [HSV] and Adenovirus [ADV] even at very low concentration (5 viral particles per 10 l). They conjugated the monodispersed

magnetic nanoparticles with virus specific antibodies. In presence of virus these nanoparticles create a super-molecular structure and their presence can be selectively detected by magnetic resonance methods like NMR and MRI. Utilizing the fact that many bacteria use the mammalian cell surface carbohydrates as anchors for attachments which subsequently results in their infection, magnetic glyconanoparticle -based system has been used to detect E. coli within 5 min, as well as remove up to 88% of the target bacteria from the medium [71]. The system is found to be capable of identifying different E. coli strains on the basis of their response patterns to MINPs.

The basis of magnetic resonance imaging [MRI] is the directional magnetic field or moment associated with charged particles in motion. Nuclei containing an odd number of protons and/or neutrons have a characteristic motion or precession. Because nuclei are charged 127

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particles, this precession produces a small magnetic moment. Clinical MRI uses the hydrogen protons and its interaction with both a large external magnetic field and radio waves to produce highly detailed images of the human body [73]. Conventional MRI scans uses gadolinium as a contrast agent which sharpens the content by changing the proton behaviours in their proximity. Advances in this technology involve replacement of gadolinium by iron oxides particles as effective contrast agents in imaging [74]. MRI contrast agents act to improve image quality by reducing the relaxation time and hence altering the NMR signal intensity of water in the body tissues containing the agent. SPIO nanoparticles represent an alternative class of NMR contrast agents that are usually referred to as T2 [transversal relaxation time] or T2 contrast agents as opposed to T1 [longitudinal relaxation time] agents such as paramagnetic gadolinium [III] chelates [75, 76]. Gandolinium makes complexes with low molecular weight chelating molecules, such as diethylene triamine pentaacetic acid [DTPA] as well as capsulated in lipsome and micelles. However, interference due to ion exchange with other metals and their complexes in the surrounding limits their application [77]. Advantages of MINPs lie in their inclusivity within tissues enabling a very large signal to be obtained from a MRI scanner. Based on the size of functionalized nanoparticle used they have been classified as supreparamagnetic iron oxides [SPIO] where particles have a size greater than 50 nm [coating included] and ultrasmall superparamagnetic iron oxides [USPIO] where particles are smaller than 50 nm [78]. The size affects their half-life in vivo as well as their bio-distribution and pharmacokinetic properties. Advances in the field of nanoparticle engineering have developed magnetism engineered iron oxide [MEIO] nanoparticle which are discussed to be better contrast agents as compared to traditional SPIO nanoparticle and cross linked iron oxide nanoparticle [CLIO]. It is because MEIO have high and tunable mass magnetization values needed to enhance the relaxation process of the proton nuclear spins for improved imaging [79]. MRI imaging using MINP depends on the effectiveness of the nano-sized contrasting agents to be captured by the cells through the endocytosis pathway so as to insert the agents in the target cells. For efficient imaging, plasma half life of particle plays an important role. In general, mechanical filtration process in spleen and liver eliminates MINP above 200nm where as particles below 10nm are cleared by opsonisation and renal clearance [80]. To ensure prolonged plasma half life, fictionalization of SPIO in terms of coating or encapsulation with amphiphilic coatings are preferred which can drastically improve plasma half life as well as targeting capabilities of the 128

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contrast agent [81]. Two of the most common coating is of polysacharride dextran [82] and poly ethylene glycol [PEG] [83]. PEG coated nanoparticles offer advantage of being escaped from macrophages leading to opsonisation thus have increased blood flow time [84, 85]. Application of SPIO in imaging can be conceptualized as passive targeting which involves SPIO probes lacking explicit molecular specificity thus allowing generalized and nontargeted cellular uptake, enhanced retention in tumors, macrophage phagocytosis, as well as accumulation in the liver, spleen, and lymph nodes. Active targeting engages SPIO functionalized against specific bio molecules. The SPIO used for active targeting may or may not have surface coating. Presence of surface coating gives additional reactive moieties for the attachment of a variety of ligand and antibodies [80]. As the trend has shifted towards active targeting using molecular specific probes, some other recent applications using this technique has helped in diagnosis and detection of diseases, tumours, infections and molecular imaging. Huang et al. [86] developed biocompatible poly-vinylpyrrolidone coated SPIO nanoparticles using high temperature process that can be efficiently labelled to Mice islet cells. These biocompatible SPIO can be used to visualize islet cells during transplantation which makes it a non-invasive technique for assessing islet engraftment and the early recognition of graft loss. The usefulness of SPIO as contrast agents in MRI extend to diagnosis of abnormalities in central nervous system [CNS]. It can be used to detect blood–brain barrier dysfunction due to tumours, neuro-inflammatory pathologies and in vivo cellular tracking in CNS disease or injury [87]. Recently Wang et al. [88] reported that non-pathogenic anaerobic bacteria can anchor and proliferate within solid tumours hypoxia area, making these bacteria a potential representative for cancer diagnosis and therapy. They have developed bio functionalized SPIO nanoparticles tagged on anaerobic bacteria for their detection and evaluation using MRI. Ease of detection and screening of these labelled bacteria have enhanced the development in the field of cancer therapy and diagnosis.

Hyperthermia is a therapeutic procedure mainly used in the treatment of cancer alongwith chemotherapy, radiotherapy or surgery. The principle of magnetically mediated hyperthermia [Figure 3b] is based on two facts: (i) Tumour cells are sensitive to temperature above 41°C than normal tissue cells [89]. At high temperature range, disruption and modification in the function 129

Apart from being used as an anti-tumour therapy. hyperthermia can be categorized into: (i) Arterial Embolization Hyperthermia [AEH]. dose of heating agents. GERMANY . H. where the particles are directly injected into the tumour and (iii) Intracellular Hyperthermia [IH]. As reported by Thomas et al. has initiated the use of this technology to a targeted tissue by conjugating the MINP with specific antibody or ligand [92]. heat generation takes place. Exploiting these two facts. (ii) Metallic objects with magnetic properties when placed in an alternating magnetic field induce currents flowing within them. magnetically mediated hyperthermia can also be employed as anti. On the basis of mode of introduction of magnetic agents. (ii) Direct Injection Hyperthermia [DIH]. As evaluated by Rosensweig [91]. [93] evaluated the efficiency of magnetically mediated hyperthermia using bimagnetic core/shell nanoparticles (Fe/Fe3O4) in order to prevent the growth of subcutaneous mouse melanomas. As the metal resists the flow of current.Dhingra. For practical implementation of this therapy the site of injection. Balivada et al. superparamagnetic materials show impressive levels of heating at low magnetic fields as compared to ferromagnetic material which require much higher magnetic field strength for effective heat generation. KG. property of tumour and tissue as well as temperature susceptibility should be accurately optimized [94]. Recent developments in the field of hyperthermia. [95] thermoablation of the bacterium Staphylococcus aureus is possible using SPIO nanoparticle biofunctionalized with carboxylic acid moiety. Their results indicated that intra-tumoral administration of surface modified MINPs can attenuate mouse melanoma after a short alternating magnetic field exposure. The amount of current and thereby heat is proportional to the size of the magnetic field and the size of the object [90]. The heating power of the particles is quantified as the specific absorption rate [SAR] and describes the amount of energy converted into heat per unit time and mass. et al. hyperthermia has proved to be an effective way of destroying tumours and thus treating cancer. DUDWELLER LANDSTR. 130 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. which engages biocompatible magnetic particles facilitating their cellular uptake by the tumour [47]. which uses the arterial supply to deliver magnetic particles into the tumour tissue. 2011 Current Topics in Biotechnology & Microbiology of many structural and enzymatic proteins occurs within cells resulting in altered growth and differentiation leading to the tumour destruction.microbial treatment.

SPIO nanoparticles do not aggregate even after removal of the external magnetic field [97]. Doxorubicin (DOX).Dhingra. tumours etc. In comparison to paramagnetic nanoparticles. proteins. serves as a healthier system for the targeted delivery of therapeutics [Figure 3a]. The overall size of MINPs must be sufficiently small to evade rapid spleen filtration but large enough to avoid renal clearance. rate of blood flow. has been delievered to tumor blood vessels and reported to suppress metastasis [102]. CNS. drug retention and side effects due to the need of higher drug dose [96]. Most imperative benefit of MINPs for drug delivery is the capacity to deliver the drug to desired area under external magnetic field [13]. The leaky vascular blood vessels and a discontinuous endothelial cell lining. et al. This dual functionality of active MINP have been successfully applied in treatment of notable malady of heart. The magnetic drug targeting although may not be the final solution to the aforesaid mentioned problems however. and vascular supply play a major role in determining the effectiveness of the drug delivery process [99]. depth of the target tissue. PEG or liposomes [98]. EPR is a phenomenon of enhanced extravasation of particles from tumour blood vessels. DUDWELLER LANDSTR. low molecular weight ligands or charged molecules [10]. Combining targeted drug delivery with MRI has been the topical inclination in current research. MINPs are vital in the treatment of cancer and other tumours [100]. KG. which is not observed in normal vasculature. uneven distribution of therapeutic agent in the body. coated on MINPs. 2011 Current Topics in Biotechnology & Microbiology DRUG AND THERAPEUTICAGENT DELIVERY The drawback of conventional drug delivery system is non-specificity. [103] reported treatment of breast cancer using DOX and Paclitaxel drugs incorporated on biocompatible MINP 131 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. To increase the blood half life of the delivery molecule and to avoid early detection by Reticulo-endothelial System [RES]. their field strength and geometry. particles are generally coated with a variety of hydrophilic polymers like Dextran. a well known anticancer therapeutic drug. Parameters such as the physicochemical properties of MINPs loaded with the drug. H. GERMANY . Molecular targeting and assisted internalization of nanoparticles can be achieved using specific ligand based delivery system involving use of cell or tissue specific antibodies. and their subsequent retention in tumour tissue. the matrix metalloproteinases (collagenases) affect disintegration of the matrix tissue surrounding blood vessels and permits the entry of particles that have limited access to normal tissue [101]. Due to the enhanced permeability and retention [EPR] effect (Figure 4). Jain et al.

During an animal trial experiment on rat and rabbit. GERMANY .and four-fold as compared to free-DOX and commercial liposome drug DOXIL® along with higher MRI sensitivity comparable to a conventional MRI contrast agent suggesting them as a promising candidate for treating liver cancer and monitoring the progress of cancer using MRI [105]. H. [104] exploited the interaction between natural receptor urokinase plasminogen activator receptor (uPAR) and its ligand urokinase plasminogen activator (uPA) by functionalizing the ligand on biocompatible iron oxide nanoparticles containing anti-tumor drug DOX. facilitate penetration of the cell membrane after complexation by targeting through external magnetic field by exploiting biological phenomenon of endocytosis and finally should unload their payloads inside of cells at appropriate time which can carefully modulated by condition sensitive interaction between bounded nucleic acid and nanoparticle. Therapeutic proteins have revolutionized the treatment of many diseases thus are potential candidate which can be loaded on naoparticles for delivery to target cells. Molecules can be attached to MINP surface either by chemical or biological contact or through electrostatic charge in view of the inherent property of DNA being negatively charged [109]. one of the most important requirements is to protect the bio molecule. Radionuclides as an alternate treatment have been targeted to the tumours in many experiments as their activity is not dependent on molecule internalization [107].Dhingra. Polyethylene amine [PEI] is the most commonly used 132 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Therefore. considering the fragility of nucleic acid. 2011 Current Topics in Biotechnology & Microbiology as anti-proliferative agents with simultaneous imaging through targeted MRI. Yang et al. et al. Success of drug delivery will only rely on the efficiency of internalization of drug molecule inside the cell. Although similar to drug delivery. KG. DOX loaded SPIO nanoparticles showed decrease in relative tumour volume up to two. the carriers must form a condensed complex with biomolecules which aid in its protection. for a successful delivery system. NUCLEIC ACID DELIVERY AND TRANSFER The success of incorporating drug on magnetic particle for targeted drug delivery has increased the attention of scientific community towards using them for delivery of nucleic acid molecules [DNA & RNA] to target cells [108]. Antioxidant enzymes such as catalase and superoxide dismutase [SOD] which are therapeutically important in disorders related to increased production of reactive oxygen species [ROS] can be delivered to the target site by loading them on MINPs [106]. DUDWELLER LANDSTR.

et al. and after systemic administration they are concentrated to the target organ [O] with the help of a magnet [M]. Advanced approach related to this method are attachment of non-viral [112] as well as viral vectors [113] containing gene of interest attached to nanoparticle. DUDWELLER LANDSTR. H. Reproduced from [47] with permission from Elsevier. which has been found to increase the efficiency of transfection by many folds and also allows possible transfection to defiant cells.or ligand-based targeting. Figure 3: In vivo therapeutic applications of distally controlled MINPs. [b] In magnetically mediated hyperthermia [MMH]. KG.Dhingra. [a] Magnetic particles [gray spheres] associated to therapeutic molecules [shown as orange spheres] act as vehicles for drug delivery. systemically administered magnetic particles [gray spheres] accumulate in a tumor [T]. The facilitation of nucleic acid delivery using superparamagnetic particle with the help of external magnetic force is termed as “Magnetofection” [111]. GERMANY . 133 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Particles in the tumor are then heated [illustrated by the change to red spheres] through the external application of an alternating magnetic field [AMF]. either through the enhanced permeability and retention [EPR] effect or upon magnetic. 2011 Current Topics in Biotechnology & Microbiology surface coating agent which is cationic in nature [110]. and this results in the death of the tumor cells.

as ATP -Raf blocks the endothelial signalling and angiogenesis in response to multiple growth factors and can result in apoptosis of the tumour-associated endothelium [116]. KG. poly-L-lysine-modified iron oxide nanoparticles [IONP-PLL] carrying the NM23H1 gene. Gene delivery imaging system for diagnosis of liver diseases has been achieved by using chitosan-linoleic acid coated SPIO nanoparticles targeted to hepatocytes and reported to be efficiently used as a carrier for gene silencing with specific siRNAs [114]. GERMANY . Cationic nanoparticle coupled to an integrin αvβ3-targeting ligand were used to deliver Raf gene selectively to angiogenic blood vessels in tumor-bearing mice. 2011 Current Topics in Biotechnology & Microbiology Figure 4: Schematic diagrams showing enhanced permeability and retention (EPR) effect of nanoparticles in tumours. DUDWELLER LANDSTR. were successfully delivered to tumour cells in vivo and have significantly extended the survival time of an experimental mouse [115].Dhingra. 134 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. et al. the first suppressor gene of cancer metastasis. Yet in another experiment. H.

For example. magnetically label to target cells were allowed to grow on a steel plate placed on a magnet [119]. Even though. et al. H. material sciences and chemistry. MINPs and magnetic force can be resourcefully employed to allocate cells on arbitrary surfaces and in this way. magnetic particle based tissue engineering show great promise for future research. 2011 Current Topics in Biotechnology & Microbiology CELL MANIPULATION AND TISSUE ENGINEERING Once tagged with MINPs or internalization of the same compels the cell to behave specifically under magnetic field. DUDWELLER LANDSTR. Even nanotechnology extends its relevance in plant world with development of nanoscale technology for improvement in crop yield qualitatively and quantitatively. At present methods such as microcontact printing and lithography have been developed but issue of complexity like requirement of specialized surfaces delimits their use. On a similar note. growth pattern and intrinsic property providing a valuable tool for studying cell science. The success in this field has opened up new opportunities in magnetic-force-based tissue engineering [Figure 5]. the complete functionality of MINPs is yet to be explored in this field but few reports are there suggesting a pivotal futuristic role of MINP in agriculture and plant sciences. A detailed overview on manipulation of cell by MINPs has been reviewed by Dobson [118]. GERMANY . Technologies for fabricating functional tissue architectures are highly attractive for tissue engineering. KG. one can generate tissue constructs of various shapes. AGRICULTURAL APPLICATIONS OF MINPs Agriculture has seen overriding revolution with the introduction of hybrid crops and use of plants as expression system. This principle can be modified in variety of ways to manipulate cell structure. magnetite cationic liposomes [MCLs]. Reports showing presence of nanoscale particle of clay. human mesenchymal stem cells [MSCs] magnetically labelled with MCLs were seeded onto an ultra-low attachment culture surface with a magnet on reverse side which facilitated growth of multilayered sheet-like structures after a 24-h culture period retaining its ability to differentiate into various cell lines [120]. With the collaboration in the fields of cell and molecular biology. To study structure and morphology of the membrane of submicronic endosomal compartments endosomal internalisation of the MINPs was carried out [117]. metal and other humic 135 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.Dhingra. The internal magnetization of endosomes allows deforming them along the direction of an applied field.

Fe. its mechanism. [125] have demonstrated the significant uptake of magnetite [Fe3O4] nanoparticles [0. hematite [α-Fe2O3]. magnetite [Fe3O4]. in the nanoscale dimension can be applied to stabilize several elements in contaminated soils [124]. pressure and temperature. Zhu et al. H. hydroxides. Zerovalent iron. translocation of nanoparticles and their interactions with plants tissues at cellular and molecular level. formed by weathering and bio-mineralization. Biomineralization provides controlled crystals of uniform size without fabrication mechanism of magnetic 136 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 2011 Current Topics in Biotechnology & Microbiology substances in the soil define their role in natural soil systems [121]. interactions between plants and nanoparticles need in depth investigations such as uptake route. The metal nanoparticles most prominently include various oxides. lepidocrocite [γ-FeOOH] and ferrihydrite [122]. and oxyhydroxides of Al. The formation of MINPs by biological systems is beneficial in terms of their properties and amenability in bio-medical application due to their uniform size distribution. phosphate] by electrostatic interactions and ligand exchange and actively promote clay flocculation and soil aggregate stabilization [123]. In a very recent study. et al. GERMANY . These nanoscale minerals play a vital role in the adsorption and retention of nutrient anions [e. thus made these nanoparticles more acceptable and compatible in biological systems.g. [126]. However. During the study.5 g/L] by pumpkin plants and their subsequent translocation and accumulation in various tissues. ADVANTAGES OF MAGNETOSOMES AND BIOSYNTHESIZED MINPs Biological methods for nanoparticle synthesis help in circumvent many of the detrimental features observed in chemical and physical synthesis methods by enabling synthesis at mild or neutral pH. and Mn. Iron derivates occur in nearly all soils in the form of Goethite [α-FeOOH]. The research group has evaluated the effect of nanoparticles in plant systems and demonstrated an outline for the safe use of agri-nanotechnology. DUDWELLER LANDSTR. This suggests that new nutrient delivery systems that exploit the nanoscale porous domains on plant surfaces can be developed (127). maghemite [γ-Fe2O3]. KG. MINP delivery in plants has been very well reviewed by Nair et al. they did not observe any toxicological effects to the plants which suggest their use in nanoparticulate delivery system in plants.Dhingra.

DUDWELLER LANDSTR. Reproduced from [47] with permission from Elsevier. Removal of the magnet allows the recovery of the construct for use. [b] Tubular structures can be generated by folding preformed cell layers. Such tubular constructs are recovered after removal of the magnet. This leads to the generation of 3D multilayered heterotypic cell sheets by magnetic-force-based tissue engineering [Mag-TE]. 2011 Current Topics in Biotechnology & Microbiology Figure 5: Uses of magnetic particles in tissue engineering. are separately labeled using magnetite cationic liposomes and sequentially seeded onto an ultra-low attachment plate under which a magnet is placed. H.Dhingra. around rod-shaped magnetic models. [a] Different cell types. indicated in purple and blue. KG. GERMANY . 137 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. obtained as shown in panel [a]. et al.

The unique and superior characteristics of magnetosomes as discussed above attracts interest for their use in magnetic separation. Biomembrane formed around the mineral core results in better stability. Magnetosomes have been reportedly used in various biomedical applications proving them better as compared to commercially available MINPs. diagnostic. dispersion and thus can be easily handled as compared to synthetic MINPs [129]. et al. which are thus far most extensively.Dhingra. drug and gene delivery and imaging. Reproduced from [10] with permission from Elsevier. GERMANY . Table 1 enlist some recent 138 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Figure 6: MNP possessing various ligands to enable multifunctionality from a single nanoparticle platform. The application of biosynthesized MINPs are presently confined to magnetosomes. KG. 2011 Current Topics in Biotechnology & Microbiology submission of extreme temperature. pH and pressure which are mostly required in physicochemical process [128]. H. The stoichiometry of membrane proteins can be engineered enhancing the applicability of biogenic MINP [131]. reviewed MINP produced by biological system. DUDWELLER LANDSTR. Advances in production strategy and bio-engineering of magnetosomes are rapidly escalating their importance. Another advantage of natural coating is that it provides an excellent target for modification and functionalization of the particle [130] [Figure 6. detection of analytes. 10].

Wacker et al. DUDWELLER LANDSTR. [B] The detection complex is concentrated using an external magnetic field. Reproduced from [138] with permission from Elsevier. charged molecules like PEI and organosilane. Although the magnetosomes themselves are of low toxicity. the associated bacterial endotoxin lipopolysaccharide [LPS] may 139 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Functionalization can be done by biotinylation.Dhingra. [138] have demonstrated the concept of magneto immuno-PCR (Figure 7). H. KG. a defined volume of the detection complex solution is transferred to a microplate containing the PCR mastermix to enable real-time PCR detection of the immobilized antigen. Subsequent washing steps permit the removal of unbound materials. 2011 Current Topics in Biotechnology & Microbiology studies showing significant biomedical applications of MINPs. polyamidoamine dendrimer as well as by advanced approaches involving genomic and proteomic level manipulation of magetosome membrane [133]. [C] After resuspending. GERMANY . et al. particles for specific application in physiological environment is important and can be performed with ease in case of magnetosome owing to presence of lipid bilayer membrane [132]. [A] HBsAg specific magnetosome-antibody conjugate and DNA–antibody conjugate are incubated simultaneously with the serum sample containing HBsAg resulting in a signal-generating detection complex. Functionalization of Figure 7: Schematic drawing of the Magneto Immuno-PCR [M-IPCR].

Figure 8: Development of nanovaccines: MINP coated with antigen interacting with a B cell. Monitoring and understanding the interaction of MINPs with cells and tissues inside the living system will constructively shape the strategies for development of biocompatible nano-magnets. CONCLUSION As can be seen in this chapter. kidney. KG. Use of MINPs in targeted drug delivery will undoubtedly improve the therapeutic potential of many water insoluble and unstable drugs. We believe that in future. considerable success has been achieved but the final objectives are still ahead. pancreas etc. more efforts are required to find out easy ways to drove these MINPs to internal body organs such as liver. [23] have excellently reviewed the advances and futuristic application of magnetosomes in biomedicine.Dhingra. In the past decade. Shifting the focus towards ecofriendly. GERMANY . et al. Adapting an interdisciplinary approach can enhance the development futuristic technologies which are more user friendly and close-to-market. 2011 Current Topics in Biotechnology & Microbiology pose adverse effect. The LPS can be easily removed as suggested by treatment with detergent retaining selective tightly bound protein on the membrane which can be used as potential anchoring molecules [129]. However. H. MINPs are one of the most extensively commercially used nanomaterial in biomedical world with diversified applications. cost effective and greener methods of nanoparticle fabrication will attract attention of the scientific community towards development of biocompatible MINPs. Arakaki et al. the trends of research will be related to Table 1: Significant biomedical applications of magnetosomes 140 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. DUDWELLER LANDSTR.

141 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Imaging Magnetosomes of size 42 nm covalently coupled to the fluorescent [136] dye DY-676 were labelled to murine macrophages J774 and examined by infrared imaging. et al. DUDWELLER LANDSTR. Detection and diagnosis HBsAg specific magnetosome-antibody conjugate and DNA–antibody [138] conjugate are incubated simultaneously with the serum sample containing HBsAg resulting in a signal-generating detection complex which is concentrated using an external magnetic field and subjected to detection by real-time PCR detection of the immobilized antigen. KG. GERMANY .Dhingra. Separation of biological molecule and cell The efficient and stable display of the immunoglobulin G [IgG]. Loaded magnetosomes were tested for their activity in EMT-6 and HL60 cell lines to evaluate the in vitro and in vivo anti-neoplastic effects on hepatic cancer. MRI. 2011 Current Topics in Biotechnology & Microbiology Application genre Key Feature Reference Detection and diagnostic.[135] binding domain of protein A on BMPs using anchor molecule. Drug delivery Bacterial magnetosomes were used as the magnetic-targeted drug [137] carrier by loading antitumor drug doxorubicin [DOX]. Imaging System for Streptavidin detection using biotin conjugated to bacterial [134] magnetic particles isolated from magnetic bacteria were used as magnetic markers for magnetic force microscopy [MFM] imaging. The anchoring properties of Mms13 onto BMPs were evaluated using luciferase fusion studies. isolated from BMPs was performed. Mms13. Evaluation of efficiency of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. confocal laser scanning microscopy and transmission electron microscopy. Gene delivery and transfer Novel non-viral delivery system based on PEI coated bacterial MINPs [139] transfecting BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase [pcD-VP1] into mice. H.

Detection and diagnostic ER ligand binding domain [ERLBD] was fused to the anchor protein. Detection and diagnostic. and MamG involved in control of the size of growing magnetite crystals. gryphiswaldense at [142] various oxygen levels and analysis of the subcellular localization of the GFP-tagged magnetosome proteins MamC. 2011 Current Topics in Biotechnology & Microbiology Separation of biological molecules and cell Magnetic separation of monocytes and B-lymphocytes from the [140] peripheral blood was achieved with high purity using magnetosome with membrane expressing protein A to reduce cytotoxicity of innate membrane. We thereby hypothesize the development of nanovaccines by antigen [epitope] presentation on nanoparticle surface [Figure 8]. and the stability of expression and fluorescence of MamC-GFPlabeled magnetosomes was studied in vitro under various conditions. KG. As far as biosynthesis of MINPs is concerned. Study of cellular mechanism Expression of GFP in the microaerophilic M. which were observed using transmission electron microscopy. Sprouting success in finding aerobic organisms capable of producing MINP will strengthen the research in this field. GERMANY . Efforts should be made to cultivate more and more MTB 142 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. DUDWELLER LANDSTR. Imaging Magnetospirillum magneticum AMB-1 were injected in mice with [143] tumour. Its specific targeted delivery will boost the commercial importance of MINPs.Dhingra. MamF. H. Bacterial growth conditions were manipulated to produce small magnetite particles. apart from MTB other microbial flora must also be evaluated. The GFP-modified fluorescent MINPs were purified from bacterial cells. [141] Mms13. Tumour targeting was confirmed using 64Culabeled bacteria and positron emission tomography and by determination of viable cell counts recovered from different organs and the tumour. newer functionalization and modification of MINPs to introduce additional functionalities thus designing active sites for incorporation of diverse utility. which are used for efficient and stable protein display onto Bacterial MINPs. To evaluate the ligand-dependent binding of GFP coactivator. et al. binding assay of GFP-coactivator to ERLBD– Bacterial MINPs was performed.

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Vinod Saharan

Department of Molecular Biology and Biotechnology, Maharana Pratap University of Agriculture and Technology, Udaipur Corresponding author e-mail: vinodsaharan@gmail.com

ABSTRACT: The successful application of various nanoplatforms in medicine under in vitro conditions has generated interest in agri-nanobiotechnology. Role of nanobiotechnology will be significant in future for agriculture. Nanobiotechnology holds the promise of controlled release and site targeted delivery of agrochemicals (fungicides, herbicides, pesticides, plant hormones etc.). Specifically, the application of nanoparticles for biotic and abiotic stress offer new ways for crop protection. Alongside, plants are become an ideal factory for synthesis of desire size and shape of nanoparticles, which are in great demanded. Nanoparticle mediated plant transformation has the potential for genetic modification of plants for further improvement and replace the old technology. Herein we reviewed in this chapter the delivery of nanoparticulate materials to plants and their ultimate effects which could provide some insights of this novel technology for the improvement of crops. Keywords: Nanoparticles, stress, biotechnology, saponin INTRODUCTION: Nanotechnology is the manipulation or self-assembly of individual atoms, molecules, or molecular clusters into structures to create materials and devices with new or vastly different properties. The definition of nanotechnology is based on the prefix “nano” which is from the Greek word meaning “dwarf”. In more technical terms, the word “nano” means 10-9, or one billionth of something. The word nanotechnology is generally referring to materials with the size of 0.1 to 100 nanometres. Nano size has different properties from bulk (or micrometric and larger) materials as a result of their size. These differences include physical strength, chemical 156

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reactivity, electrical conductance, magnetism, and optical effects. Combining nanotechnology with biology evolve nanobiotechnology. Nanobiotechnology has the potential to revolutionize the agricultural and food industry with new tools for treatment of plant diseases, enhancing the bioavailability of agrochemicals and enable plant cells as nanoparticle synthesizer. Improvement in these areas through nanobiotechnology interventions can make agricultural systems more producible and organized. Application of target delivery system for agrochemicals is most imperative field of research in agriculture. “Smart Delivery Systems” should possess combinations of time controlled, specifically targeted and multifunctional characteristics to avoid biological barriers for successful targeting plant cells. Nanoformulation could increase the efficiency of agrochemicals and, allowing lower doses to be used to achieve desire effects. Thus, nanobiotechnology would protect the environment through control use of agrochemicals. Besides these significant properties, plants provide a biological synthesis route of several metallic nanoparticles which is more ecofriendly and allows a controlled synthesis with well defined size and shape. Development of nanoparticle / nanovesicle mediated genetic transformation also an important area to be exploited. This chapter presents recent advances in nanobiotechnology for agriculture (A) Nano-augmented delivery system for agrochemical: The successful application of various nanoformulations in drug delivery in medicine has raised concern in agriculture. Processes such as nanoformulation /nanoencapsulation/ nanoemultions show the benefit of more efficient use and safer handling of agrochemicals. In the past few decades, the use of adjuvant / surfactant has become common practice to enhance the delivery of agrochemicals to the inner tissue of the plant. The long- term fates of adjuvant/ surfactant in soils and elsewhere in the environment are also largely unknown because of the lack of long-term monitoring data [1]. Therefore, environmental questions have recently been raised about these traditional products. Available methods for delivery of agrochemicals are not specifically targeted; hence, 50-70 % of agrochemicals like fungicides, herbicides, pesticides, plant growth regulators etc. are deposited in environment without reaching the required sites. The activity of agrochemical gets further reduced by UV light, temperature, humidity etc. The agrochemicals loaded into the inner core of nanoparticles would show a typical sustained-release pattern, protection from external environment (UV, temperature, humidity etc.). Hence, such nanocarriers have a promising future in application of agrochemicals. Adjuvant /surfactants are 157

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Oil based, Polymers+ lipid based Film-forming materials Inorganic salts Silicone-based nonionic Amine ethoxylate & nonylphenol ethoxylates Adjuvant /surfactants are partially or completely synthetic materials i.e. Raising environmental questions Non-degradable Less efficient and costly Severe necrotic damage to plant cells Toxic to soil & aquatic organisms
Not universal (need to change with different agrochemicals )

(a) Phytosaponin as a delivery system: Saponins are a highly diverse group of glycosides of plant origin. They contain either a steroidal or a tri-terpenoid aglycone to which one or more sugar chains are attached. The sugars are usually glucose, galactose, glucuronic acid, xylose, or rhamnose. The diversity of saponins is a result of the variability in the sugar side chains. The main biological activity ascribed to saponins is their membrane permeabilizing property. Saponins have additionally been reported to exhibit adjuvant-active properties. Based on these observations, an open cage-like immunostimulating complex (ISCOM) of cholesterol, lipid, immunogen, and saponins from the bark of Quillaja saponaria (soap bark tree) has found successful application as an active adjuvant for vaccination [2]. Synthesis of nanosaponin formulation with chitosan and other components for evaluation of drug delivery system for animal system has established successfully. Nanosaponin formulation with chitosantripolyphosphate cross linking found promising for encapsulation of biological active compounds [3,4]. The physico-chemical characteristics of saponins indicate its suitability for delivery of biological active compound. Saponins assemble in micelle structures in aqueous environment. The property of making novel micelles structure enable saponins to encapsulate various biomaterials. Microscopic studies of saponin extract from Quillaja and Balanites plants revealed their self-assembled properties [5,6]. Biological activity of saponins is due to their binding activity with sterols components in the biological membranes and causes partial pour formation [7,8]. Plant based adjuvant; Quillaja saponin has been used abundantly in animal system for delivery of drugs [9]. In this fashion, saponin molecules could play a vital role in delivering agrochemicals in a targeted manner in crop plants. 158

development. Currently more efforts have been concentrated to cope with adverse effect of global warming in agriculture. fruit size. possess a variety of functions in vegetative and reproductive phases of plant life cycle such as seed germination. etc [14. Study further conclude that saponin at predetermine concentration as ability to bind to lipid molecules and make safe passage for plant growth regulators. 2011 Current Topics in Biotechnology & Microbiology Nanosaponin delivery system Nano size Efficient in encapsulation of target material Biodegradable & non-toxic to soil & aquatic organisms Cheap and easily available UV protection of encapsulating materials Natural binding ability to biological membranes Quillaja and Balanites saponin has been successfully tested to cross cuticle membrane [10. film. Plant hormones like 2. it is used extensively in drug delivery applications [13]. The primary amine groups render special properties that make chitosan very useful in carrier/delivery system for animal/plant cells. Both are made by linear β-(1-4)-linked monosaccharides. Novel formulation of saponin with plant growth regulator has been evaluated and found excellent for enhancement of in vitro plant growth at lower concentration of plant growth regulators. DUDWELLER LANDSTR. 1) [11]. fiber. paste. Plant growth regulators have been used over the past decades for several purposes in crop production. GERMANY . Therefore. BAP. ABA.Dhingra. It is suggested that chitosan can be 159 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Agrinanobiotechnology could bring the revolution in this respected by designing target specific delivery system for plant growth regulators. Studies gave hypothesis that saponin encapsulates the plant growth regulators through ultrasonication and deliver encapsulated material efficiently to the surface/inner part of plant cell (Fig. Chitosan is reported to influence the production of substances related to stress response. flowering. Compared to many other natural polymers.4-D. an important difference to cellulose is that chitosan is composed of 2-amino-2-deoxy-β-d-glucan combined with glycosidic linkages. et al. Chitosan is relatively reactive and can be produced in various forms such as powder. stem elongation. chitosan has positive charges which render chitosan to bind with biological membranes [12]. 18]. GA etc. 11]. metabolism and morphogenesis of higher plants. (b) Chitosan as a delivery system: Chitosan is a polysaccharide. Foliar and root application of nutrients and plant growth regulators improve yield characteristic. However. 15]. grain yield and nutrient use efficiency. Plant hormones are involved in controlling the growth. H. KG. such as phytoalexins [16] and chitinases [17. leaf expansion. similar in structure to cellulose.

Recently. KG. et al. (2003) [22] reported that combination of S. melanosporofaciens EF-76 and chitosan represents a promising method of biocontrol against common scab in potato crop. Hydrophilic head Hydrophobic tail (Saponin molecule) (Saponin micelle structures) (Encapsulation of agrochemical into saponin) Figure 1: Saponin encapsulation of agrochemicals 160 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The activation of protective mechanisms in plant tissues with chitosan inhibited the growth of taxonomically different pathogens [20]. It has been considered as an alternative to chemical fungicides [21].. GERMANY . resulting in improved yields and plant health in numerous crops and fruits.Dhingra. Chitosan treatments have plant growth promoting effects. Beausejour et al. chitosan nanoformulations found promising for encapsulations of various components and applied in animal cells. 2011 Current Topics in Biotechnology & Microbiology used commercially for controlling various plant disease under field conditions [19]. It could be predicted that nano scale chitosan formulation will act for effectively than normal chitosan on cell membrane either thought proper exposure with receptors or cross easily via membrane pores. DUDWELLER LANDSTR. H.

Microscopic and X-ray absorption studies confirmed the nucleation and growth of nanoparticles inside plants. Hence. The mechanism of formation of nanoparticles. 34. The application of fluorescent labelled starch-nanoparticles as plant transgenic vehicle was reported in which the nanoparticle biomaterial was designed in such a way that it bind and transport genes across the cell wall of plant cells by inducing instantaneous pore channels in cell wall [45]. 44]. 32]. 37. GERMANY . This process has similarity with microinjection method of gene delivery [42. this method provides a better and safe means of nanoparticle production with defined size and shape of our interest. H. 24]. 35. 2011 Current Topics in Biotechnology & Microbiology (B) Nanobiotechnology as Agronanofactories: Various physical and chemical methods used for synthesis of nanoparticles. 30].Dhingra. The amount of nanoparticle accumulation in plants also varies with reduction potential of ions and the reducing capacity of plants that depends on the presence of various polyphenols and other heterocyclic compounds present in plants [31. (C) Delivering genetic materials into plants: Nano-fibres/nanoparticles/nanovesicles with cationic nature have ability to precipitate negative charged DNA molecules and bring into cell through nonporous of cell membrane. compared to the chemical synthesis of nanoparticles. 29. The formation and growth of gold nanoparticles [Au NPs] inside live alfalfa plants and sesbania seedlings grown in gold enriched media was reported [25]. Recently the use of biological systems for the synthesis of nanoparticles is gaining interest since it is more ecofriendly compared to existed system [23. et al. KG. plants provide an easy and safe green route for the synthesis of various metals. Different studies regarding the extracellular biological synthesis of nanoparticles using leaf extracts [33. The formation of nanoparticles of an alloy of gold–silver-copper using plant was also reported [27]. The uptake of silver ions and their reduction and distribution as Ag NPs within cellular structure of some metallophytic plants has also been investigated [26]. Copper biomineralisation with some wetland plants that transform copper into metallic nanoparticles at soil-root interface with the help of some endomycorrhizal fungi were also reported that could reduce copper toxicity in contaminated soils. It is possible to integrate different genes on the nanoparticle at 161 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 41] were also reported. The uptake and translocation of nanoparticles across root cells depends on the type of metal ions and plant species. 38] and dead tissues of various plants [39. DUDWELLER LANDSTR. 36. 40. 43. whether they are formed outside in the media and then translocated to plants or whether they are formed by the reduction of metal salts within the plants itself still needs more clarification [28. This opens up a great scope of using plants for the production of mixed metal nanoparticles of specific compositions. Among various biological systems.

Particles used for bombardment are typically made of gold since they readily adsorb DNA and are non-toxic to cells. Experiments showed that the plasmid DNA transferred by gene gun method using gold-capped MSNs was successfully expressed in intact tobacco and maize tissues. KG. After entering the protoplasts. 162 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. This efficient delivery method has pronounced applications in various protoplast-based gene expression studies. This problem was solved by capping MSNs with gold nanoparticles which increased their momentum after acceleration by the gene gun. nucleotides and chemicals in plant biotechnology. This is how the nanoparticle mediated plant transformation differs from the conventional genetic engineering methods (like electroporation. etc). DUDWELLER LANDSTR. microinjection. et al. GERMANY . 2011 Current Topics in Biotechnology & Microbiology the same time and the imaging of fluorescent nanoparticle is possible with fluorescence microscope thus understanding the movement of exterior genes along with the expression of transferred genes. H. Nowadays gene gun or particle bombardment is one of the popular tools to deliver DNA into intact plant cells [47]. Such modification of MSNs also allowed plasmid DNA to adsorb on MSN surface. The major advantage is the simultaneous delivery of both DNA and effectors molecules to the specific sites that results in site targeted delivery and expression of chemicals and genes respectively. It was found that surface modification of MSNs with triethylene glycol was necessary to penetrate the cells. Hence successful generation of pores on cell wall and cell membrane by suitable agents help in nanoparticle mediated DNA transfer that might be more successful in regenerative calli and soft tissues.Dhingra. In this method the minimum amount of DNA that is required to detect marker expression was 1000-fold less than that required for the conventional delivery method. In preliminary experiments protoplasts were incubated with fluorescently labelled MSNs. the plasmid DNA was released from the MSNs and the green fluorescent protein (GFP) marker encoded in the DNA was expressed in the cell which was detected by microscopy. The ability of surface functionalized mesoporous silica nanoparticles (MSNs) to penetrate plant cell walls also opens up new ways to precisely manipulate gene expression at single cell level by delivering DNA and its activators in a controlled fashion [46]. Future scopes include pore enlargement and multifunctionalization of MSNs which provide even better possibilities in target-specific delivery of proteins. it is difficult for delivering foreign DNA attached on MSNs by gene gun method. Since MSNs are too light.

X. H. P. & Osbourn A. R. 7. GERMANY .. 63. Agric. 3. Environmental fate of mineral. S. J. 114-128. A.P.Dhingra. Preparation and characterization of saponin-loaded chitosan-tripolyphosphate microspheres by spray drying. Sun. M. (2009). Sci. and Mol. 173-178. Designed nanoparticles can be successfully used to deliver agrochemicals or other substances (elicitors. Y. vegetable and transesterified vegetable oils. Plant mediated synthesis of metal nanoparticles provide a safe synthesis route with better control over morphology of nanoparticles. KG. J. J.. B. Nanotechnology. Q..E. Battersby. 18. Morrissey & Morrissey. 54. (2007). 163 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Chitosans and pectic polysaccharides both induce the accumulation of the antifungal phytoalexin pisatin in pea pods and antinutrient proteinase inhibitors in tomato leaves. Fungal Resistance to Plant Antibiotics as a Mechanism of Pathogenesis. Su. H. (1993). Biol. C. Food Chem.X. Huang. 5. J. et al. & Ye.A. nucleic acids etc.. H. Lu. 2. Z. L. Cornish. Phyto-Saponins as a Natural Adjuvant for Delivery of Agromaterials through Plant Cuticle Membranes. 4. DUDWELLER LANDSTR. 6. Li. Chapagain.. (1999). D. D. 37. & Watkinson.. 64.Y. REFERENCES 1. 1787-1796. Microbiol. Drug Development Research. Walker-Simmons. & Hyun. 708724. & Ryan. Revi. 2011 Current Topics in Biotechnology & Microbiology CONCLUSION: Nanobiotechnology is heading to the expansion of a range of economical nanotech applications for enhanced plant growth.Y. N. & Wiesman. Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf. Advances in saponin-based adjuvants Vaccine.Y. (1983). J. Nanostructures endow with an efficient means to distribute agrochemicals in a controlled fashion with high site specificity. 5104-5114.. Biochemical and Biophysical Research Communications. (2006). 110. 27. G. Sun.P.. &Yang. 6277-6285. On other hand nanocarrier would be a leading tools for gene transformation in a variety of plants. P. Xie . 194-199. Pestic.) into localized areas of plant tissues.. Hadwiger. (2005). Kashappa & Kashappa.

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the CTB gene sequences differs among the two biotypes. a filamentous bacteriophage. Based on non-random base variations. This bacterium after passing the stomach acid barrier adheres to and colonizes in the intestinal epithelial cells leading to production of cholera toxin.Dhingra. have been described. Institute of Life Sciences. 46 and 68. New ctxB variants showing additional polymorphism at amino acid positions 28 and 34 have recently been described in V. cholerae O139 strains. H. Email: turabe_fazil@yahoo. DUDWELLER LANDSTR. In this chapter. *Corresponding Authors: MHU Turabe Fazil. Bioinformatics Centre. Cholera toxin is a hexameric A-B5 type toxin and encoding genes lies on CTXΦ. Efficient binding to GM1 could potentially raise the uptake of antigen across the mucosa and lead to an improved presentation of conjugated molecule to the immune system. KG. Although the action of CT is conserved among classical and El Tor strains. Nalco Square.com ABSTRACT: Vibrio cholerae is the causative agent of the deadly diarrheal disease cholera. Bhubaneswar.* and Durg V singh1 1 2 Infectious Disease Biology. Sunil Kumar2. three types of ctxB genes due to change in the deduced amino acid sequence positions at 39. Bhubaneswar. India. 2011 Current Topics in Biotechnology & Microbiology Chapter VIII IN SILICO ANALYSIS OF SEQUENCE VARIATIONS IN CHOLERA TOXIN B SUBUNIT AND THEIR BINDING WITH CARBOHYDRATE LIGANDS MHU Turabe Fazil1. India. GERMANY .*.com Sunil Kumar. The toxicity is attributed by enzymatic activity of the A-subunit whereas B subunit stimulates mucosal antibody responses. These differences can be employed in designing paratopes that can determine specificity of either a precise biotype or a standard anti-cholera toxin monoclonal antibody and could be exploited for designing drugs or vaccines. Bioinformatics Email: skybiotech@gmail. Institute of Life Sciences. et al. We found subtle variations in hydrogen bond binding abilities of variants of CTB with the carbohydrates ligands constituent of GM1 receptor. The mechanism of CTB binding to the GM1 receptor of epithelial cells has not been fully defined. we discussed the possibility of variable binding efficiency of CTB variants to constituent carbohydrates of GM1 ganglioside through molecular and computational approaches. 168 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Nalco Square.

The fluid loss may be so rapid that the patient will be at risk of death within a few hours of onset of the disease. Infectious disease processes. Cholera gravis caused by V. DUDWELLER LANDSTR. the size of the inoculums.the disease V. has helped us to understand natural science and added tremendous inputs to existing knowledge of biology. like new generation sequencers. Understanding pathogenesis is not only important to find immediate therapy but also for gaining knowledge on the evolution in a species. cholerae is the causative agent of the deadly diarrheal disease cholera. The severity of the infection depends on several factors like local intestinal immunity arose from previous bacterial infection or vaccination. we describe the use of gene sequencing and computational protein modelling to address questions on pathobiology of Vibrio cholerae. the surviving bacteria reaches to intestine. cholerae is classified into more than 200 serogroups of which only O1 and O139 serogroups have the potential to cause epidemic or pandemic cholera. adhere to and 169 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. which had elucidated the imagination of biologists. 1. often with several foci of onset.int/mediacentre/factsheets/fs107/en/index. About 3–5 million cholera cases have been reported per annum with a mortality rate of 20-30% that could increase if not treated in time (http://www. The most distinctive and salient features of cholera is epidemiologic behaviour that tend to cause explosive outbreaks. cholerae have been reported infecting populations throughout the world but to specific endemic regions including Asia and Africa. In case of severe cholera. KG. crystallographic structure elucidation methods. H. 2011 Current Topics in Biotechnology & Microbiology INTRODUCTION Food security and increase in average life expectancy can be attributed to biotechnological developments in the last century. resulting in epidemics and subsequently pandemics. Cholera can be transmitted after consuming contaminated water and food. GERMANY . et al. microarrays.Dhingra. In this chapter. The advent of modern molecular techniques. These bacteria after passing the stomach acid barrier.html). the rate of purging may quickly reach up to 500–1000 mL/h leading to acute dehydration. have been unravelled to an understandable level owing to biotechnology. Cholera.who. V. the temporal inhibitions of understanding miniscule proceedings of molecular machinery have been dampened to a good extent. The most distinctive feature of the cholera is the painless purging of voluminous stools resembling rice-water and fishy odour. adequacy of the stomach gastric-acid barrier and the patient’s blood group. Since introduction of computers to investigate biological problems.

The RS1 element is very similar to the RS2 but contains an additional gene. Acquisition of CTXΦ is a key event in the emergence of V. The CTXΦ is integrated in the large chromosome of toxigenic V. et al. Basically three distinct CTXΦ variants have been described. DUDWELLER LANDSTR. is one of the principal virulence factors of the diarrhoeal pathogen V. cholerae. The CTXΦ. cholerae [2]. Since RS1Φ lacks the genes for its morphogenesis. cholerae chromosome. a ~6. it cannot independently packaged into the genome for transmission. The GC content of ctxAB also differs from the other CTXΦ genes that led to the hypothesis that ctxAB are acquired after the evolution of a pre-CTXΦ that lacked these genes [4]. related to coliphage M13. The CTXΦ prophage is often flanked by a related genetic element known as the RS1.Dhingra. a filamentous bacteriophage. KG. infects host bacteria by adsorbing to a ‘toxin-coregulated pilus’ (TCP). 2011 Current Topics in Biotechnology & Microbiology colonize in the intestinal epithelial cells leading to production of cholera toxin (CT) and causing symptoms of cholera. cholerae. the organization of the CTXΦ core region is similar to other filamentous phage genomes that infect E. namely CTXETΦ derived from V. Genetics of CTXΦ The genes encoding for cholera toxin lies on CTXΦ. The RS1 element is the genome of a satellite phage of ~2. CTXCalcΦ derived from O139 V. This single stranded DNA phage.9 Kb element. and can replicate and transmit vertically as a plasmid. cholerae El Tor and O139 strains whereas O1 classical biotype isolates had prophages in both large and small chromosome. RS1 replicates by the mechanism similar to CTXΦ. GERMANY . However. utilizing the CTXΦ morphogenesis genes to produce RS1Φ particles [3]. With the exception of the toxin genes (ctxAB). coli and V. rstC which acts as antirepressor. cholerae as a pathogen. cholerae O1 El Tor. often as an array of tandemly repeated copies.7-kb. capable of both autonomous replication and chromosomal integration. The gene for the pilus protein is expressed under control of the ToxR regulatory system that also regulates the transcription of the cholera toxin genes. This model proposes the existence of a common pre170 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. cholerae [1]. Virulence factors are frequently encoded within mobile genetic elements such as phages and plasmids. H. CTXΦ phage was shown to horizontally transmit a virulence factor that results in lysogenic conversion of a bacterium to toxigenicity. can integrate into V. CTXclassΦ derived from classical V. The ctxAB genes do not have any role to play in the CTXΦ life cycle. 2.

Cholera toxin Cholera toxin is a hexameric A-B5 type toxin. Dukoral (R) is the only vaccine recommended and registered by WHO for vaccination of populations at risk of cholera epidemic region. This vaccine consists of killed cells of V. Cholera toxin B subunit (CTB) and the related Escherichia coli heat-labile enterotoxin B subunit (LTB) are competent carrier molecules for the stimulation of mucosal antibody responses and oral tolerance. cholerae strain CVD103HgR. It has also been shown that CTB induces major histocompatibility complex (MHC) class II expression on B cells and enhances antigen presentation by macrophages in the absence of increased MHC II expression. H. KG. given in two doses within 1–6 weeks. The B subunit is considered as molecular recognition unit and delivery vehicle for A-subunit [6]. et al. CTA1 confers CTmediated toxicity. DUDWELLER LANDSTR. would stimulate antibacterial and antitoxic immunity. cholerae of both toxigenic biotypes and cholera toxin B subunit.ion and water ultimately leading to profuse diarrhea. given as a single-dose. whereas CTA2 acts as a linker between CTA1 and cholera toxin B subunit (CTB). GERMANY . has also been found providing antibacterial and antitoxic immunity. The other lyophilized oral vaccine. Efficient binding to GM1 could potentially raise the uptake of antigen across the mucosa and lead to an improved presentation of conjugated molecule to the immune system. The mechanism behind CTB's efficiency as a mucosal carrier molecule has not been fully defined but believed to be associated with binding to the GM1 receptor present on the epithelial cells. leading to activation of adenylate cyclase and a concomitant elevation of cAMP levels causing hyper-secretion of Cl. Cholera toxin B subunit is used as an oral cholera vaccine candidate. Like several other toxin-encoding phages. The toxicity is attributed by enzymatic activity of the A-subunit that catalyzes ADP ribosylation of the α-subunit of GTP binding protein. 2011 Current Topics in Biotechnology & Microbiology CTXΦ ancestor for the currently known CTXΦ variants [5]. ctxAB is adjacent to the CTXΦ attachment site indicating that an imprecise excision of the pre-CTX prophage can generate a new phage by acquisition of ctxAB sequences by yet uncharacterized mechanism. 3. Cholera toxin A subunit (CTA) consists of two polypeptide chains. CTB triggers the polyclonal activation of B cells and selective 171 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. CTA1 and CTA2.Dhingra. The five B-subunits of the toxin bind principally to monosialoganglioside (GM1) receptor present on the surface of the intestinal epithelial cells. GS. Orochol an avirulent mutant of V.

Because of presence of variants of CTXΦ in V. we employed a phage specific amplification of cholera toxin B subunit [12]. The DNA sequence between rstR gene and the 172 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. GERMANY . and in determination of relationships between variants of CTB and severity of disease apart from defining the requisite binding sites of drug like molecules. 2011 Current Topics in Biotechnology & Microbiology apoptosis of CD8 cells from both the Peyer’s patches and intraepithelial lymphocytes. The therapeutic applications of CTB-mediated oral tolerance include immunoglobulin E (IgE)mediated allergic reactions. the treatment of T-cell-mediated autoimmune diseases and infection related pathological inflammatory conditions [7. H. CTB sequence variation and binding with carbohydrate ligands Although the action of CT is conserved among classical and El Tor strains. not involved in receptor binding. N-acetyl galactosamine and glucose were employed individually for docking simulations. 4. However.S. Mutation analysis of CTB revealed variations in immunoreactivity. cholerae strains.Dhingra. can be a reason for epitope variation of CTB and. a close homolog of CT. haemolysis and GM1 binding ability for the toxin subunit [11]. the role of natural variations in CTB and their advantage if any. Gulf Coast strains. sialic acid. in survival or pathogenesis is not yet explored. genotype 2 in El Tor biotype strains from Australia. monosachharide components of GM1 receptor namely galactose. Genotype 1 is found in classical biotype strains worldwide and in U. The total gene sequence of the cholera toxin B sub-unit is comprised of 375 bases. therefore. In this regard. coli heat labile toxin. dynamics simulations and docking studies for all natural CTB variants. and genotype 3 in El Tor biotype strains from the seventh pandemic and the Latin American epidemic. if any. et al. molecular modelling. DUDWELLER LANDSTR. 46 and 68. KG.8]. New ctxB variants showing additional polymorphism at amino acid positions 28 and 34 have recently been described in V. The information generated would be useful in establishing enterotoxic potential of the CTB variants. Previous studies indicated that amino acid sequence variations at positions. cholerae O139 strains [9. 10]. We carried out a gene sequencing. Based on nonrandom base variations. need be considered for development of synthetic and natural vaccine against cholera. Monosaccharide libraries had been earlier used for exploration of binding sites to develop simple and easily synthesizable molecules against E. the CTB gene sequence differs among the two biotypes formed the basis for ctxB genotyping. three types of ctxB genes due to change in the deduced amino acid sequence positions at 39. have been described.

cross over-95. KG. designated as genotype 1.Dhingra.000. Crystal structure of the cholera toxin B-pentamer complexed with GM1 pentasachharide (PDB code: 2CHB) was selected as template for all modelling procedures. were 173 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. designated genotypes 2 and 3. N-acetyl neuraminic acid.ac. All calculations were performed by using ACCELRYS DS Modeling 2. number of operations-1. both variants of CTB produce classical type of cholera toxin B. whereas the Calcutta type phages comprise mostly of genotype 5. The experimentally generated crystal structures for all known variants in V. cholerae are not yet available. number of docking 10. comprising complete coding sequence. Sequencing data indicates that a majority of V.php). et al. All atom MD simulations of CTB protein in explicit water was carried out using GROMACS 4. USA). The sequence generated was used in obtaining translated amino acid sequence of all the possible CTB variants.cam. The classical type of CTB. we built molecular models for all the reported variants of ctxB gene (Table 1) by means of comparative modelling using MODELLER software.9 kb.gov).uk/~sdm/sdm. The O139 V. 2011 Current Topics in Biotechnology & Microbiology ctxB were selectively amplified using forward primers of rstRET or rstRCalc and reverse primer of ctxB that yielded amplicons of ~6. as the crystal structure 2CHB represented classical cholera toxin. 5 and 6 (Table 1). The primary model generated was that of classical CTB. GERMANY . Three dimensional co-ordinates for these molecules were generated using CORINA program. cholerae O139 El Tor phages belong to genotype 4.nlm. was produced by the V. DUDWELLER LANDSTR. However. galactose. El Tor type of CTXΦ prophages produces El Tor type of cholera toxin that includes two variants of CTB. H.nih. The chemical structures of monosaccharides were extracted from pubchem (http://pubchem. Structures of all four carbohydrate ligands (glucose. 00.0 software suite (Accelrys Inc. VERIFY 3D and ERRAT analysis was performed. CA 92121. This PCR product was then used as template in nested PCR to amplify ctxB gene of 449 bases. In order to assess the stereo-chemical qualities of the three dimensional models.bioc. N-acetyl galactosamine) included in binding to CTB were retrieved in two dimensional MDL/SDF format. cholerae harbouring classical type of CTXΦ prophages.ncbi.6 program and the GROMOS96 force field for a time scale of 1ns.0. Therefore. The standard default settings. migrate-10. PROCHECK.1. niche size-2. The in silico mutations performed using the mutate _model command of MODELLER was used to generate the rest of the variants. structure and function of the protein were calculated by SDM server (http://mordred. cholerae possesses both El Tor and Calcutta type of CTXΦ prophages includes genotypic variants 4. consisting of population size-100. Amino acid variations affecting the stability. selection pressure-1. San Diego.

Considering the chronology of evolution in ctxB genotypes. The results of this study thus indicated that there is decrease in binding efficiency of El Tor CTB compared to the classical cholera toxin. The ligand showing maximum interactions with the protein were plotted using the program LIGPLOT. et al. sialic acid and N-acetyl galactosamine in comparison to all other known CTB genotypes (Fig. while the El Tor type phage replicated in the host to produce functional phage. cholerae O139. produces CT of the genotype 3. cholerae O139. while the classical biotype produces CT belongs of genotype 1. cholerae belonging to classical biotype are known to cause more severe cholera. cholerae O1 and O139 strains showing amino acids variations of ctxB and their respective genotypes Strains ctxB Genotypes Amino Acid Position Variation 28 34 39 46 68 Classical. Table 2 indicates respective variations in percentage solvent accessibility at mutation position and differential free energy of unfolding for all modeled CTB variants. V. (2002) Type 5 A H H F T V. Australia Type 2 D H H L T El Tor. H. Thus. (2005) Type 6 D P Y F T V. generated by SDM software in comparison to crystal structure of Genotype 1 (classical). shows most impressive bonding characteristics with galactose. similar to 174 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. KG. while El Tor biotype strains survive better in aquatic environments. 2011 Current Topics in Biotechnology & Microbiology adopted for GOLD docking. Table 1. cholerae biotype El Tor. The CT genotype of the El Tor strains currently associated with cholera in the Indian subcontinent has shifted from genotype 3 to genotype 1. cholerae O139.Dhingra. The data generated suggests that new ctxB genotypes of V. cholerae. The phage that encoded this CTB genotype was found integrated in host chromosomes but unable to replicate independent of the host. cholerae O139. GERMANY . 569B Type 1 D H H F T El Tor. type1 of CTB was the first reported genotype in classical V. DUDWELLER LANDSTR. cholerae O1 biotype El Tor. Among the CTB variants V. cholerae O139. (2005) Type 4 D H Y F T V. V. among various carbohydrate ligands. the current circulating El Tor strains causing cholera have characteristics of El Tor biotype but possess classical CTB. N16961 Type 3 D H Y F I V. causative organism of the ongoing seventh pandemic. 1). the most recent known type 6. Docking simulations studies suggested that the ability of the type1 CTB to interact with galactose and N-acetyl galactosamine were considerably superior to its counterpart type 3 found in V.

9.7. and thus in forming stable complexes with respect to seventh pandemic clone of biotype El Tor.429 Kcal/mol Figure 1: Molecular interactions of CTB variants with lignd galactose 175 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.Tyr) .055 Kcal/mol 6 34 (His – Pro. DUDWELLER LANDSTR. Table 2. Recent variations in CTB employed by the microbe indicate that V. GERMANY .786 Kcal/mol 3 39 (His .040 Kcal/mol 4 39 (His . 68 (Thr .4.Tyr).Leu) + 0.1 2. Genotypes Position variations % age solvent accessibi-lity Overall differentat difference at position of tial free energy of mutation unfolding 2 46 (Phe .8 and 39 by – 4. cholerae produce a potent toxin more proficient in hydrogen bonding. 2011 Current Topics in Biotechnology & Microbiology classical CTB were more profound in potential to bind carbohydrate ligands. KG. This could be a reason to worry because current strains of V.7 3. 39 (His – Tyr) 34 by + 21.82 Kcal/mol 5 28 (Asp – Ala) .Ile) 39 by . et al. cholerae O1 and O139 are host to CTX prophages that had acquired capabilities of independent replication and infection and can lead to rapid spread of severe cholera. H.Dhingra.7 0.9 3.7 1. Results obtained using SDM software for CTB variants in comparison to crystal structure of type 1 CTB.9 and 68 by + 28.

M. 2011 Current Topics in Biotechnology & Microbiology CONCLUSION The rate of change in the genetic profile of toxigenic V. Mekalanos. J.. G. to the Institute of Life Sciences.. variations in CTB have been studied of late. the data presented could be exploited for designing drugs or vaccines. Nusrin. 272:1910-1914. Hamabata. KG. Science. 2. et al.. (2004). Journal of Clinical Microbiology. GERMANY . D. ACKNOWLEDGEMENTS This work in part was supported by the funds contributed by the Department of Biotechnology. (1996).A. Bhubaneswar.. Of the various markers used to monitor genetic changes. Ansaruzzaman. Nair. we explored the possibility of variable binding efficiency of CTB variants to constituent carbohydrates of GM1 ganglioside.. Turabe Fazil are gratefully acknowledged. Safa. G. Hossain. with respect to competence in hydrogen bonding.. M..5854-5856. M. 42. A. Y. Senior Research Fellowships awarded by the Indian Council of Medical Research. New Delhi. Khan.K. M. As CTB is an attractive drug candidate for autoimmune diseases as well as differentiation therapy. 176 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Takeda. T. These differences can be employed in designing paratopes that can determine specificity of either a precise biotype or a standard anticholera toxin monoclonal antibody. S. India and Institute of Life Sciences. H. New Delhi.. The results of this study indicate subtle variations in the binding abilities among variants of CTB with the carbohydrates ligands constituent of GM1 receptor. Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic in Bangladesh. Waldor. R. an important step in causing severe cholera through molecular and computational approaches. cholerae has been a reason for concern due to their ability to cause epidemic and pandemic cholera.Dhingra.A. S. Bhuiyan.Y.. Though there had been no phenotypic differences among strains harbouring CTB variants. the data provides clues to potential molecular discrepancies in binding of CTB to its receptor... to M.J. Khan.H.. Faruque. In this study. N. DUDWELLER LANDSTR.U.B. Sack.A. REFERENCES 1. Lysogenic conversion by a filamentous phage encoding cholera toxin.

I. K. H. 5530–5538. Waldor.. A.. Immunological memory after immunization with oral cholera B subunit--whole-cell vaccine in Swedish volunteers. Heilpern.D. (2000).B. T. Mantri. B. and epitopes of cholera toxin B (CTB) subunits and CTB/heatlabile enterotoxin B subunit chimeras. Infection and Immunity. GERMANY .. S. et al. Bhanumathi.K. J.. Ramamurthy.. A. K.. (2001).S. (2000). 7. 69. M.G.P. Jobling. B. southern India. Moyer. M. 4.. (2009). Davis. Turabe Fazil. A. Morita.V.. 177 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. (1994).. Research in Microbiology.K. M.. pentamer formation. A. Holmgren.F. Holmes. El Tor and Calcutta CTXΦ precursors coexisting with intact CTXΦ copies in Vibrio cholerae O139 isolates. M. E. Fando. Holmgren. Pandey. FEMS Immunology and Medical Microbiology.. 4125-4128. Nordstrom. (2002). 159. E. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.U. Kilander. H. 1260-1271.. R. B.. S. N. Characterization of Vibrio cholerae O139 belonging to multiple ribotypes and isolated from diarrhoeal patients in Kerala. T. Infection Immunity. Vaccine. 57. DUDWELLER LANDSTR. 81-87. R. Jertborn.. Watanabe. G. 8... 10. 11. 5. Infection Genetics and Evolution. M.. Rodriguez. Boyd. H. C. (1992).H. Alam.M. Spangler.M.. M. Journal o f Bacteriology. 454-459 11. D. 12. 10. Bhuiyan. 70. Cravioto.V.. (2010). KG.CTXΦ and CTXΦ from Vibrio cholerae O139 strains.622-647. Changing genotypes of cholera toxin (CT) of Vibrio cholerae O139 in Bangladesh and description of three new CT genotypes. Singh. Effect of storage and sodium chloride on excision of CTXΦ or pre. 6.. Boyd. Mutational analysis of ganglioside GM (1)-binding ability. J. Singh.K. M. (2008). 182. Nair.. Jertborn. 1078-1082.. Molecular analyses of a putative CTX precursor and evidence for independent acquisition of distinct CTXs by toxigenic Vibrio cholerae..136-141. Microbiology Review 56. 9. Suzarte.. 6992-6998.K.. 2011 Current Topics in Biotechnology & Microbiology 3.. Marrero. Journal of Bacteriology. Campos. R.E. 182. M. CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes. Svennerholm.. 12. J.. (2011). Nusrin. Czerkinsky. Waldor. E. C.J. Infection Genetics and Evolution.A. D.Dhingra.. Mohapatra. Ledon.F. Local and systemic immune responses to rectal administration of recombinant cholera toxin B subunit in humans. 925-930.

N. huge man power and millions of dollars in order to come up with a single effective novel drug. Computer-aided drug design (CADD) represents more recent applications of computers as tools in the drug design process. GERMANY . Virtual screening is performed by docking method in which ligand databases are screened for a target. and develop compounds into clinical trial candidates. H.g. In structure based drug design approach one should have reliable 3-dimensional structural information for the receptor or the ligand-receptor complex which is available. new ligand molecules are built up within the constraint of active site of the target. Chandrabhan Seniya2. India Corresponding Author: Pramod K. M. Madhav Institute of Technology & Science Gwalior. efficacy and safety. In de novo ligand design method. Structure-based ligand or inhibitor design aims to identify chemical compounds that bind strongly to key regions of biologically relevant molecules. Bihar. Khan3 1 2 Department of Computational Biology & Bioinformatics. SBDD is a powerful method and it is an integral part of drug discovery today. et al. India. The approach used in CADD is dependent upon the amount of information that is available about the ligand and receptor. NMR or homology modeling techniques. L. design compounds for selectivity. KG. Yadav. Yadav1. consuming several years. 178 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The ligand-based approach is applicable when the structure of the receptor site is unknown. e. Allahabad-211007. Singh2. but when a series of compounds have been identified that produce the activity of interest.P. India Department of Biotechnology.. Atul K. G. as from X-ray diffraction. E-mail: ABSTRACT: The traditional method of discovering drugs has always been a tedious process. SHIATS. 3 Department of Biotechnology. 2011 Current Topics in Biotechnology & Microbiology Chapter IX STRUCTURE-BASED DRUG DESIGN APPROACHES IN DRUG DISCOVERY Pramod K. J. The current methods for structure-based drug design are virtual screening and de novo ligand designing.Dhingra. Most common strategies used in drug designing are structure or target based drug designing (SBDD) and ligand or analogue based drug designing (LBDD). enzymes or receptors and should be able to inhibit or stimulate the biological activity of their target molecules. CADD uses a variety of computational methods to identify novel compounds. Satendra Singh1. Darbhanga-846008.Mithila University. DUDWELLER LANDSTR.

The understanding about this quantitative relationship heralded in a new era in drug discovery process in the form of computer aided drug design. clinical trial testing and pharmacogenomic optimization [1]. ligand-based drug design. 2011 Current Topics in Biotechnology & Microbiology 1. target selection. consuming several years. 2. Sometimes CADD is interchangeably used as rational drug design (RDD).Dhingra. It was not until the 1960's that some understanding began to develop about the quantitative relationship between the structure of a drug and its biological activity. The pipeline of drug discovery from idea to market consists of seven basic steps: disease selection. with the advent of information technology. the ability to rationally design drugs using protein structures was an unrealized goal for many structural biologists. efficacy and safety. It is a process used in the pharmaceutical industry to discover and develop new drug compounds. During the early 1980s. H. CADD approaches have contributed to the 179 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. also called computer-aided molecular design (CAMD). lead compound identification. DUDWELLER LANDSTR. Today. and by the early 1990s the success stories started pouring in. et al. INTRODUCTION Evidences for the use of medicines and drugs can be traced back to as far as the first Egyptian dynasty during 3100 B. their discovery has always been a relied on trial-and-error method testing of chemical substances on cultured cells or animals. even though there is still quite a bit of fine-tuning necessary to perfect the process. The first projects were underway in the mid80s. lead optimization. and matching the apparent effects to treatments. KG.C. preclinical trials. (Figure 1). huge man power and millions of dollars in order to come up with a single effective novel drug. The conventional way of synthesizing drugs has always been a tedious process. and develop compounds into clinical trial candidates. Majority of the time when drugs have been used. thereby saving huge funds and man power. design compounds for selectivity. RDD uses a variety of computational methods to identify novel compounds. COMPUTER AIDED DRUG DESIGN Computer-aided drug design (CADD). structure-based drug design is an integral part of most industrial drug discovery programs and is the major subject of research for many academic laboratories. These methods fall into several categories – structure-based drug design. and represents more recent applications of computers as tools in the drug design process. However. GERMANY . de novo design and homology modeling – depending on how much information is available about drug targets and potential drug compounds. it can be done efficiently in silico.

CADD was used to predict the binding affinity of an inhibitor designed from a lead compound prior to synthesis [2]. In each case. et al. 2011 Current Topics in Biotechnology & Microbiology successful discovery of novel numerous enzyme inhibitors. HIV-1 Protease and purine nucleoside phosphorylase inhibitors. KG. Binding of ligand 180 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. DUDWELLER LANDSTR. including inhibitors of thymidylate synthase. GERMANY . Disease selection Target selection Lead compound identification Lead optimization Pre-clinical trials Clinical trial testing Pharmacogenomic optimization Figure 1: Overview of drug discovery process In most current applications of CADD.Dhingra. H. attempts are made to find a ligand (the putative drug) that will interact favorably with a receptor that represents the target site.

there is no a priori reason to exclude higher energy conformers as the source of activity. 2011 Current Topics in Biotechnology & Microbiology to the receptor may include hydrophobic. In the opposite extreme. all of the structures are superimposed to generate the pharmacophore. In applying this strategy. in which case computational methods must be applied without the constraints that the experimental data would provide. Conformational searching on the more flexible compounds is then done while applying distance constraints derived from the structures of the more rigid compounds. The ideal is seldom realized. an attempt is made to identify a pharmacophore.Dhingra. electrostatic. or homology modeling. One strategy is to find the lowest energy conformers of the most rigid compounds and superimpose them. conformational analysis will be required. KG. With the availability of the receptor site. one would have 3-dimensional structural information for the receptor and the ligand-receptor complex from X-ray diffraction or NMR. To be used most effectively. The approach used in CADD is dependent upon the amount of information that is available about the ligand and receptor. H. Ideally. DUDWELLER LANDSTR. as from X-ray diffraction. In fact. This approach to CADD optimizes the fit of a ligand in a receptor site. Based on the information that is available. one can apply either ligand-based or receptor-based molecular design methods [3]. et al. NMR. the extent of which will be dependent on the flexibility of the compounds under investigation. one must recognize that one is assuming that it is the minimum energy conformers that will bind most favorably in the receptor site. and hydrogen-bonding interactions. GERMANY . The receptor-based approach to CADD applies when a reliable model of the receptor site is available. which is accomplished by docking procedure [4]. This template may then be used to develop new compounds with functional groups in the desired positions. The ligand-based approach is applicable when the structure of the receptor site is unknown. and with a range of intermediate activities. In recognition site mapping. one should have structurally similar compounds with high activity. one may have no experimental data to assist in building models of the ligand and receptor. It is represented as a collection of functional groups in three-dimensional space that is complementary to the geometry of the receptor site. the problem is to design ligands that will interact favorably at the active site. with no activity. which is a template derived from the structures of these compounds. Ultimately. 181 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. but when a series of compounds have been identified that produce the activity of interest. In applying this approach.

We will focus on structure-based drug design in this chapter and describe a few of its salient features. et al. The methods of this approach are applied when the three dimensional structure of macromolecule-target is unknown. quantitative structure properties relationship (QSPR) etc.g. and then the structure of the ligand guides the ligand design process. In such cases. Together with advances in structural determination techniques such as nuclear magnetic resonance. For example. These methods are based on analysis of sets of ligands with known biological activity [5]. KG. Structure or target based drug designing (SBDD). Designed compounds should be able to inhibit or stimulate the biological activity of their target molecules. They include: design of pharmacophore models. The rapid progress of the human genome project is providing an ever-increasing number of potential protein drug targets. many important receptors are membranebound proteins (transmembrane proteins). analysis of quantitative structure-activity relationship (“classic” QSAR) [6] and its modification 3D-QSAR (which takes into account spatial structure of compounds). 2. 2.2 Structure or target based drug designing (SBDD): Structure-based ligand or inhibitor design aims to identify chemical compounds or peptides that bind strongly to key regions of biologically relevant molecules. a lead compound or active ligand must be found. The “classical” QSAR is effective for development of close analogues of known compounds [7]. structure-based design of ligands or inhibitors has emerged as an important tool in drug discovery and pharmaceutical research [8]. 2011 Current Topics in Biotechnology & Microbiology Drugs can be designed computationally by using two strategies: 1. Pharmacophore model represents a set of points in space with the certain properties and distances between them. 2. DUDWELLER LANDSTR. H. Ligand or analogue based drug designing (LBDD). which define binding of given group of ligands with target. enzymes or receptors. Once we have an active compound. we can begin to refine the biological activity. 182 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.1 Analogue or Ligand based drug designing (LBDD): Many receptors are not readily amenable to receptor-based drug design. which are very difficult to crystallize. Ligand-based design techniques use information about one or several known active compounds (ligands) as a basis for the designing of lead compounds. crystallography and even homology modeling. e.Dhingra. for which three-dimensional structures are known. and there is difficult to design its reliable model. GERMANY .

et al. In the second cycle. After several cycles of the drug design process. and further optimization of the lead compound.Dhingra. DUDWELLER LANDSTR. The Process of Structure-Based Drug Design: The process of structure-based drug design is an iterative one and often proceeds through multiple cycles before an optimized lead goes into phase I clinical trials. These compounds are scored and ranked based on their steric and electrostatic interactions with the target site and the best compounds are tested with biochemical assays. purification and structure determination of the target protein or nucleic acid by one of three principal methods: X-ray crystallography. the target site is a pocket or protuberance with a 183 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. (i) Selection of a drug target The choice of a drug target is primarily made on a pharmacological and biochemical basis. NMR. often. H. GERMANY . one with at least a micromolar inhibition in vitro. The first cycle includes the cloning. Ideally. and homology modeling. the optimized compounds usually show marked improvement in binding and. crystal structures determined with data extending to beyond 2. Crystal structures are the most common source of structural information for drug design. (ii) Evaluating a target for structure-based drug design Once a target has been identified. structure determination of the target in complex with a promising lead from the first cycle.5 Å are acceptable for drug design purposes [9]. Additional cycles include synthesis of the optimized lead. since structures determined to high resolution may be available. Using computer algorithms. it is necessary to obtain accurate structural information. There are three primary methods for structure determination that are useful for drug design: Xray crystallography. (iii) Active site identification in the target Structure-based design begins with the identification of a potential ligand binding site (active site) on the target molecule. and the method is useful for proteins that range in size from a few amino acids to 998 kDa. reveals sites on the compound that can be optimized to increase potency. KG. The target molecule usually has a well-defined binding pocket or active site. compounds or fragments of compounds from a database are positioned into a selected region of the structure. structure determination of the new target-lead complex. or homology modeling. Typically. specificity for the target. 2011 Current Topics in Biotechnology & Microbiology 3. The ideal target macromolecule for structure-based drug design is one that is closely linked to human disease and binds a small molecule in order to carry out a physiological function. NMR.

DUDWELLER LANDSTR. there are several paths to developing a good lead based on structure of the target.Dhingra. a large number of molecules are screened to find those fitting the binding pocket (active site) of the receptor. Both ligand and protein atoms need to be classified and their atomic properties should be defined. The ligand binding site can be the active site. This knowledge is then exploited to create new ideas on ways to improve existing ligands or to develop new alternative bonding skeletons for a target [10]. (c) H-bond acceptor: Oxygen and sp2 or sp hybridized nitrogen atoms with lone electron pair(s). phosphorus. (iv) Drug Design Methods Once the three dimensional structure of the target is evaluated and the active site in the structure is identified. halogen. All aspects thought to be responsible for binding affinity and selectivity is collected. GERMANY . an assembly site with another macromolecule. into four atomic types: (a) hydrophobic atom: all carbons in hydrocarbon chains or in aromatic groups. (d) Polar atom: Oxygen and nitrogen atoms that are neither H-bond donor nor H-bond acceptor. which is usually referred as database searching [11]. or a communication site necessary in the mechanism of the molecule. De novo ligand designing The first category is about “finding” ligands for a given receptor. In this case. KG. Current methods for structure-based drug design can be divided roughly into two categories. Virtual Screening 2. 1. sulfur. Some researchers call this “virtual screening” in analogy to the bioassay screening procedure employed in the traditional drug discovery process. 184 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. H. as well as their atomic properties. basically. (b) H-bond donor: Oxygen and nitrogen atoms bonded to hydrogen atom(s). 2011 Current Topics in Biotechnology & Microbiology variety of potential hydrogen bond donors and acceptors. AUTODOCK [13] which are freely available for non-commercial use to researchers. The basic inputs for this step are the 3D structure of the protein and a pre-docked ligand in PDB format. The earliest programs for performing 3D database searching are DOCK [12]. and sizes of molecular surfaces. et al. as in an enzyme. hydrophobic characteristics. metal and carbon atoms bonded to heteroatom(s).

The earliest de novo design method began with GRID [14] and many groups have been actively involved in this field since then. Two search approaches can be classified: • geometric or combinatorial and • stochastic or energy driven 185 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. et al. SPROUT [18]. In the first steps of a drug design project the goal is to find a so-called lead structure. a small molecule which binds to a given target protein and can be further developed to a drug. GERMANY .Dhingra. LUDI [15]. Current popular de novo design programs include GROW [14]. Goal is to find the orientation and conformation of the binding partners corresponding to the minimum of free energy of binding. ligand molecules are built up within the constraints of the binding pocket by assembling small pieces in a stepwise manner. KG. In this case. The two main questions arising are what the complex between a protein and a potential leads looks like and how strong the binding affinity of the lead is with respect to other candidates [20]. H. fit or interact together in 3D space (Figure 2). Searching refers to the fact that any docking method must explore the accessible configuration space of the interacting molecules. DUDWELLER LANDSTR. LEAPFROG [17]. 3. Usually a docking method can be classified by the way it handles the receptor and ligand and by the type of search algorithm that is applied [21]. These techniques are raising much enthusiasm to the drug design community. 2011 Current Topics in Biotechnology & Microbiology Another category of structure-based drug design methods is about “building” ligands. which is usually referred as de novo design. Searching and scoring are the two components that typically make up a computational docking. If the threedimensional structure of the target protein is known. LIGBUILDER [16]. These pieces can be either atoms or fragments. docking algorithm can be applied to virtually search for potential leads.1 Docking: Molecular docking is a study of how two or more molecular structures. PROLIGAND [19] etc. Full unrestricted degrees of freedom of translation and rotation are not permissible because they present a high number of potential solutions resulting in need of giant computational resources. for example drug and enzyme or receptor.

Thus. This is not readily available for computation. Usually knowledge of interaction energies and forces flows into an assigned score. the binding free energy difference between the bound and unbound states of the ligand and protein [23]. ∆Gbind . KG. more theoretical value. 2011 Current Topics in Biotechnology & Microbiology Active site of receptor Ligand Figure 2: A ligand (WR99210) docked in the active site of receptor (PFDHFR-TS) All configurations generated during a search process need to be evaluated and ranked. Preferred would be the actual free energy as a score.Dhingra. H. DUDWELLER LANDSTR. one of the most important principles for designing or obtaining potential new ligands is to predict the binding affinity of a certain ligand to its target and use it as 186 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Structure-based drug design attempts to use the structure of proteins as a basis for designing new ligands by applying accepted principles of molecular recognition. The basic assumption underlying structure-based drug design is that a good ligand molecule should bind tightly to its target. Scoring [22] functions achieve this. so scoring functions must approximate binding free energies with sufficient accuracy. which ranks the searching results and selects out the best binding geometry based on the energies of the of the complexes or. Scoring functions can be classified as either force field based or empirical based on a weighted sum of interactions. GERMANY . et al.

The Master Equation is the linear combination of these components. Docking method may be of three types: protein-ligand docking. The sub models of empirical functions differ due to the consideration of researchers. DUDWELLER LANDSTR. et al. GERMANY . KG. In other words flexibility is not provided to the interacting molecules. H. the ligand has only 6 degrees of freedom. the bond angles. Depending on the modification of them.1.e. The target can be represented via a pre-computed grid. With respect to computational costs. the relation between dissociation equilibrium constant. Kd and the components of free energy alternation was built. 25 and 26]. sampling all orientations of the ligand versus the target with 187 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. It has long been a scientific challenge to design the sub models. A historical research was done to develop a general-purposed empirical function in order to describe the binding energy. full rigid docking is invincible. protein-protein docking and protein-DNA docking which exclusively depend upon the types of macromolecules or small molecules chosen for the study of molecular interaction. However.Dhingra.1 Rigid body docking In rigid body docking. A subject of speculation is whether or not rigid-body docking is sufficiently good for most docking. the empirical scoring function is improved and continuously consummated [24. bond lengths and torsion angles of the components are not modified at any stage of complex generation. According to Gibbs free energy equation. The basic idea is that the overall binding free energy can be decomposed into independent components which are known to be important for the binding process. it is also called as “lock and key model”. Each component reflects a certain kind of free energy alteration during the binding process between a ligand and its target receptor. i. Two approaches are often used for the docking studies. The concept of the “Master Equation” was raised. while the ligand is regarded as a rigid body. 3. 2011 Current Topics in Biotechnology & Microbiology a criterion for selection.

3. HEX: HEX is a protein docking and molecular superposition program. However. GOLD: GOLD is a program for calculating the docking modes of small molecules in protein sites and is provided as part of the GOLD suite. scoring all possible conformational changes is computationally expensive and procedures must intelligently select small subset of possible conformational changes for consideration. It is designed to predict how small molecules.1. DOCK: The DOCK suite of programs is designed to find favorable orientations of a ligand in a “receptor. 188 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. iv. such as substrates or drug candidates. GERMANY . iii.2 Flexible docking Docking procedure in which conformational change or flexibility allowed in interacting molecules is called as flexible docking. FlexX: FlexX is a fast computer program for predicting protein-ligand interactions two main applications: complex prediction (create and rank a series of possible protein-ligand complexes) and virtual screening. 3. it is also called as “induced-fit model”. It uses spherical polar Fourier correlations to accelerate docking calculations. The induced-fit mechanism proposes that the protein has to undergo usually minor but however considerable changes to bind with the ligand. have been added over the years. This approach of docking gives more reliable docked complexes and is widely employed by majority of docking programs. v. KG.3 Tools for docking Variety of tools are available for docking studies which are given below: i. It uses matching algorithm for rigid body docking and an algorithm for flexible ligand docking. ii. AutoDock: AutoDock is a suite of automated docking tools. bind to a receptor of known 3D structure. It has been shown that protein and ligand flexibility is crucial for binding. The same is applied for the ligand.1. H. Allowing flexibility in both the receptor and ligand molecules in the computational model virtually increases the conformational search space. This approach is particularly useful in the case of virtual screening of large number of compounds to a target. 2011 Current Topics in Biotechnology & Microbiology exhaustive search algorithms demands considerable computational effort. DUDWELLER LANDSTR. et al. It uses genetic algorithm (GA) for protein-ligand docking and full ligand flexibility is provided.Dhingra.

fundamentally. Drug design using molecular modeling techniques involve screening a very large number of ligand records or molecules of compounds in a chemical database (CDB) to identify those that are potential drugs. peptides. GLIDE: GLIDE is a high-throughput ligand-receptor docking program for fast library screening. ix. FT Dock (Fourier Transform Dock): FTDock performs rigid-body docking on two biomolecules in order to predict their correct binding geometry outputs multiple predictions that can be screened using biochemical information. PatchDock: PatchDock is an automatic server used for molecular docking. drugs. DUDWELLER LANDSTR. where no information on the protein is necessary – instead. 189 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. which requires knowledge of the 3D structure of the target protein binding site to prioritize compounds by their likelihood to bind to the protein. KG. viii. Docking each molecule in the target chemical database is both a compute and data intensive task [28]. two approaches to the general problem: ‘VS by docking’. DNA. this criterion should regard molecules that bind tightly to the same proteins as similar [27]. FlexiDock: FlexiDock is a simple. such as substrates or drug candidates. 2011 Current Topics in Biotechnology & Microbiology vi. vii. bind to an enzyme or a protein receptor of known threedimensional (3D) structure. It uses fast genetic algorithm for generation of configurations. et al. Program identifies the best binding mode through Monte Carlo sampling and provides an accurate scoring function for ranking of binding affinities. There are. The output is a list of potential complexes sorted by shape complementarity criteria. It helps researchers to predict how small molecules. The screening procedure extracts compounds from the database according to an appropriate similarity criterion. H.Dhingra. and ‘similarity-based VS’.2 Virtual Screening: Virtual screening uses computer-based methods to discover new ligands on the basis of biological structures [11]. GERMANY . Virtual (database) screening (VS) is an increasingly important component of the computer-based search for novel lead compounds. The input is two molecules of any type: proteins. flexible docking of ligands into binding sites on proteins. 3. This process is called molecular docking. In order for the screening procedure to be effective. one or more compounds that are known to bind to the protein are used as a structural query (Figure 3). Its algorithm is based on shape complementarity principles.

This database is available for free download (http://zinc. A Web-based query tool incorporating a molecular drawing interface enables the database to be searched and browsed and subsets to be created. Within certain limits. 3D SDF. et al. using catalogs of compounds from vendors (the size of this library continues to grow). GERMANY . and number of rotatable bonds. H. mol2.Dhingra. Acquire and screen 190 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Users can process their own molecules by uploading them to a server [29]. calculated LogP.org) in several common file formats including SMILES. 2011 Current Topics in Biotechnology & Microbiology Modelled structure XRay/NMR Structure Known active sites Rank Hits Commercial databases Virtual screening In-house database In silico Visual selection of Ligands Figure 3: Overview of virtual screening process 3. DUDWELLER LANDSTR. and DOCK flexibase format. the molecules are prepared in multiple protonation states and multiple tautomeric forms. In one format.1 ZINC . Each molecule in the library contains vendor and purchasing information and is ready for docking. The molecules have been assigned biologically relevant protonation states and are annotated with properties such as molecular weight. KG. each with 3D structure.docking. multiple conformations are available for the molecules.Database of Commercially Available Compounds for Virtual Screening: ZINC is a library of very huge number molecules.2.

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3.3 De novo drug Designing: In de novo drug design method, ligand molecules are built up within the constraints of the binding pocket by assembling small pieces in a stepwise manner. These pieces can be either atoms or fragments. The key advantage of such a method is that novel structures, not contained in any database, can be suggested.

De novo Ligand design Methods

Active Site Analysis Methods

Whole molecule Methods

Connection Methods

Sequential Build-up Methods

Fragment Connectio n Methods

Site Point Method

Random connectio n Methods

Figure 4: Overview of De novo Ligand Design Methods

As shown in Figure 4, de novo ligand design methods fall into a few different categories, depending on the approach used: 1. Site-point connection methods: In this method desirable locations of individual atoms which play crucial role in the active site of the target (“site points”) are determined and then suitable fragments at those locations are placed. 2. Fragment connection methods: This is started with the previously positioned fragments and then “Linkers” or “scaffolds” are determined to connect those fragments without moving them. 3. Sequential buildup methods: In this method a new ligand is constructed within the constraint of active site using the atom-by-atom or fragment-by-fragment buildup approach. The set of building block is generally small, and the construction process may be random.


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4. Random connection methods: A special class of techniques combining some of the features of site point, fragment connection, and sequential buildup methods, along with bond-disconnection strategies and other methods to introduce randomness. Among all four methods described above, sequential buildup and fragment connection methods are frequently used in de novo ligand designing. There are two strategies to build up ligand molecules: one is growing strategy while the other is linking strategy. GROW and LINK is designed to carry out them, respectively. The concept of these two strategies is illustrated in Figure 5. With the growing strategy, the building-up process starts from a ”seed” structure that has been pre-placed in the binding pocket. The user can assign certain”growing sites” on the seed structure and then the program will try to replace each growing site with a candidate fragment. The newly formed structure will serve as the seed structure for the next growing cycle. With the linking strategy, the building-up process also starts from a pre-placed seed structure. However, in this case the seed structure consists of several separated pieces that have been positioned to maximize the interactions with the target protein. The growing of fragments happens simultaneously on each piece and the program will always try to link these pieces in an acceptable way. This process continues until all the pieces have been integrated into one molecule [16]. Future Prospects of Structure-based drug design Structure-based drug design is a powerful method, especially when used as a tool within an armamentarium, for discovering new drug leads against important targets. After a target and a structure of that target are chosen, new leads can be designed from chemical principles or chosen from a subset of small molecules that scored well when docked in silico against the target. After a preliminary assessment of bioavailability, the candidate leads continue in an iterative process of re-entering structural determination and reevaluation for optimization. Focused libraries of synthesized compounds based on the structure-based lead can create a very small promising lead which can continue to phase-I clinical trials. As structural genomics, bioinformatics, and computational power continue to explode with new advances, further successes in structurebased drug design are likely to follow. Each year, new targets are being identified, structures of those targets are being determined at an amazing rate, and our capability to capture a quantitative picture of the interactions between macromolecules and ligands is accelerating.


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Structure-based drug design is an integral part of drug discovery today. While it can't be said that the approach is full proof and sufficient to getting a drug to market, no single technology can claim that ability. The reason behind this is that SBDD is in fact a collection of technologies that includes molecular biology, computational chemistry and bioinformatics. After designing the new drug molecules, ultimately we will have to go to wet laboratory experiments to verify the pharmacological properties of designed drugs. Above all, the approach hinges on people working together in interdisciplinary teams to attack a disease target through customdesign of a new drug. It is clear that targeted strategies to find new drugs are a successful approach that is saving lives every moment. As more drugs derived from structure-based drug design come to market, many more success stories will be waiting to be told.

Figure 5: Growing strategy (the left) and linking strategy (the right) for building up ligands

1. Augen, J. (2004). Bioinformatics in the Post-Genomic Era: Genome, Transcriptome, Proteome, and Information-Based Medicine. Addison-Wesley Edition: 1st edition, 388pp. 2. Reddy, M.R. and Erion, M.D. (2005). Computer Aided Drug Design: strategies Used in the Discovery of Fructose 1, 6-Bisphosphatase Inhibitors, Current Pharmaceutical Design, 11, 283-294. 193

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3. Anderson, A.C. (2003). The process of structure-based drug design. Chemistry & Biology, 10, 787–797. 4. Bevan, D.R. (1997). QSAR and drug design, Network Science.


5. Veselovsky, A.V. and Ivanov, A.S. (2003). Strategy of Computer-Aided Drug Design, Current Drug Targets - Infectious Disorders, 3, 33-40. 6. Kubinyi, H. (1994). Variable Selection in QSAR Studies I. An Evolutionary Algorithm, Quantitative Structure-Activity Relationships, 13, 285-294. 7. Fujita, T. (1997). Recent success stories leading to commercializable bioactive compounds with the aid of traditional QSAR procedures. Quantitative Structure-Activity Relationships, 16, 107-112. 8. Zeng, Jun. (2000). Computational Structure-Based Design of Inhibitors that Target Protein Surfaces, Combinatorial Chemistry and High Throughput Screening, 3, 355-362. 9. Nissen, P., Hansen, J., Ban, N., Moore, P. and Steitz, T. (2000). The structural basis of ribosome activity in peptide bond synthesis. Science, 289, 920–930. 10. Gerhard, Klebe (2000). Recent developments in structure-based drug design, Journal of Molecular Medicine, 78,269–281. 11. Meng, E.C., Shoichet, B.K. and Kuntz, I.D. (1992). Automated Docking with Grid-Based Energy Evaluation, Journal of Computational Chemistry, 13, 505-524. 12. Schoichet, B. K. and Kuntz, I. D. (1993). Matching chemistry and shape in molecular docking, Protein Engineering, 6, 723-732. 13. Morris, G.M., David, S.G., Robert, S.H., Ruth, H., William, E.H., Richard, K.B., Arthur, J.O. (1998). Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function, Journal of Computational Chemistry, 19, 1639–1662. 14. Goodford, P.J. (1985). A computational procedure for determining energe- tically favorable binding sites on biologically important macromolecules, Journal of Medical Chemistry, 28, 849- 857. 15. Bohm, H. J. 1994). "The development of a simple empirical scoring function to estimate the binding constant for a protein-ligand complex of known three-dimensional

structure". Journal of Computer Aided Molecular Design, 8, 243–56. 16. Wang, R., Gao, Y. and Lai, L. (2000). LigBuilder: A Multi-Purpose Program Based Drug Design, Journal of Molecular Modeling 6, 498 – 516. 194

for Structure-

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17. Cramer, R. D., DePriest, S. (1996). Implemented in the SYBYL program, Tripos Associates, St.Louis, MO, USA. 18. Gillet, V. J., Newell, W., Mata, P. (1994). Sprout: recent developments in the de novo design of molecules Journal of. Chemical Information and Computer Science, 34, 207-217. 19. lark, D. E., Frenkel, D., Levy, S. A. (1995). PROLIGAND: an approach to de novo molecular design. Apllication to the design of organic molecules. Journal of .Computer-Aided Molecular Design, 9, 13-32. 20. Rarey, Matthias (2002). Protein- ligand docking in drug design. In: Thomas Lengauer (ed.) Bioinformatics- from genomes to drugs, Vol I (Wiley-VCH) 7: 315-318. 21. Wilke, Christoph (2004). Molecular conformation and ligand- receptor docking, Seminar: Algorithms in drug design, WSI Computer Science Department. 22. Kuntz, I.D., Meng, E.C. and Shoichet, B.K. (1994). Receptor-Based Molecular Design, Accounts of Chemical Research, 27, 117-123. 23. Tao, P. and Lai, L. (2001). Protein ligand docking based on empirical method for binding affinity estimation, Journal of computer-aided molecular design, 15, 429-426. 24. Gohlke, H., Hendlich, M., Klebe, G. (2000). Knowledge-based scoring function to predict protein-ligand interactions. Journal of Molecular Biology, 295, 337–56. 25. Clark, R.D., Strizhev, A., Leonard, J.M., Blake, J.F., Matthew, J.B. (2002). Consensus scoring for ligand/protein interactions. Journal of Molecular Graphics and Modeling, 20, 281–95. 26. Wang, R., Lai, L., Wang, S. (2002). Further development and validation of empirical scoring functions for structure-based binding affinity prediction. Journal of Computer Aided Molecular Design, 16, 11–26. 27. Lengauer, T., Lemmen, C., Rarey, M. and Zimmermann, M. (2004). Reviews, Novel technologies for virtual screening, Drug discovery today, 9,1- 27. 28. Buyya, R., Branson, K., Giddy, J. and Abramson, D. (2003). The Virtual Laboratory: a toolset to enable distributed molecular modeling for drug design on the World-Wide Grid, Concurrency and Computation: Practice and Experience, 15,1–25. 29. Irwin, J.J. and Shoichet, B.K. (2005). ZINC – A Free Database of commercially available Compounds for Virtual Screening, Journal of Chemical Information and Modeling, 45, 177182. 195

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Chapter X


Pradeep kumar L and 2Vani B*


Birla Institute of Technology and Science, Pilani, Rajasthan (India)

Corresponding Author: Vani B., Assistant Professor, E.mail: bvani70@gmail.com

ABSTRACT: Cyanobacteria are photoautotrophic prokaryotes and constitute a large taxonomic
group within the domain of eubacteria. Different species of cyanobacteria utilize / assimilate nitrogen available to them in different forms. Based on their nitrogen acquisition ability, they are divided as N2-fixing and non N2-fixing. Some of the filamentous species are subdivided into those with and without a heterocyst which is a differentiation from vegetative cells for fixing nitrogen. The aim of this chapter is to highlight facts and ideas on the physiology of nitrogen utilization, which have not received much attention in the past. Keywords: Nitrogen fixation, Cyanobacteria, nif

Cyanobacteria belong to the oldest group of organisms on earth [1]. They are photosynthetic prokaryotes that resemble eukaryotic green algae, making them classified as "blue green algae". They are found in very diverse habitat, from oceans to fresh water to bare rock to soil. They also provide an extraordinarily wide-ranging input to human daily life [2] and are of economic importance [3]. Further, they are important primary producers and their nutritive value is very high. They contribute globally to soil and water fertility [4]. The optimal growth of a cyanobacterial species depends on the availability of nitrogen. Nitrogen (N) can represent as much as about 11% of the dry weight of a cyanobacterial cell [5]. Cyanobacteria use numerous nitrogen containing compounds as sources of nitrogen. Nitrogen fixing cyanobacteria fixes atmospheric nitrogen to cope up with nitrogen depletion. The non N2-fixing group can use nitrate, nitrite, ammonium, organic compounds like urea, amino acids, and some nitrogencontaining bases as nitrogen source (Fig. 1). Cyanobacteria shows preference over some nitrogen sources than other, such that more easily assimilated source of nitrogen is available to the cells. 196

The P cluster may have an 197 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. an iron protein (Fe-protein) and an iron-molybdenum protein (MoFe-protein).protein chemistry Cyanobacterial (di) nitrogen fixation is catalyzed by the enzyme complex nitrogenase. Nitrogen acquisition in all these organisms consume photosynthetically generated ATP and reducing power (ferredoxin) [6]. GERMANY . The Fe-protein homodimer is composed of a single Fe4S4 cluster bound between identical 32–40 kDa subunits. because several of them. Each α2β2 dimer of the larger nitrogenase protein binds one FeMo cofactor and one P cluster. They are the best studied organism on the subject. et al. H. capable of obtaining more than two oxidative states [9] and transfers electrons to the MoFeprotein [10]. The Fe4S4 cluster is redox-active [8]. Nitrogenase . 2011 Current Topics in Biotechnology & Microbiology This phenomenon was called nitrogen control [6]. The MoFe-protein is a α2β2 heterotetramer of 250 kDa. DUDWELLER LANDSTR. can be easily genetically modified by molecular techniques. Figure 1: Core pathways of nitrogen assimilation [6]. The ammonium production in the process is defined with the equation [7]: Cyanobacterial nitrogenase consists of two components. KG.Dhingra.

Figure 2: Schematics representation of different adaptations enabling nitrogen fixation in cyanobacteria and its light and dark regulation [17]. whereas nitrogenase resides in heterocyst.13].Dhingra. as a means to protect their nitrogenases against atmospheric O2 [12. In turn. and homocitrate. gets immediately consumed by high respiratory activity and 198 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. H. The FeMo cofactor consists of 1 Mo atom. Residual O2. Reproduced with permission of Elsevier Adaptation in N2 fixation: heterocyst differentiation Some of the cyanobacterial genera undergo cell differentiation. 9 S atoms. GERMANY . DUDWELLER LANDSTR. et al. KG. These differentiate from vegetative cells by cell division and extensive metabolic changes. their vegetative cells perform photosynthetic O2 evolution. 7 Fe atoms. so that they cannot perform the photosynthesis [14. plus an asyet-unidentified light atom (or ion) at its center [7]. the O2 problem is enhanced by the photosynthetic evolution of this gas [7]. 2011 Current Topics in Biotechnology & Microbiology N2 fixation specific role forcing reversibly bound N2 to the irreversible reduction pathway [11]. called the heterocysts (Fig. as O2 reversibly damages Fe4S4 cluster and P cluster. if reaches the inside of the heterocysts. heterocysts provide nitrogen as glutamine formed via N2 fixation and glutamine synthetase/glutamate synthase (GS/GOGAT) [16]. In cyanobacteria. The FeMo cofactor is the site of substrate binding and reduction [11].15]. During aerobic growth conditions. including Photosystem II (PSII) degradation. Nitrogenases are more susceptible to O2. 2).

NtcA is the main 2-oxoglutarate (2-OG) sensor [22]. which binds to NtcA-dependent promoter. In Anabaena species heterocyst formation takes about 24 h of deprivation. H. N2 fixation through temporal separation Without cell differentiation. The organism has nitrogenase in all cells in equal amounts [33]. 19. Plectonema temporally separates oxygenic photosynthesis and nitrogen fixation [34]. 2-OG senses C-N balances in cyanobacteria. controlling heterocyst formation. In cyanobacteria. However. HetR.Dhingra.Thus the organism fix nitrogen under microaerobic conditions [35]. 20. NtcA belongs to the catabolite activator protein (CAP) family. NtcA comprises of a helix-turn-helix motif at its C-terminal end. a serine-type protease. they need oxygen for respiratory processes that are a crucial for ATP and reductant [34]. perfect enough for nitrogenase to function. 27]. DUDWELLER LANDSTR. organisms like Plectonema boryanum has evolved to function in low oxygen environments to cope up with O2 problem in nitrogen fixation (Fig. 2-OG thus formed. They temporally separate transcriptional regulation 199 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. are found to be the key regulators of the process [18. et al. NtcA and PII mediated regulation in nitrogen acquisition is detailed in the later part of this chapter. Other than NtcA. Thus heterocysts provide an anaerobic environment. unlike higher plants. and NtcA. . cyanobacteria lacks 2-oxoglutarate dehydrogenase [24. signal protein PII is also involved in regulation of heterocyst formation[32]. Under these conditions. strain PCC 6803 [30]. Heterocyst formation is complex process controlled by many genes [15]. 20]. 2011 Current Topics in Biotechnology & Microbiology also other reactions in the heterocysts. 29]. is used by GS-GOGAT pathway as the carbon skeleton for the incorporation of ammonium [28. Its principal role as a carbon skeleton for ammonium incorporation would make 2-OG. 25] leading to NADP1-isocitrate dehydrogenase catalyzed formation and accumulation of 2-OG [26. an ideal sensor of the C-N balances [31]. KG. The expression of the icd gene encoding isocitrate dehydrogenase was found to be highest under nitrogen stress in Synechocystis sp. Nitrogen deprivation upregulates the expression of hetR and the expression is regulated by NtcA is the global nitrogen regulator [21]. it activates the transcription of heterocyst formation genes in response to nitrogen step-down [22]. The reason for it is explained by the fact that. 2). a transcription factor. GERMANY . Plectonema is unable to fix nitrogen when grown anaerobically.

DUDWELLER LANDSTR. 47]. 43]. some of the non-heterocystous cyanobacteria can undergo temporal separation without the need for a microaerobic environment. GERMANY . protection against oxygen was found to involve a complex interaction between spatial and temporal segregation [42. maintaining a phase difference of 12 h from the peak of photosynthetic activity. In Trichodesmium. In these organisms high nitrogenase activity coincides with high respiration rates. H. with a temporal phase difference of ~6 h [45]. Nevertheless. a filamentous cyanobacteria [37] and the unicellular Gloeothece and Cyanothece belongs to the same category of N2-fixers [38. On the contrary. Phormidium. some non-heterocystous species of cyanobacteria can fix nitrogen during the day time (Fig. They fix nitrogen at night and nitrogenase is typically found in all cells. 2011 Current Topics in Biotechnology & Microbiology nitrogenase and photosynthetic genes [36]. Different research groups have recently explained this complex phenomenon. These cells are arranged consecutively along the trichome [42] with active photosynthetic components [43. Hence. in heterocystous species the 200 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. A circadian clock controls the transcription of nitrogenase and the expression of photosynthetic genes essential to the activation of photosystems I and II (PSII). Thus. Such pattern is also reflected at the transcriptional level. 2). Symploca. Genetic structure of nif gene In general nitrogen fixation genes have two basic structural components a) genes encoding for nitrogenase reductase (nifH). Thus. and some species of Pseudoanabaena also undergo such temporal separation as that of Plectonema [9]. 39]. 41]. spatial aggregation of nitrogenase into these cells is not sufficient to protect against photosynthetic oxygen evolution. et al. Further. KG. Non-heterocystous cyanobacteria (both unicellular and filamentous) have a contiguous nifHDK operon [48. This enables the cells to turn photosynthetic activity on or off. alpha and beta subunits of nitrogenase (nifDK) and b) genes encoding the two subunits of the NifNE protein complex involved in biosynthesis of the FeMo-cofactor [46. these organism have adapted a circadian control to cope up with C:N balance [40. 44]. 49]. a significant fraction of nitrogenase would be inhibited by O2. involve compartmentalization of nitrogenase in a fraction of the cells. without damaging the photosynthetic machinery [17]. The mechanism of protection from photosynthetically evolved oxygen in Trichodesmium. and observed under either continuous light or darkness.Dhingra.

Unlike N2-fixing group. Oscillatoria). The products of nifN and nifE function as a 200-kDa α2β2 tetramer (NIFNE) that has been purified. two transmembrane subunits and two ATPase subunits that are located in the cytoplasmic side of the membrane. uses the photosynthetically generated assimilatory power namely. GERMANY . gets metabolized to ammonium. Two 201 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. and found to contain what appears to be a single 4Fe-4S center [53]. ATP and reduced ferredoxin. Nitrate assimilation is nitrogen acquisition for most of these non-nitrogen fixing cyanobacteria [55] It enters the cells by an active transport system and gets sequentially reduced to ammonium by the action of nitrate reductase (NarB) and nitrite reductase (NirA) [56]. 2) (e. DUDWELLER LANDSTR. KG. and incorporated into carbon skeletons through the GOGAT pathway. where some organisms were able to adapt to an oxic world. The cyanobacterial ATPbinding cassette (ABC)-type permeases are involved in nitrate uptake [6]. 3 represents the nitrate assimilation system as found in some of these fresh-water cyanobacteria. while others were not (Fig.. partially characterized. They are composed of a periplasmic substrate-binding protein that is anchored to the membrane. Nitrogen acquisition from other sources of nitrogen The loss of cyanobacterial nif genes was also an adaptation strategy existed early. This transposon gets removed during the heterocyst formation. Fig.g. 58]. Interestingly. these genes nifN and nifE exhibit striking similarity to nifK and nifD [54]. The structural elements of nitrate uptake and assimilation include [58]: • ABC-type permease • Ferredoxin–nitrate reductase (Fd–Nar) • Ferredoxin–nitrite reductase (Fd–Nir) ATP-binding cassette (ABC)-type uptake transporter Combined nitrogen sources are taken up through permeases. This process is well established in Anabaena. The excision leads to the restoration of the nifD coding sequence and of the entire nifHDK transcription unit [52]. required for nitrate reduction [57. the nifD gene is interrupted by an 11 000 base pair DNA element. During heterocyst differentiation of this element is excised by site-specific recombination. H. 2011 Current Topics in Biotechnology & Microbiology structural nif operon of most is interrupted by a large transposon [51]. nitrate uptake and reduction to ammonia in these organisms. In vegetative cells of Anabaena. et al.Dhingra.

Figure 3: The nitrate assimilation system as found in fresh-water cyanobacteria. nirate is reduced to nitrite by nitrate reductase (NR). NrtB bears six transmembrane segments. Ferredoxin–nitrate reductase Once inside the cell. represents a conserved component of these permeases [58]. They couple ATP hydrolysis to the transport of solutes across cell membranes. Spirulina platensis genes encoding elements of ABC-type uptake permease are arranged in a operon (NrtA-B-C-D) (Fig. They are monomer products of narB gene of 80 kDa. NrtD. ATP binding and hydrolysis induce conformational changes in the membrane spanning domains of the permease. which are of very high conservation in sequence throughout cyanobacterial genera [58]. Cyanobacterial nitrate reductases catalyzes the 2-electron reduction of nitrate to nitrite. which is highly hydrophobic protein of about 280 amino acids [67].Dhingra. They are partially associated with the thylakoid membrane and uses photosynthetically reduced ferredoxin as physiological electron donor [70 – 202 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. GERMANY . NrtA is a periplasmic substrate-binding lipoprotein that can bind both nitrate and nitrite. abundant in the cytoplasmic membranes [59-62]. 69]. DUDWELLER LANDSTR. H. 3) [63. which further mediate the transport process [65]. NrtA is of about 440 amino acids that is anchored to the cytoplasmic membrane [66]. They were first identified in Synechococcus elongatus as an ammonium-repressible protein of about 48 kDa. 2011 Current Topics in Biotechnology & Microbiology cytoplasmic subunits of ABC-type uptake transporter powers the transport reaction. containing a [4Fe– 4S] cluster and a Mo-cofactor [68. which is the first step in the nitrate assimilation. et al. a protein of about 275 amino acids. 64]. KG.

through the GOGAT cycle. GERMANY . 2011 Current Topics in Biotechnology & Microbiology 71]. thus necessary for maintaining nitrogen and carbon equlibrium [76]. DUDWELLER LANDSTR. gene inactivation studies have revealed that Fd-GOGAT directly utilizes photoreducing power for the catalytic reaction. is used up as the final N acceptor (Fig. Ferredoxin–nitrite reductase Cyanobacterial nitrite reductases participate in 6-electrons reduction of nitrite to ammonium. with prosthetic groups including a [4Fe–4S] cluster and a siroheme [58]. The interaction of nitrite reductase with ferredoxin seems to be stabilized by electrostatic forces. et al. Their molecular weight is between 52–56 kDa and are monomers products of nir gene.Dhingra. cyanobacteria can take ammonium from external medium as well. The electron donors for the enzyme are the photosynthetically reduced ferredoxin or flavodoxin [71]. Two regions in nitrite reductase that are rich in positively-charged amino acid residues are actively involved in regulating its interaction with ferredoxin [73]. From Plectonema. However. H. 4) [74]. Further. Glutamine synthetase-glutamate synthase: The final step completing nitrate assimilation is incorporation resulting ammonium into amino acids by the GS/GOGAT pathway. Incidentally. The process consumes ATP and reducing power and 2-OG. The glnA gene encoding a typical type I glutamine synthetase have been cloned and characterized from numerous cyanobacteria [75]. any hindrance in the generation of reductant via the photolysis of water and the reduction of the plastoquinone pool or imbalance 203 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. KG. is electrostatically stabilized by the interaction of lysine and arginine residues of NR [72]. Figure 4: Cycle involving ammonium incorporation using 2-OG carbon skeleton. genes encoding a ferredoxin-dependent (glsF) and an NADH-dependent (gltB and gltD) glutamate synthase were cloned [76]. The complex between NR and negatively charged residues on ferredoxin.

strain PCC 7120 also posses a similar gene cluster for nitrate assimilation operon [83-84]. 2011 Current Topics in Biotechnology & Microbiology in the utilization of such reductants for carbon assimilation or by nitrate assimilation causes physiological stress in vivo. KG. as well as of other genes involved in nitrogen assimilation [88 -90]. nrtA. Anabaena sp. The 2-OG binds to the EBD at a pocket similar to that used by cAMP in catabolite activator protein. This gene cluster is conserved throughout cyanobacteria genera. When the cells perceive a high C to N ratio. and narB. It ensures production of a balanced amount of the different proteins of the nitrate assimilation system [84]. NtcA mediated regulation of nitrogen control depend on modifications of both enzyme activity and gene expression [77]. Gene cluster involved in nitrate assimilation In both nitrogen fixing and non-nitrogen fixing cyanobacteria. induced by 2-OG maintains the proper distance between the two Fhelices for DNA recognition [87]. et al. nrtB. but with a different pattern [87]. The Synechocystis sp. The recently determined crystal structure of NtcA has given an insight into its mode of action [87]. a global nitrogen-control regulator [22]. 80.65]. Structural analysis have revealed that a tighter coiled-coil conformation of the two C-helices of NtcA. NtcA activates expression of the nir operon. nrtC and nrtD. The expression of the operon is induced in the absence of ammonia [79. encoding nitrate reductase (Fig. 204 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. GERMANY . 86]. this operon is transcribed from a complex promoter that includes binding sites for NtcA. 5) [64. nitrate assimilation operon bears the following genes: nir encoding nitrite reductase [78. 85. NtcA and PII meditaed regulation of gene expression NtcA is a dimer of two monomeric unit (~222 amino-acid) present in almost all cyanobacteria [77. The transcriptional activity of NtcA is regulated by binding of an effector molecule (2-OG) to the N-terminal effector binding domain (EBD). DUDWELLER LANDSTR. H.Dhingra. Figure 5: Map of the nrt operon of the genome of Synechocystis sp. nitrate assimilation genes are commonly found in an operon [77]. 5) [64. the binding affinity of NtcA towards NtcA promoter increases [22]. 87]. 79]. 81]. encoding a nitrate/nitrite transporter (Fig. When 2-OG bind to EBD.

(Fig. However. presence of a nitrogen source induce medial PII phosphorylation. Figure 6: PII phosphorylation cycle in response to cellular 2-oxoglutarate levels [92]. Presence of ammonium initiates dephosphorylation. DUDWELLER LANDSTR. GERMANY . 2011 Current Topics in Biotechnology & Microbiology The next level of control in nitrogen assimilation in cyanobacteria is mediated by signal transduction protein. recognizing ATP and 2-OG [91-94]. Phosphorylation at ser49 in response to the cellular nitrogen and carbon supply is the key factor determining its activity (Fig. ATP and 2-OG controls the reactivity of PII towards various targets [95-96]. which are modulated by the inorganic carbon supply to the cells. 205 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. PII signalling mediates the NtcA-activated gene expression under conditions of nitrogen starvation [101-102]. Dephosphorylation of PII-P depends on a protein phosphatase. KG. forms a tight complex with nonphosphorylated PII. et al. They are yet another central molecule for perception and signalling of the cellular nitrogen status. other direct targets of interaction with PII are still to be revelaed. H. PII [91]. NAGK. Elevation in 2-OG levels (discussed earlier in this chapter) signals this phosphorylation [97-98]. 103]. Reproduced with permission of John Willey & Sons. which is highly sensitive to 2-OG (even in submillimolar range)[92. 6) [92-100]. 99]. N-acetyl-L-glutamate kinase (NAGK) was recently identified as one of the target of PII signalling [91. Nitrogen starvation induces highest levels of PII phosphorylation (Fig. PphA. the key enzyme in arginine biosynthesis. enhancing the catalytic activity of this enzyme [103-104]. 6) whereas. 6).Dhingra.

nitrite. a serine-type protease. an ideal sensor of the C-N balances. ACKNOWLEDGEMENT: The work done in the authors’ laboratory was supported by grant from the Department of Science and Technology (DST). Other than NtcA. other direct targets of interaction with PII are still to be revelaed. et al. and promises to yield new insights for many years to come. GERMANY . NtcA activates expression of the nir operon. uses the photosynthetically generated assimilatory power namely. Cyanobacteria use numerous nitrogen containing compounds as sources of nitrogen. N2 fixation is catalyzed by the enzyme complex nitrogenase. 2011 Current Topics in Biotechnology & Microbiology CONCLUSION Cyanobacteria are important primary producers and their nutritive value is very high. enhancing the catalytic activity of this enzyme. forms a tight complex with nonphosphorylated PII. KG. a transcription factor are found to be the key regulators of the process. New Delhi. and some nitrogen-containing bases as nitrogen source. signal protein PII is also involved in regulation of heterocyst formation Some cyanobacteria temporally separate transcriptional regulation of nitrogenase and photosynthetic genes Unlike N2-fixing group. 2-OG senses C-N balances in cyanobacteria. However. NAGK. Some of the cyanobacterial genera undergo cell differentiation. LPK thanks UGC for a fellowship. NtcA mediated regulation of nitrogen control depend on modifications of both enzyme activity and gene expression PII signalling mediates the NtcA-activated gene expression under conditions of nitrogen starvation.Dhingra. as well as of other genes involved in nitrogen assimilation. Nitrogen (N) can represent as much as about 11% of the dry weight of a cyanobacterial cell. called the heterocysts. required for nitrate reduction . Nacetyl-L-glutamate kinase (NAGK) was recently identified as one of the target of PII signalling . organic compounds like urea. Nitrogen fixing cyanobacteria fixes atmospheric nitrogen to cope up with nitrogen depletion The non N2-fixing group can use nitrate. the key enzyme in arginine biosynthesis. HetR. ammonium. DUDWELLER LANDSTR. nitrate uptake and reduction to ammonia in these organisms. They contribute globally to soil and water fertility. India. as a means to protect their nitrogenases against atmospheric O2 Heterocyst formation is complex process controlled by many genes. 2-OG. We therefore conclude with the confidence that nitrogen acquisition in cyanobacteria as a research topic is still alive and well. is used up as the final N acceptor. 206 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. ATP and reduced ferredoxin. and NtcA. amino acids. H. Its principal role as a carbon skeleton for ammonium incorporation makes 2-OG.

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2011 Current Topics in Biotechnology & Microbiology Chapter XI PLASTIDS IN Plasmodium – SIGNIFICANCE IN MALARIA TREATMENT *Vishal Saxena and Shilpi Garg Center for Biotechnology. The symptoms of 217 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.com ABSTRACT : Malaria remains one of the most serious infectious diseases in the world. the functional characterization of the genes identified in the circular genome of plastid still remains hypothetical. Thus. This chapter thus focuses on the origin of this organelle in the Plasmodium species and related genus. Over 108 countries are affected by this disease over decades but there is no permanent cure available for the disease. vivax accounts for most (upto 85%) of the malaria infections all over the world including Sub-Saharan Africa. GERMANY . Human malaria caused by Plasmodium falciparum and P. inflicting acute illness on more than 300 million people and leading to more than two million deaths annually. Deadly fever-probably malaria has been recorded since the beginning of the written word (6000-5500 B. a detailed insight is required to understand and decipher the functions of the circular genome and the metabolic pathways.C. Four metabolic pathways specific to prokaryotes are also reported in this organelle and are considered important for the survival of the parasite.). Birla Institute of Technology & Science. The enzymes functional in these pathways are encoded by the nuclear genome of the parasite and are considered as putative drug targets. INTRODUCTION Malaria is probably one of the oldest diseases known to mankind. KG. H. Southeast Asia and Latin America. et al. A plastid like organelle present in the Plasmodium species found to be indispensable for the survival of the parasite is being looked upon as a putative drug target. The situation has become more complicated with the increasing reports on drug resistance in the parasite.Dhingra. the circular genome and the metabolic pathways of the plastid. Email: vishalsaxena12@yahoo. The organelle harbors a circular genome similar in constitution to that of chloroplast DNA. its importance in the survival of the parasite. Department of Biological Sciences. However. Pilani Corresponding author: Vishal Saxena. DUDWELLER LANDSTR.

However.ovale is mainly localized in tropical West Africa where as P. However. Recent reports detail few cases of human malaria by P. P. falciparum and P. malaria causes significant morbidity and mortality. malariae has a very patchy distribution worldwide. it was only in the 5th century B. Details of this disease can be found even in the ancient Indian medical literature like Charaka Samhita and Sushrutha. Although preventable and treatable. malariae and P ovale. P.C. Prevalence of Malaria Malaria is caused by species of the apicomplexan parasites Plasmodium. The disease is transmitted to humans by species of female Anopheles mosquito. falciparum on the basis of microscopic examination 218 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. falciparum. knowlesi (a primate parasite). The P. which even associated these fevers with the bites of the Mosquitoes. tertian (alternate days) and quartan (fever three days a part).Dhingra. accounting for most malaria deaths globally. GERMANY . He classified the fever types as quotidian (daily). Four species of this protozoan parasite are known to cause human malaria viz. vivax and P. Half of the world’s population is at risk for malaria. Malaria is one of the world’s most common and serious tropical diseases. KG. which is endemic (where a constant. 2011 Current Topics in Biotechnology & Microbiology malaria were described in ancient Chinese medical writings.C. P. Laveran and Ross discovered that the disease was caused by a protozoan parasite transmitted by anopheline mosquitoes. In 2700 B. has however been responsible for resurgence in malaria cases. Children are at particular risk.. It was only at the turn of this century.. DUDWELLER LANDSTR. respectively.. Significant control of the disease was accomplished in the 1930s and 1940s due to the advent of synthetic antimalarial drugs and modern insecticides. data from intensive studies undertaken in 1930 [4] show an almost equal prevalence of P. and parts of Asia and Latin America also face significant malaria epidemics [2]. that malaria was clinically recognized and described by Hippocrates [1]. The Canon of Medicine. et al. several characteristic symptoms of what would later be named as malaria were described in the “Nei Ching”. measurable number of new cases and natural transmission occurs over time) in over 108 countries. The rise in incidence of drug resistant parasites and insecticide resistant vectors. P. P. particularly in resource-poor regions. Sub-Saharan Africa is the hardest hit region in the world.vivax accounts for over half of all malaria infections outside Africa and an estimated 75 million acute episodes per year in Africa [3] and has been traditionally considered to be the dominant species in India. vivax. H.vivax are observed worldwide in tropical and some temperate zones. Among the four species.

with abnormal behavior. impairment of consciousness. including cerebral malaria. falciparum malaria. all of which are commonly associated with P. Severe manifestations including organ dysfunction are well documented in P. falciparum infections [57]. vivax malaria. H. and jaundice. which may occur even after the parasite counts have decreased in response to treatment • Abnormalities in blood coagulation and thrombocytopenia (decrease in blood platelets) • Cardiovascular collapse and shock Other manifestations that should raise concern are: • Acute kidney failure 219 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. falciparum. KG. et al. DUDWELLER LANDSTR. recent reports have also clearly established the occurrence of severe manifestations in P. 2010): • Cerebral malaria. The clinical data from the patients infected with P. The manifestations of severe malaria include (World Health Organisation.Dhingra. As of 2006. It is well known in causing the varying degrees of severity of malaria with the most complicated form of the disease often termed Severe Malaria. hemoglobinurea. falciparum is known to cause most dangerous of malaria and has the highest rates of complications and mortality. seizures. However. or other neurological abnormalities • • • Clinical jaundice plus evidence of other vital organ dysfunction Hemoglobinuria (hemoglobin in the urine) due to hemolysis Pulmonary edema (fluid buildup in the lungs) or Acute Respiratory Distress Syndrome. severe anemia. GERMANY . in most African countries. abnormal bleeding. It is more prevalent in sub-Saharan Africa than in other regions of the world. it accounted for almost 90% of the deaths. After the control and eradication programmes of the 1950’s and 1960’s the number of cases of both species went down considerably. renal failure. more than 75% of cases were due to P. acute respiratory distress syndrome (ARDS). coma. vivax in the studies done in Bikaner. P. vivax can cause both sequestration-related and non sequestration related complications of severe malaria. 2011 Current Topics in Biotechnology & Microbiology of blood smears. circulatory collapse. India and some other parts of the world has strongly indicated that P.

GERMANY . These include all residents of areas with low or no malaria transmission. which penetrates through the stomach wall and forms an oocyst. In all the Plasmodium sp. A female mosquito may ingest male and female gametocytes along with the blood meal. the female gametocyte matures to form a macrogamete. The sporozoites injected into the blood stream leave the blood vascular system and invade the parenchymal cells of the liver within a very short time. Simultaneously. resulting in the production of thousands of merozoites. which on fertilization forms a zygote. where more than 5% of the red blood cells are infected by malaria parasites • Metabolic acidosis (excessive acidity in the blood and tissue fluids i. (c) schizont (the stage at which the parasite begins to segment into multiple daughter parasites) and 220 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The oocyst after further maturation produces a large number of slender threads – like sporozoites. viz. Some of these enter the mosquito salivary glands and may be inoculated into the blood stream of the intermediate host (man) during the next meal. Severe malaria occurs most often in persons who have no immunity to malaria or whose immunity has decreased. et al. 2011 Current Topics in Biotechnology & Microbiology • Hyperparasitemia. KG. Life Cycle of Plasmodium The life cycle of Plasmodium is carried in two stages in different organisms. one in humans acting as host for asexual stage and second in mosquito acting as vector and site for sexual stage (Figure 1). asexual multiplication occurs in the liver. exo-erythrocytic (when sporozoites infect liver cells. The male gametocyte matures inside the mosquito resulting in the production of a number of slender spindle shaped microgametes. Hypoglycaemia may also occur in pregnant women with uncomplicated malaria. infecting man. DUDWELLER LANDSTR. (b) tropohozoite. often in association with hypoglycemia • Hypoglycemia (blood glucose < 2. and young children and pregnant women in areas with high transmission. H. or after treatment with quinine. Various species of anopheline mosquitoes form definitive hosts for the malaria parasites. there are two sub-stages of parasite division during its asexual life cycle.2mmol/l or <40mg/dl).e plasma bicarbonate <15mmol/l ).Dhingra. To elaborate further. develop into schizonts and further into merozoites which infect red blood cells) and erythrocytic stages (includes (a) ring (the earliest stage after initial infection of red blood cells). The zygote elongates and becomes active forming an ookinete.

These are released into the circulation and infect the erythrocytes in the blood. Figure 1: Schematic representation of the life cycle of malaria parasite Chemotherapy for Malaria The fight against malaria began some 100 years back. GERMANY . A few of these merozoites may subsequently form male and female gametocytes. KG. Antimalarials containing quinoline components are the most effective drugs for malaria chemotherapy. formed with in 48-72 hrs and travel through the plasma from the spent blood cell and invades a new host erythrocyte) stage. This group of compounds 221 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. During this period. various drug molecules have been formulated in way of trials to combat malaria. DUDWELLER LANDSTR. 2011 Current Topics in Biotechnology & Microbiology (d) merozoite (4-36. Rupture of the RBC releases products of metabolism of the parasites as well as the cells into circulation. the volume of toxic material thrown into the blood stream may be sufficient to bring about a malarial paroxysm.Dhingra. It is believed that if a large number of infected cells rupture simultaneously. et al. H.

arteether) are most effective antimalarials and seem to have effect on protein synthesis by the malaria parasite. The second line drugs include dihydrofolate reductase inhibitors like proguanil. falciparum genome project are enzymes from the Apicoplast — a plastid (or chloroplast) that is a legacy of the malaria parasite’s distant non-photosynthetic ancestry. The other useful antimalarials are Artemisinin compounds synthesized from the plant Artemisia annua. a member of Apicomplexan family of parasites. making them potential drug targets [9]. sadly the cheapest and most effective first line chemotherapies like chloroquine. The plastid in Plasmodium is believed to have a red algal origin and is postulated to have appeared in the parasites due to secondary endosymbiosis (Figure 2) [10.Dhingra. This cyanobacterial heritage of the apicoplast means that many of its bacteria-like enzymes are fundamentally different from the mammalian host equivalents. artemether.11]. H. safe and commonly available drug. due to its presence in apicomplexans and similarity to the plastids as indicated by various workers in the past based upon presence of plastidic genome and various enzymes characteristic of plastids. etc. KG. Many of the more exciting new targets to be revealed by the P. with malaria mortality rates redoubling in many areas. cheap. Apicoplast A three – four membrane organelle was reported some 40 years back in the genus Plasmodium. sulfamethoxazole and sulfadoxine. was discovered as first anti malarial in 17th century but in use from very old times) and includes 4-aminoquinoline compounds such as chloroquine and mefloquine of which former is more effective. pyrimethamine and trimethoprime and sulfa drugs like dapsone. sulfalene. These compounds (artesunate. These are used in combination therapy for the treatment of severe malaria and have shown very rapid parasite clearance in comparison to quinine compounds [8]. et al. However. Clearly. Even continuous trials to develop a successful vaccine against malaria have been impeded due to the antigenic variations shown by the parasite. used to fight malaria for decades are now losing efficacy due to emergence of drug resistance in the parasite population. DUDWELLER LANDSTR. chloroproguanil. 2011 Current Topics in Biotechnology & Microbiology has evolved from the structural modification of quinine (originally extracted from Cinchona tree bark but now also chemically synthesized. there is a need for new antimalarials or identification and exploitation of new therapeutic targets. This has led to the resurgence of the disease. pyrimethamine. GERMANY . 222 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. These drugs are normally used in combinations. Tetracycline and its derivatives such as doxycycline are very potent antimalarials and are used for both treatment and prophylaxis. The organelle was termed Apicoplast (apicoplast).

This generally well-supported argument proposes that a red algal cell engulfed by an ancestral alveolate protist was enslaved after the transfer of many of its genes to the host cell nucleus and their subsequent inheritance. DUDWELLER LANDSTR. Following these ancient secondary endosymbiotic events in the red and green lineages. 223 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. haptophytes. Some apicomplexans (Cryptosporidia) may even have lost the plastid organelle altogether. KG. initiated the formation of monophyletic group of chromalveolate protists including the cryptophytes. et al. the visible remains of the secondarily acquired algal cell are restricted apparently to a multi-membraned plastid organelle. Figure 2: Hypothetical evolutionary scheme outlining sequential endocytobioses carrying primary and secondary plastids. although a store of laterally transferred genes from the algal symbiont has yet to be discounted in such cases [11].Dhingra. H. 2011 Current Topics in Biotechnology & Microbiology The latest hypothesis on the origin of Apicoplast has proposed that a single endocytobiotic event approximately 1300 million years ago. A nucleomorph. various algal structures and functions became incorporated into chimeric ‘meta-algal’ cells that underwent considerable diversification as they evolved. heterokonts and apicomplexan parasites. In apicomplexans. This is no longer photosynthetic and is bereft of much of its former genic content. GERMANY . the remnant of a red algal nucleus still persists in cryptophytes whereas chlorarachniophytes have a green algal version (Figure 3).

They had a contour length of 10. Gardner and co – workers isolated the counterpart from the human parasite P. falciparum with an estimated size of 35 kb and substantiated the organellar origin of the molecules by obtaining a sequence corresponding to a fragment of a prokaryotic small-subunit (SSU) ribosomal RNA gene [16-18]. lophurae. The circular molecules were first isolated in usable quantity from the simian parasite P. knowlesi as a low-density minor satellite in CsCl gradients. They were 11. berghei and P. GERMANY . knowlesi [15].5 mm in contour length and showed a cruciform structure just like that of Toxoplasma. KG. Similar low density satellites were also reported in two rodent malaria parasites. DUDWELLER LANDSTR.3 mm (31 kb) and a buoyant density linking them to the previously noted satellites. H. chabaudi [12]. These genes helped 224 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 2011 Current Topics in Biotechnology & Microbiology Figure 3: Schematic representation of various stages of hypothetical endosymbiotic events leading to formation of apicomplexans. P. berghei and Toxoplasma gondii (another Apicomplexan) stated this circular DNA to be of mitochondrial origin but also raised concern on the large cruciform structures seen in the electron micrographs of the DNA [12. Kilejian was the first person to “see” the plastid – DNA circles in extracts of isolated “mitochondria” of the avian parasite P. et al. Some similar studies following this in P. the plastid-like DNA (Figure 4) was first noted in P. She understandably assumed them to be of mitochondrial origin [13]. Various genes and open reading frames similar to the genes of red and blue-green algae were found in the 35 kb circular DNA. In the initial findings. The discovery of apicoplast is related with the discovery of its circular DNA.Dhingra. 14].

Inverted Repeats The inverted repeat (IR) has a total length of approximately 10.Dhingra. The remaining two–third circle is a single copy region which includes ribosomal protein genes. falciparum. It is thus suggested that it may be an ancestral apicomplexan character derived from numerous rearrangements and deletions in an ancient progenitor of the apicomplexan clade [12]. supercoiled architecture. Both forms of the rRNA genes are flanked by tRNA genes for met [M]. DUDWELLER LANDSTR. ala [A]. falciparum plastid DNA circle is packed with over 60 genes. 2011 Current Topics in Biotechnology & Microbiology researchers to relate the prokaryotic origin of the organelle and pointed to its plastid like provenance [19]. The Plastid DNA Plastid genomes are similar to those of their bacterial progenitors in that they are super – coiled DNA circles.5 kb and encodes duplicated genes for the small subunit (SSU) [17] and large-subunit (LSU) rRNAs [18] and nine different tRNAs [21. arg [R]. There are some specific genes encoding elongation factor Ef–Tu. The identity and arrangement of the genes on the 35-kb circle overwhelmingly favor its evolution from a plastid genome. et al. the Plasmodium plastid DNA has high A+T content (~82-86%) [20. IRA and IRB. The genes are usually separated by only a few nucleotides and in some cases even have small overlaps. falciparum is depicted in Figure 4. leu [L]. KG. About one–third of the circle includes inverted repeat regions. caseinolytic protease ClpC. An outline of genes associated to the circular plastid genome in P. H. The P. the apicoplast genome is the smallest known plastid genome. At 27–35 kb. 22]. asp [N]. and SufB proteins. 7 unidentified ORF’s and RNA polymerase genes. GERMANY . These include tRNA and ribosomal RNA (both small and large) genes.12]. arg [R]. the DNA molecule number goes upto 25 per cell. There are about eight to fifteen copies of 35-kb circles per cell in P. In other apicomplexans like Toxoplasma gondii. but it appears to have retained a circular. The order of this IR is conserved in Toxoplasma gondii (another apicomplexan) while it differs from other known plastids. val [V]. Iso [I] and Thr [T]. 225 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. As compared to plastid DNA from their counterparts in prokaryotes.

DUDWELLER LANDSTR. respectively. rpoC1 and rpoC2. They are designated as rpoB. RNA polymerase genes and ribosomal protein genes are given in the text.Dhingra. which encode subunits β. 2011 Current Topics in Biotechnology & Microbiology Figure 4: An outline of the gene map of the circular DNA of Plasmodium falciparum. 1. The Inverted Repeat regions can be divided into two parts – IR-A and IR-B based upon the gene positions. The two halves (A and B) of the inverted repeat (IR) are indicated. 226 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.2. H.4 denote 7 unidentified ORFs of varying lengths [20]. Another plastid-like feature of the diminutive malarial circular genome is the subdivision of the E. Immediately downstream of the IR-A region encoded on the same DNA strand as the LSU rRNA and tRNA lay ORF470/ SufB which is understood to be a homologue gene encoding SufB protein essential for the growth of cyanobacteria Synechocystis species. Downstream to SufB/ORF470 are two unassigned ORFs (ORF101 and ORF51). β’. Their recognition was one of the first clues to the plastid ancestry of the circle [19. et al. The rpoB gene is the closest to the plastid sequence of the protist Euglena gracilis while based on some phylogenetic analysis it seems to have diverged considerably from other plastid sequences [12]. GERMANY . and rpoC2. KG. The details of inverted repeats. The above three ORFs are followed by three larger genes of an RNA polymerase similar to that found in cyanobacteria and chloroplasts and typically not in mitochondria. It is proposed that the gene might be functioning as an aid in iron metabolism in the apicoplast [23]. 24]. rpoC1. coli rpoC-like gene into two genes. and β”. sufB.3. clpC and tufA genes are denoted.

et al. which encodes the elongation factor Tu (EF-Tu). a member of the hsp100 gene family. This process has been extensively detailed in apicomplexans like T. gly (G). a series of ORFs for ribosomal proteins are present downstream to the tRNA genes. falciparum. within the anticodon. The ribosomal proteins are followed by a tufA gene. ORF129. which divides into as many plastids as there are daughter cells in the maturing schizont. and finally a identified as clpC. one plastid is present in each parasite daughter cell. 2011 Current Topics in Biotechnology & Microbiology Immediately downstream to the IR-B region on the same DNA strand as of rRNA and tRNA genes is present a ribosomal protein gene rps4. a G-protein crucial in the elongation step of protein synthesis. There appears an intimate linkage between the P. the apicoplast starts forming a reticulate. This is followed by three tRNA genes. Apicoplast Division The division of apicoplast occurs in coordination with the division stages of the parasite.. leu [L]. and trp (W). every merozoite contains one copy of slightly elongated plastid ([25]. gondii and P. 23]). falciparum nuclear division and segregation of parasitic stages and the plastid division. branched structure. Thus. During schizont stage. H. rounds up into a sphere in early trophozoites and grows in size. ser [S]. This is followed by a cluster of 10 tRNA genes (his [H]. 2000 where they have depicted the morphology of plastid division with the help of Green fluorescent protein targeted to different stages of 227 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. glu [E] and pro [P]). It is notable that tufA occurs on the plastid genome of many algae but not on that of higher plants. asp [D]. similar to other plastid genomes. gln (Q). 20. KG. These three codons mark the end of repeat regions on both sides. The single apicoplast is crescent shaped during the ring stage (as discussed before in life cycle) of parasites. located.Dhingra. met [M]. Similar to other plastid genomes. as a thumb rule like all the other organelles. as with other plastid homologs. DUDWELLER LANDSTR. The leucine gene holds the only intron so far recognized on the circle. which shares the same first three codons as ORF470/ SufB at the other end of the IR-A. a short tentative ORF. another tRNA genes tRNA[F] lies distinctly on the complementary strand. Downstream of tufA. lys [K]. these genes encode ubiquitous heat shock or stress proteins that act as molecular chaperones with diverse functions ( For more details on this genome readers are requested to refer to [12. GERMANY . After red blood cell rupture. The readers are suggested to study the reports by Waller et al. cys [C]. The apicoplast follows maternal inheritance whereby only those merozoites which develop into female gametocytes carry the copy of apicoplast with them. tyr [Y].

Immediately after apicoplast disruption (either pharmacological or genetic). transcription or translation [29. whatever the apicoplast provides for the parasite is crucial for a viable infection process. GERMANY . Apicoplast is the site for metabolic pathways that are specific to the parasite and are not found in its vertebrate host. DUDWELLER LANDSTR. This has been proven by the recent plastid isolation studies whereby it was shown that plastid alone can not be isolated from the parasite but is always accompanied by the mitochondria [26-28]. Parasites die after treatment with drugs that interrupt apicoplast genome replication. but subsequently arrest and die after infecting a new host cell. 9]. H. Presumably.Dhingra. The fact has been proven by various experimental data. All the pathways are of prokaryotic origin. It has also been observed that the division of apicoplast is immediately followed by division of nucleus and throughout its life cycle in parasite the plastid remains mysteriously attached to the mitochondria. Some of these drugs have also been shown to effect the parasite survival (Table 1) [36]. The only gene that has been studied in detail from another human malaria parasite P.30]. parasites continue to grow normally in the host cell. due to this fact the plastid is also considered as putative drug target. or perhaps a resource that is usually replenished at the time of host-cell invasion [32-34. vivax is of tufA gene [35] where the scientists have detailed the structural and functional aspects of the Ef-Tu A protein encoded by tufA. mutant parasites lacking an apicoplast are not viable [31]. Apicoplast Functions The importance of the apicoplast has been an ongoing issue of debate since its discovery. KG. Moreover. algae and bacterial systems. falciparum sequences to orthologues identified from different bacterial databases. This could be a component of the parasitophorous vacuole. The enzymes involved in these pathways are encoded by the nuclear genome and are targeted 228 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The plastid genome sequences from other Plasmodium species are only countable and have been studied to understand the evolutionary position of the parasites. 2011 Current Topics in Biotechnology & Microbiology apicoplast division. et al. The organelle is considered indispensable for the survival of the parasite. The functions of the genes present in the apicoplast genome have been only detailed based upon homology of P. which surrounds parasites in the host cell. A number of enzymes present in these pathways are proven to have drug binding sites in the equivalent enzymes present in plant.

However. algae (within their plastids) and many bacteria. Initial drug inhibition studies indicated to the presence of this FASII pathway in the blood – stage parasites. recent reports suggest that FASII pathway is only essential in the late liver stages of the parasite [25. 2011 Current Topics in Biotechnology & Microbiology from the nucleus to the apicoplast. to be secreted from the cell into the parasitophorous vacuole [25. One of the four major metabolic pathways for which the apicoplast has been convincingly shown as the site is the de novo Type II fatty acid biosynthesis (FASII) pathway. reduction. Isoprenoids are diversified group of natural products such as sterols. 37] while removal of the signal peptide alone lead to accumulation of the protein in the cytosol [25].e. while the remainder of the N-terminal extension represents a plastid transit peptide. animals. The former is characteristic in fungi. The N-terminus starts with a typical hydrophobic signal peptide. et al. Isopentenyl diphosphate (IPP).The signal peptides are believed to mediate traffic across the outermost membrane of the apicoplast.Dhingra. GERMANY . Apart from the much studied FASII pathway. 229 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. dehydration and reduction reactions to form a 10 – 16 or upto 18 carbon chain long compounds. The discovery of the pathway may be attributed to the identification of nuclear genes which encode for enzymes of this pathway and are targeted to apicoplast. the precursor of isoprenoids can be synthesized in cells by two different types of pathways i. H. These include b-ketoacyl-ACP synthase I/II (FabB/F) and b-hydroxyacyl-ACP dehydratase (FabZ) enoyl-ACP reductase (FabI). the parasitic plastid also houses isoprenoid biosynthesis [44. certain bacteria and protozoa while the later one is usually found in plants. The antibiotic/ herbicide fosmidomycin (and its derivatives) have been shown to block the DOXP pathway by targeting DOXP reductoisomerase in certain rodent Plasmodium species and in P. This transport of proteins takes place by a two–fold mechanism and includes a secretory pathway [9].falciparum as well. while the transit peptides mediate traffic across the inner two membranes. Mevalonate – dependent pathway and the Non-mevalonate or Deoxyxylulose phosphate (DOXP) pathway. The apicoplast targeted proteins are tagged at the N– terminal with a bipartite leader sequence. that now only contained an N-terminal signal peptide. 45] pathway. DUDWELLER LANDSTR. The pathway includes conversion of acetyl-CoA to malonyl-CoA by acetyl CoA carboxylase (ACCase) which includes addition of malonyl moiety to the acyl chain in repeated rounds of condensation. KG. Deletion of just the transit peptide causes proteins. 38 – 43]. carotenoids and terpenoids.

H. and sufS (in the nuclear genome) which are member of the Suf family indicates to some functionality of this pathway in the parasite apicoplast. both photosynthetically active and inactive. falciparum. GERMANY . The fourth pathway. Tetracyclin Thiolactomycin b-ketoacyl-ACPsynthase III (fabH) Fatty acid biosynthesis Plastid 16S rRNA Protein translation Plastid 23S rRNA Protein translation RNA transcription Metabolic Activity affected DNA Replication Apicoplast also appears to harbour a ferredoxin-based electron transport system [46] which is characteristic of plant plastids. 48] is required by the P. in non-photosynthetic plastids. Chloramphenicol Doxycyclin. Thiostrpton. KG.Dhingra. Azithromycin. et al. DUDWELLER LANDSTR. sufC. Erythromycin. sufD.. This complete ferrodoxin based electron transport system is yet to be proved in P. This whole system and this complete pathway still remain to be proven in the malaria parasite. the Heme Biosynthesis pathway [47. 2011 Current Topics in Biotechnology & Microbiology Table 1: Drugs acting on plastid targets and affecting Plasmodium parasite growth and survival Drug/ herbicide Putative target in plastid Ciprofloxacin Plastid DNA type II topoisomerase Rifampicin Plastid RNA polymerase bsubunit Clindamycin. the electrons derived from splitting of water are transferred back to ferrodoxin by cofactor NADPH. viz. The presence of genes. i. The reduced ferrodoxin may serve as reductant for various plastid specific processes. sufB (in plastid genome) and sufA.e. The Suf system is well known to be involved in the bacterial ferrodoxin based electron transport. falciparum for synthesizing heme which may be acting as a precursor in synthesis of certain 230 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Micrococcin. As per analogy.

the task has become much easier and new studies can be designed to get the details on the function of cluster of genes present in the plastid genome and on function of as yet uncharacterized hypothetical proteins targeted to apicoplast. ALAD has also been found in these parasites. The enzymes functional in the metabolic pathways specific to this organelle have been identified as potential targets to various drugs and chemotherapeutic agents. There arises a need to get insight into the reported metabolic pathways and their products as whole that might be helping the parasite to cause infective of varying grades in its vertebrate host. While some evidence indicates that large amounts of the mammalian ALAD enzyme are imported from the red blood cell into the parasite cytoplasm [50 – 52]. the plastid organelle in apicomplexan parasite Plasmodium has paved the way for the identification of novel drug targets in our fight against malaria. a number of other enzymes involved in the complete heme biosynthesis pathway have now been deduced and have been found to be localized in the apicoplast. other authors have found that the Plasmodium genome encodes its own ALAD. 231 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 48]. DUDWELLER LANDSTR. 2011 Current Topics in Biotechnology & Microbiology proteins. which is synthesized by two different biosynthetic pathways. the close contact between these two organelles observed in various life stages supporting this scenario [47. The enzyme delta-aminolevulinate dehydratase (ALAD) leads to condensation of two ALA from the above two pathways running simultaneously in respective compartments. KG. 49]. to yield porphobilinogen. The second pathway C5 or Glutamate pathway synthesizes ALA from glutmate and is found mainly in plastids of algae and plants and in some eubacteria. It is thought that Plasmodium uses ALAS and the Shemin pathway in its mitochondria [20. It has been speculated that the malarial parasites synthesize ALA in the mitochondria and continue the biosynthetic pathway of heme with ALAD in the apicoplast. GERMANY . The Shemin pathway (characteristic to animals and fungi & located in mitochondria) synthesizes ALA by action of deltaaminolevulinate synthetase (ALAS) on glycine and succinyl –CoA. falciparum and plastid genome. et al.Dhingra. With the availability of the complete nuclear genome data of P. One crucial precursor for heme biosynthesis is delta-aminolevulinate (ALA). Still some more enzymes remain to be identified in apicoplast to completely prove this pathway. However. H. CONCLUSIONS Apicoplast.

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colonizes the human stomach and is estimated to inhabit at least half of the world’s human population. 1Shailesh K. which can progress to a variety of other gastroduodenal diseases. et al. INTRODUCTION: About 25 years ago. such as peptic ulcers. a Gram-negative. Varanasi. 2011 Current Topics in Biotechnology & Microbiology Chapter XII THE ENIGMA OF Helicobacter pylori INFECTION AND GASTROINTESTINAL DESEASES 1 Sushil Kumar. microaerophilic. India * Coressponding author: Dr.mail: kasokbt@rediffmail. Notably.Dhingra. GERMANY . H. The bacterium has been recognized as the causative agent of chronic gastric inflammation. Varanasi. mucosa associated lymphoid tissue lymphoma. *1Ashok Kumar and 2V. KG.K.com ABSTRACT: Helicobacter pylori. School of Biotechnology. Banaras Hindu University. perhaps depending on factors such as host characteristics that are governed by genetic polymorphisms. Banaras Hindu University. Banaras Hindu University. DUDWELLER LANDSTR. Dixit 1 School of Biotechnology. Institute of Medical Sciences. Varanasi. or even gastric cancers. India E. Ashok Kumar. It is a highly successful bacterial pathogen that persistently colonizes the mucosa of the human stomach and infection occurs mostly in early childhood. only a fraction of colonized individuals develop peptic ulcer and gastric cancer. Barry Marshall and Robin Warren described the successful isolation and culture of a spiral bacterial species from the human stomach [1]. frequently leading to persistent infection lifelong. Shahi. India 2 Department of Gastroenterology. differentially represented bacterial determinants and/or specific interactions between a particular strain and its host that occur during decades of coexistence. During that period (198283) the conventional thinking was that no bacterium can survive in the human stomach as the 237 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Professor of Biotechnology. spiral-shaped bacterium.

and gastric cancer. preventive vaccination strategies still do not exist. such as chronic gastritis. et al. pylori in the Western world is decreasing. pylori persistence and pathogenesis is thus mandatory to aid the development of novel intervention and prevention strategies. [3] and Morris and Nicholson [4] and later experiments with volunteers (5). pylori experiments by Marshall et al. 1. gastric colonization by H. which was resolved after sequential therapy. DUDWELLER LANDSTR. which showed that gastric colonization with H.The genus Helicobacter belongs to the ε subdivision of the Proteobacteria. Infection with H. order Campylobacterales. in spite of the pathogenicity of H. The organism was initially named “Campylobacter-like organism. the increase in antibiotic resistance is hindering the efficacy of treatment. MICROBIOLOGY (i) Genus description and phylogeny of Helicobacter . pylori remains widespread in the developing world. first with doxycycline and then bismuth subsalicylate. Self-ingestion of H.” “Campylobacter pyloridis. This knowledge had a major clinical impact with regard to the management of these diseases.” and “Campylobacter pylori” but is now named Helicobacter pylori in recognition of the fact that this organism is distinct from the members of the genus Campylobacter [2]. These initial data strongly stimulated further research. KG. Unfortunately. pylori together with various types of gastrointestinal diseases caused by its infection and factors involved in disease appearance in the host. H.” Although the prevalence of H. This discovery resulted in the award of the 2005 Nobel Prize in Physiology and Medicine to Robin Warren and Barry Marshall for their “discovery of the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease.Dhingra.” “gastric Campylobacter-like organism. pylori. 2011 Current Topics in Biotechnology & Microbiology stomach produced excessive amounts of acid which was similar in strength to the acid found in a car-battery. pylori can lead to variety of upper gastrointestinal disorders. demonstrated that these bacteria can colonize the human stomach. GERMANY . peptic ulcer. Marshall developed a transient gastritis after ingestion of H. pylori. The most important stage in the development of the taxonomy was proposed by Goodwin and 238 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. A better understanding of H. pylori can be diagnosed by a variety of tests and can often be successfully treated with antibiotics. gastric mucosa associated lymphoid tissue (MALT) lymphoma. and. family Helicobacteraceae. thereby inducing inflammation of the gastric mucosa. the case described by Morris developed into a more persistent gastritis.. This paper deals with the basic biology of H. In addition. the persistence of a pathogen in an environment long thought to be sterile also resulted in insights into the pathogenesis of chronic diseases.

H. majority of them are catalase and oxidase positive. To date. (iii) Genome. (ii) Morphology . 23S.587 genes.5 to 1 m in width. which do not seem to carry genes responsible for virulence or antibiotic resistance [9]. 26695 and G27) genome is approximately 1. KG. This results in every H.7 Mbp. pylori is genetically described as a gram-negative bacterium. Pathogenicity Island (cag 239 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. with a G+C content of 35 to 40%. The reasons of genetic heterogeneity are possibly an adaptation of H. Although usually spiral-shaped or curved rod with one to three turns when observed in vivo. et al. Bacterial cells are mostly actively motile in viscous solutions such as the mucus layer overlying the gastric epithelial cells. non-culturable state.The genome (chromosomal) DNA of H. and 5S rRNA genes. the genus Helicobacter consists of over 20 recognized species but many of them is awaiting formal recognition [7]. GERMANY . the coccoid shapes appear after prolonged in vitro culture or antibiotic treatment [8]. measuring 2 to 4 m in length and 0.491 genes. The H. pylori infection [11]. pylori should be transferred to the genus H. Many strains (ca 45%) carry one or more cryptic plasmids. pylori is a single circular molecule. 2011 Current Topics in Biotechnology & Microbiology colleague [2] to establish a new genus called Helicobacter. pylori to the harsh gastric conditions of its host. The bacterium has 2 to 6 unipolar (lophotrichate) sheathed flagella of approximately 3 m in length. J99. pylori strain 26695 genome includes 1. pylori is genetically heterogeneous. plasmids and strain diversity . A striking example of this is the cytotoxic associated gene. as well as to the distinct patterns of the hostmediated immune response to H. HPAG1. pylori (Shi 470. Members of Helicobacter genus are microaerophilic. suggesting a lack of clonality. pylori. These coccoids loose their ability to be cultured in vitro and are thought to represent dead cells [8]. pylori-positive subject carrying a genetically distinct strain [10] although differences within relatives may be small. whereas the genome of strain J99 includes only 1. The latter usually have an aberrant G+C content and often carry genes involved in virulence. Genetic diversity is thought to occur via several methods of DNA rearrangement and the introduction and deletion of foreign sequences [12]. H. The size of the five sequenced H.Dhingra. which often carry a distinctive bulb at the end of flagellum. The creation of the new genus was argued mainly on the basis of the 16S rRNA (small subunit) sequence data [6]. although it has been suggested that coccoid forms may represent a viable. in contrast. Generally bacterial pathogens are highly clonal (such as Shigella dysenteriae and Mycobacterium tuberculosis). DUDWELLER LANDSTR. They proposed that C. and many but not all species are urease positive. Genomes of J99 and 26695 contain two copies of the 16S. H.

DISEASES ASSOCIATED WITH H. H. However. but other plasticity regions have also been suggested to play a major role in the pathogenesis of H. pylori infection [13]. and oipA (outer membrane protein-encoding genes).Dhingra. it frequently develops into chronic atrophic gastritis with loss of epithelial glands over a period of decades. pylori infection is more common in childhood and persists for life long unless specifically treated.It has been elegantly demonstrated that infection with H. Overall. pylori prevalence is low in early childhood and slowly rises with increasing age in industrialized countries. Prevalence of H. This event shows a reversible phenotypic diversity with only minor genetic variation. new H. At the molecular level. pylori always causes chronic active gastritis. 2011 Current Topics in Biotechnology & Microbiology PAI). hopZ. DUDWELLER LANDSTR. resulting in a shift in translation of the affected gene. the prevalence of H. 240 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. EPIDEMIOLOGY The prevalence of H. namely sabA. Often phase variation occurs via reversible slipped-strand mispairing in homopolymeric G or C tracts. thereby causing phase variation via a single mutation. H. Within particular geographical areas. KG. Several virulence genes. The association between gastrointestinal (GI) symptoms and chronic superficial gastritis [16]) is uncertain. pylori is inversely proportional with socioeconomic status. as do lipopolysaccharide (LPS) biosynthetic enzymes [10]. pylori INFECTION (i) Gastritis . and neutrophils (a marker for the activity of gastritis). pylori is considerably lower in children and adolescents than in adults and elderly people [14]. 2. pylori infection rates rise rapidly in the first 5 years of life in developing countries and remain constantly high thereafter. indicating that H. pylori provokes invasion of the mucosa by lymphocytes/plasma cells (a marker for the grade of gastritis). Gastritis commonly refers to inflammation of the lining of the stomach. pylori positive [10]. GERMANY . pylori prevalence generally remains under 40% of the population. H. the diversity in genome may be seen via several mechanisms. In the industrialized countries. pylori is acquired early in childhood. However. pylori shows wide geographical variations in different parts of the world. H. display such phenotypic variation. et al. this is due to living standard during childhood [15]. sabB. More than 80% of the population of various developing countries are H. Infection with the H. including transcriptional and translational phase variation and mutation. 3.

pylori. may be.Gastric or duodenal ulcers (peptic ulcer) is defined as a defect in the gastric mucosa of at least 0. quickly feeling 'full' after eating. which is at least 3-4 times higher than for uninfected individuals [19. 17]. Seroepidemiological evidence from different populations revealed that the lifetime risk for peptic ulcer disease among H. Ulcer development in the presence of H. et al. 30 to 60% of patients with nonulcer dyspepsia carry H. pylori infection has dramatically changed the understanding of the pathogenesis of ulcer disease and also its treatment. 241 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.Dyspepsia is simply. a third well-designed trial reported a benefit of eradication treatment in a small subgroup of patients [21].Dhingra. Symptoms are often related to eating. Peptic ulcer (PU) disease is strongly associated to H. pylori seroprevalence of 60100% in gastric ulcers cases [17]. especially in elderly. DUDWELLER LANDSTR. (iii) Nonulcer dyspepsia . The main symptom of dyspepsia is usually pain or discomfort in the upper abdomen. The discovery of H. GERMANY . pylori is influenced by variety of host. heartburn. in addition. pylori eradication and relief of symptoms [20]. However. Non-ulcer or functional dyspepsia is defined as the presence of symptom of upper gastrointestinal distress without any identifiable structural abnormality during diagnostic work-up. however. 17]. pylori infected individuals is 620%. pylori infection has.5-cm in diameter and penetrating through the muscularis mucosa. bacterial and environmental factors. KG. Serological confirmation of H. Occurrence of most ulcers is at sites where mucosal inflammation is most severe. Two randomised double-blind placebo controlled studies in adults with nonulcer dyspepsia did not find association between H. pylori worldwide [18. verified an H. The remaining DUs and PUs. The corresponding prevalence of infection in the case of gastric ulcer patients has been more complicated to establish since biopsy based diagnosis generally shows false-negative results due to association between gastric ulcers and atrophic changes of the mucosa. H. H. are due to drugs like NSAID use and other causes (rarely) such as Zollinger-Ellison syndrome and Crohn’s disease [18]. 2011 Current Topics in Biotechnology & Microbiology (ii) Peptic ulcer . feeling sick (nausea) or vomiting. but this prevalence is not much different from that in the unaffected population [10]. in particular upper gastrointestinal tract. pylori infection. bloating. pylori infection is diagnosed in 95% of duodenal ulcer (DU) where as 85% of gastric ulcer occurred in the presence of the infection of H. a term which includes a group of symptoms that come from a problem in upper gastrointestinal tract. belching. with or without bleeding and perforation [17].

pylori infected cases (1-2%) is likely to develop gastric carcinoma. pylori negative subjects [10].Gastric adenocarcinoma is the second most common cause of cancer related death world wide and 14th cause of overall death [22]. other pathophysiological disturbances seem to play a role in its pathogenesis. GERMANY . GERD has long been considered to occur independently of H. KG. DUDWELLER LANDSTR. including interaction with other infectious agents and dietary factors. stricture. pylori infection. pylori in gastric carcinogenesis [25]. Even though GERD is primarily a motility disorder. pylori plays a critical but major role in the development of both types of gastric adenocarcinoma [24]. overall 1520% of infected patients will develop gastric or duodenal ulcer disease and only a fraction of all H. pylori infection in DU patients may provoke reflux esophagitis. pylori significantly decreased the rate of gastric carcinoma development in H.Gastroesophageal reflux disease (GERD) is defined as chronic symptoms of mucosal damage produced by the abnormal reflux of gastric contents into the esophagus [27]. Gastric adenocarcinoma is histopathologically sub-divided into intestinal-type and diffuse type. pylori than in the H. geographical location. i. pylori-infected patients without precancerous lesions. to occur with the same frequency and severity in H. Eradication of H. Spectrum of the esophagus injury includes esophagitis. 2011 Current Topics in Biotechnology & Microbiology (iv) Gastric cancer . However. diet and ethnicity. Intestinal-type gastric adenocarcinoma.e. The bacterium colonizes over half of the population worldwide. et al. providing evidence for the causative role of H. the majority are asymptotic. Though the risk varies with age. based on 276 DU patients randomised to either eradication 242 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. H. A 2-year follow-up. affect the immune response to H. Infection is usually acquired in childhood and in the absence of antibiotic therapy and therefore persists for the lifelong of the host. further studies suggested that the curing of H. pylori-positive and H. pylori and may impact disease presentation over the life of the host [26]. pylori-uninfected population. A multitude of host and environmental factors. (v) Gastroesophageal reflux disease . Although most infected patients will develop a chronic active gastritis. A large-scale prospective study also revealed that the risk of development of gastric carcinoma was much greater in the population infected by H.. the development of columnar metaplasia in place of the normal squamous epithelium (Barrett’s esophagus) and adenocarcinoma [28].Dhingra. A small subset of diffuse type adenocarcinoma is of familial origin. caused by mutations in the Ecadherin gene [23]. is more common than diffuse type. Epidemiological studies as well as infection studies using animal models have indicated that H. which occurs in older people.

migraine. presumably from its host. 243 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 4. GERMANY . pylori has been observed in many patients [10]. pylori prevalence in children with chronic diarrhoea has been observed. autoimmune thyroid disease and thromophenomenon. pylori and Vibrio cholerae. pylori scavenge iron. bacterial antigens or urease activity all being indirect methods are preferred.The invasive tests for H. The non-invasive methods based on peripheral samples such as blood. pylori infection and the reversal of iron deficiency anaemia after eradication of H. pylori is linked to a variety of extragastric diseases. The choice of a specific test for an individual patient depends on severity of disease. histology. pylori can detect very small numbers of bacteria and characterize the various genes and this being direct methods.Till date there is a general belief that H. local experience. association of H. dermatological disorder such as rosacea and idiopathic urticaria. However. Furthermore. 31]. iron deficiency anemia and Guillain-Barre syndrome [10. 2011 Current Topics in Biotechnology & Microbiology therapy or long-term omeprazole treatment. (vi) Extragastric manifestations . revealed that both regimens were equally effective in controlling dyspeptic symptoms and GERD in patients with a healed DU [29]. a positive association has been noted between H. The observations might be due to subclinical bleedings from the GI tract but are also likely to be due to the genetic factors indicating that H. scleroderma. confirmation of these observations would need further studies taking into account various factors such as genetic and socio-economic/nutritional conditions. DUDWELLER LANDSTR. the healing of ulcers was not found to increase the risk of GERD. DIAGNOSES Invasive and non-invasive diagnostic methods . pylori infections are based on gastric specimen for culture. KG. In some paediatric studies. Among children in developing country. Sidropenic anaemia has been reported to be associated with H.Dhingra. urine or saliva for detection of antibodies. stools. rapid urease assays and polymerase chain reaction (PCR) which by amplifying segments of DNA from H. a population-based study of German pre-school children found an inverse association between H. they are generally not considered suitable for paediatric epidemiological investigations. pylori infection” field [30]. In contrast. Since these methods require upper endoscopy. pylori infection with short stature has been suggested. pylori and diarrhoea [32]. et al. H. for ethical reasons and for the cost. A higher H. breath samples. These include coronary heart disease. thereby opening the new “extragastric manifestations of H.

(i) Culture . In hospital-based care. pylori is fastidious and microaerophilic organism. may vary between laboratories since the method is most technically demanding. However. H. In this situation immunohistochemical staining [35] and other technique such as in situ hybridization. The organisms are sensitive to temperature. Even though the sensitivity of histology is generally high.H. 2011 Current Topics in Biotechnology & Microbiology clinical setting and cost.This test uses the high concentration of pre-formed urease enzyme activity in H. In addition. pylori. et al. DUDWELLER LANDSTR. Difficulties arise when patients use anti-Helicobacter treatment where the bacterial count may be much reduced but infection not completely eradicated. sensitivity can be improved by examining additional samples from the antrum and corpus. oxygen and medium which create problem during transportation and the culture itself requires special conditions and 3-7 days of incubation. its culture is not always successful in many laboratory. pylori is one of the microorganisms that can be confidently recognised by histology.e. Genetic diversity study can also be easily made by culture of bacteria. this method may be affected by observer related factors and topographical changes in the stomach may introduce sampling errors. Poor orientation or small biopsy samples will lower sensitivity. The main advantages of culture are the excellent specificity and the opportunity to test for susceptibility to antibiotics in patient management [33]. (ii) Histology . H. Also. (iii) Rapid urease test . although this investigation is limited to selected research centres. gastritis and in adults premalignant (dysplastic) changes or neoplastic lesions such as carcinoma. Although a single biopsy from the lesser curvature (2–3 cm from the pylorus) will yield a positive result in over 90% of infected stomachs [34]. KG. If H. i. lymphoma or carcinoid. GERMANY . pylori adopts a coccoid form then confident identification becomes impossible. and thus the sensitivity of the test. pylori infected gastric biopsy samples. the histopathologists are uniquely placed both to identify infection and to assess the disease process associated with it. many patients undergo endoscopy. Specificity is generally not a big problem. The ability to culture the organism.Dhingra. [36] or PCR [37] may resolve the issue. pylori infection. The most important factor affecting sensitivity is the experience and consciousness of the observer.. the genotype of the isolate with regard to cytotoxin associated antigen gene (cagA) and the vacuolating cytotoxin antigen gene (vacA) and other pathogenic gene status can be determined. The increasing alkalinity resulting from the 244 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. which is then combined with an invasive test of H.Culturing of bacterial colonies from gastric biopsies is regarded as a definitive proof of H.

it may not be reliable in assessing patients who have had gastric surgery. pylori present in human gastric juice and mucosa may give false results [39]. 13 C-labeled urea is becoming increasingly popular because this non- radioactive isotope is innocuous and can be safely used in children and pregnant women. which is produced by H pylori in large quantities. which diffuses into the blood through mucus layer of the gastric mucosa via the systemic circulation and is excreted in the expired air through lungs. The test gives a direct measure of an active gastric infection [40]. pylori infection within the gastric mucosa. at 24 hours false positive results may be obtained by other urease producing bacteria [38].The urea breath test (UBT) is one of the most important non-invasive methods for detecting H. et al. In this situation this test will give false positive for H.Dhingra. Also. many urease positive microbes other than H. Thus. Occasionally. The sensitivity of the test depends on the number of bacteria present in the sample. 2011 Current Topics in Biotechnology & Microbiology production of ammonium ions (NH4+) is detected by a colour change in the pH indicator and urea containing medium. H. The test can be carried out using small quantities of standard urea broth for in-house methods but commercial kits are also available. oral urease producing bacteria may cause false positive results [43]. It has been calculated that 104 microbes are required for a positive result. bismuth salts and proton pump 245 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. GERMANY . thus providing a gold standard against which the accuracy of other diagnostic tests (both invasive and noninvasive) can be validated [42]. Low levels of colonisation are to be expected if patients used anti-Helicobacter treatment where the bacterial count may be much reduced but infection not completely eradicated. KG. antibiotics. One of the most important advantages of the UBT is that it samples the whole stomach and there is no chance of sampling error unlike biopsy based tests (invasive test). (iv) 13C-Urea breath test . The test is easy to perform and can be repeated as often as required in the same patient. Increased sensitivity can be achieved by using two biopsy specimens and incubation at 37°C. but declines with the length of the incubation. Recently many urease positive microbes other than H. also reflecting the bacterial load and grade of histological gastritis [41]. pylori. Additionally. DUDWELLER LANDSTR. The UBT is a very sensitive and specific method. which can be influenced by the patchy distribution of H. and a proportion of patients will harbor densities lower than this threshold [33]. The test exploits the rapid hydrolysis of orally administered labelled urea by the enzyme urease. pylori in human gastric juice and mucosa have been discovered [39]. The specificity of this rapid urease test is very good when the test is read at one hour. pylori infection. However. Urea is hydrolysed to ammonia and carbon dioxide.

but also due to the occurrence of maternal antibodies up to the age of 3-7 months. It has been suggested that the cut-off value needs to be corrected specially in the case of children since they produce lower antibody responses than adults. such as Campylobacter sp. pylori passive infection in individuals not undergoing gastroendoscopy and this test is based on the principle of detecting the circulating antibodies against H. caused by the need for expensive mass spectrometric equipment. the test is not affected by sampling errors. allowing for the development of preventive infection early in life [45]. 2011 Current Topics in Biotechnology & Microbiology inhibitor (PPI). and minimal patient discomfort thereby making it suitable also for large-scale screening [46]. may introduce false negative results. The cost of producing 13 C-urea is high.Dhingra. in children it has shown variable sensitivity and specificity [48.Serological test is also one of the non-invasive tests. H. Another major disadvantage is the high initial cost for the test. pylori. Advantage of this test is that the result is not affected by sampling errors like other invasive or non-invasive tests. pylori infection. pylori infection and have been helpful in epidemiological studies of prevalence. The ELISA format has the advantage that many serum samples can be tested in parallel and the process can be completely automated [45]. Serology is the easiest way to identify H. Although ELISA has proven to be highly accurate for the diagnosis of H. which reduce the extent of H.e. These tests are used for the detection of H. i. However. (iv) Serology . reservations have been made since the test is less reliable in children <5 years and it is not yet evaluated among the youngest (<2 years) [44]. DUDWELLER LANDSTR. 49]. [47]. It has been demonstrated that initially lower specificity of the method was due to false positive results mainly due to nonspecific crossreaction with other microorganisms. but it may be possible to reduce the dosage further by administering it in capsule. Common designs are based on antibody detection tests which include the Western Blot (WB) and enzyme-linked immunosorbent assay (ELISA) technique. et al.. It has shown 246 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Recommendation of UBT is for monitoring the effect of eradication treatments and it is also a suitable test for epidemiological study. particularly in children. one of the major drawbacks of this test is that a positive test does not necessarily indicate a current (active) infection however similar to the UBT. This is the most widely used method due to its simplicity and cost effective features. pylori infection in adults. However. needs to be evaluated for each assay system and in the population being investigated. speed. KG. not only due to delayed and low antibody production. Many different types of antigen preparations can be used in the ELISA and the upper limit of normal values. the cutoff level. GERMANY .

If the target is too low. pylori DNA or organism. Among the drawbacks of PCR are. pylori but conserved in all strains of the species. quantification and detection of specific genes relevant to pathogenesis such as cagA. To improve DNA extraction. ERIC-PCR etc and specific mutations associated with antimicrobial resistance. Recently. H. formerly named ureC [51]. Target choice is the most important factor in the primers designing which must be specific for H. pylori. pylori and as many strains of other bacterial species as possible. Therefore. however. pylori gene used was the genes of the urease operon: ureA [50] and glmM. (i) existence of possible Taq and other themostable polymerase inhibitors which can decrease the sensitivity of the reaction. which alters the specificity. This method revolutionized the study of DNA. AFLP. KG. DUDWELLER LANDSTR.5%) and specificity (83%) than saliva based assay. GERMANY . this was quickly applied to the detection of H. vacA. babA etc. However. In the case of direct detection of H.Soon after the discovery of PCR. Methods such as the reverse dot blot line probe assay (LiPA) [53] and liquid 247 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. it has been demonstrated that use of an internal control helps in the test of possible PCR inhibitors. it is necessary to know the DNA sequence of the target in as many strains of H. The first target of H. pylori from specimen it is also necessary to know host DNA sequence. To date several methods have been proposed to improve sensitivity and specificity of PCR. as well as in diversity study based on RAPD. especially after the introduction of a thermostable DNA polymerase obtained from Thermophilus aquaticus (Taq polymerase). and the 16S rRNA gene [52]. the use of nested or heminested PCR increases the risk of airborne contamination by amplicons and must be discouraged. H. (vi) Polymerase chain reaction (PCR) (a) Standard PCR assay for the detection of H. use of a nested or seminested PCR has been suggested to escape false positive as well as false negative result [10]. pylori concerns not only the detection of the bacterium but also its genome sequencing. pylori-specific IgA and IgG antibodies detection in saliva from general population. phenol extraction. Most IgG diagnostic tests are serum-based. et al. CTAB mini prep method or the use of many commercial kits for DNA extraction and purification to eliminate the possible PCR inhibitors in target DNA have been employed. pylori. 2011 Current Topics in Biotechnology & Microbiology limited sensitivity (80%) and specificity (70%) of H. 16S rRNA gene is highly conserved in bacteria and exhibits sequences which are specific for different species. iceA. Its application in the field of H. and (ii) the specimen could be contaminated by exogenous H. pylori-specific IgG in urine has shown more sensitivity (95. it is also advantageous in checking false negative.Dhingra.

iceA) and adherence (babA2. Other genes involved in pathogenicity (oipA. When detection for both cagA and vacA is performed.e. the possibility of false positives arises if grinding apparatus in the lab. endoscopes [54] or biopsy forceps are not carefully cleaned. et al. 2011 Current Topics in Biotechnology & Microbiology phase (DNA-enzyme immunoassay) have also been proposed. This method does not require cultivation of bacteria. directly from gastric biopsy samples. KG. a strong association is seen between the presence of cagA and vacA s1. simple and fast [37].An important application of standard PCR is the genotyping and detection of specific pathogenic factors of H. recently developed method based on multiplex PCR assay seems very useful [37]. This method is based on mRNA. Several authors have reported the use of reverse transcription-PCR (RT-PCR) assay. corresponding to strains with the highest production of cytotoxin [55] as well as more severe pathology and disease. saliva. Template for amplification is obtained simply by vortexing the biopsy samples. A significant improvement in sensitivity of PCR was obtained with the introduction of real-time PCR method. the development of real-time PCR has facilitated more rapid. This method is cost effective. extraction of genomic DNA. The specificity of PCR is not usually a problem when dealing with gastric biopsy specimens. the cag PAI and the polymorphism of the vacA gene. dental plaque and faeces has been emphasized. pylori . Some of the investigators developed strip. boiling of biopsy samples or frequent steps of centrifugation. pylori in different types of biological samples for invasive as well noninvasive tests. However.. There are two most important pathogenic factors i. As is always the case when a new method is more sensitive than the reference method. dupA.More recently. Usefulness of PCR for detection of H. 248 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. (b) PCR assay for the detection of pathogenicity genes of H. vacA signal sequence (s1 and s2) and mid-region (m1 and m2) alleles as well as the presence or absence of a cagA gene can be detected in a single reaction. Using this assay the prevalence of type I and/ or type II strains. it is necessary to prove that the results of new methods are not giving false positive results. DUDWELLER LANDSTR. (c) Real-time PCR . pylori genes are amplified with a biotin-labelled PCR primer and subsequently analyzed by a single-step reverse hybridization on the strip [53]. but no improvement in sensitivity has been shown in this method.based detection by which H. such as gastric juice. specific and quantitative estimation of gene targets as they are amplified in real-time [56]. GERMANY . sabA) can also be detected by PCR. H.Dhingra. pylori. However. Although sensitivity and specificity of this test have high practical consideration but due to cost it has limited use. it determines the viability of the bacteria present.

GERMANY . pylori infection parameters Available reports suggest that the causes of different outcomes of H. 2011 Current Topics in Biotechnology & Microbiology Advantage of this technique is not only detection of H. The amount of unknown amount of wild template can be calculated on standard curve. pylori have been suggested as virulence factors. et al. pylori in gastric mucus was correlated with other invasive tests as well as with the urea breath test (UBT) in a study made by Furuta et al. Infection scenario of H. followed by real-time PCR which has considerably facilitated the process. pylori infection are governed by host and environmental factors as well as differences in bacterial virulence factors [60]. A visual comparison allows the detection of bands with the same intensity indicating the amount of target DNA present [58]. pylori initially 249 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. however. KG. H. This has been done by using an intercalating agent.PCR is also used for the quantitation of H. the exponential nature of DNA amplification is prone to burden the experimental data with significant standard error due to the tube-to-tube inherent variations also known as tube effect in amplification efficiency. H. pylori is very important to distinguish between colonization and virulence factors. The principle of this method is quantification of the amplicons formed in real time.. The colonization factors are those that are necessary for the survival of H.e. However. However. [59]. Several different characteristics and products of H. quantity of H. DUDWELLER LANDSTR.g. SYBR green I dye. pylori in a quick and precise manner but also its quantification and the detection of point mutations associated with antibiotic resistance. Therefore it necessitates the introduction of a variable amount of internal standard (PCR mimic) of different sizes for the reliable quantitation of DNA or RNA templates by PCR and co-amplification of a constant amount of target DNA sequence with the same set of primers. e. 5. they also used this method for post-eradicated patient follow-up with a high predictive value [59]. pylori in the human stomach at the very first step/acute infection when the H. The validation of this method i. (d) PCR for quantification . a detection by colorimetric method is also possible. or the fluorescence resonance energy transfer (FRET) principle [57]. pylori initially by using a competitive PCR assay.. The standard curve is constructed on the basis of band intensity as well as colorimetric data of internal standard templates (ordinate) against the log of the ratios of the PCR products amplified from the known amounts of wild-type template and standards (abscissa).Dhingra.

pylori is to enter the stomach and survive in the acidic milieu. pylori is well equipped to survive in strong acid but it is not an acidophile and needs to leave the gastric lumen. pylori colonization factors (a) Urease . Ottemann and Lowenthal [64] showed that motility of bacterium is required for colonization of H. H. Most probably protection of the flagella from acidinduced damage is by the flagellar sheath. H. (b) Flagella . pylori chromosome [63]. UreI. This enzyme is a 550 kDa protein complex. These consist of two structural subunits: a major 53 kDa FlaA and a minor 54 kDa FlaB encoded by genes located at the H. H. et al. pylori is equipped with the urease enzyme and expression of this enzyme is a universal feature among all H. pylori strain which has non-motile flagella was less virulent in comparison to its wild type strain in the infectious dose. which is an extension of the outer membrane. pylori strains.Motility is also important for colonization. (i) H. allowing the bacterium a precise level of control over its pH environment. DUDWELLER LANDSTR. The ammonia increases the pH in periplasmic space and protects the bacteria from the acidic environment. but is also important for persistence of the H. KG.Dhingra. which neutralizes gastric acidity [10]. which is open at low pH and closed at neutral pH conditions. Movement of flagella is guided by chemotactic factors. GERMANY . which may help the pathogen to move freely in the mucus layer. 2011 Current Topics in Biotechnology & Microbiology enters the stomach. A urease mutant was shown to be deficient in colonizing gnotobiotic piglets as compared to the wild type [61] emphasizing the importance of this enzyme for survival and successful colonization. pylori such as toxins and factors causing tissue inflammation in the host. which aid in persistence of the infection of H. pylori has a unipolar set of 4-6 flagella which makes this bacterium highly motile [62]. Motility is not only essential for bacteria to escape the acidic surrounding and reach the gastric mucus. H. It has been demonstrated in mouse animal model that mutant H. H. also to avoid discharge of the urease in the intestine gastric epithelium as it needs neutral pH environment. H. the cytosolic urease enzyme hydrolyses urea into ammonia and carbon dioxide. pylori in gastric mucosa of mice. pylori has been shown to possess the enzymatic ability to disrupt the oligomeric structure of mucin. pylori uses the polar flagella so that it can encounter the surface of the gastric epithelium. which include bicarbonate ions and urea. The virulence factors are those. pylori 250 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. To colonize the stomach. Activity of urease enzyme is tightly controlled by a pH-gated urea channel.The first and most crucial requirement for the pathogen such as H. to move through the viscous gastric mucus layer.

(c) Adhesion . However. approaches the epithelial lining and adheres to the Lewis b antigen on the surface of the epithelial cells. Bacterial strains which possess the babA2 gene adhere more tightly to epithelial cells and promote a more aggressive phenotype. pylori to Lewis b (α-1. The mucus layer always secretes mucus toward the lumen for protecting gastric epithelium from acid since motility is required for movement of the bacteria into the newly formed mucus.A final step in colonization of the stomach is the adhesion of H. Adhesion of the bacteria to the epithelial layer of host is mediated by a large family of 32 related outer membrane proteins (Hop proteins) that include the adhesions [65].Dhingra. pylori can bind to a variety of different receptors on the host tissue and possibly adopts the properties of adhesin. 66]. which encode homologous proteins. the frequency of incidence of peptic ulcer is more in Lewis b antigen secretor than non-secretor individuals. H. KG. Only the babA2 gene product is necessary for Lewis b binding activity [66]. have polymorphic mid region sequences but have conserved sequences in the 5’ and 3’ regions [10. Three bab alleles namely babA1. babA1 and babA2 are identical alleles except that babA1 has a 10-bp deletion of the signal peptide sequence that leads to the elimination of the translational initiation codon. where it has been reported that the incidence of peptic ulcer is higher among individuals of blood group ‘O’ phenotype. BabA is a 75-kDa adhesion protein molecule that mediates the attachment of H. The relation between development of gastrointestinal disease and the blood groups have been demonstrated half a century ago. and babB have been identified [66]. adhesion and interaction with host cells induce inflammation and finally sialylation of tissue cells takes place. The bacteria also adapt to the new environmental conditions in the tissue and express a new adhesin allowing it to bind to sialylated structures for 251 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. It is prerequisite that the bacteria strongly adhere to the gastric tissue to accomplish colonization against the peristalsis movement of the gastric wall combined with the fast turnover rate of the epithelial cells. et al. Moreover. pylori to the gastric surface epithelium. It has been reported that BabA2 is associated with a higher incidence of gastric adenocarcinoma. 2011 Current Topics in Biotechnology & Microbiology infection. The babA2 and babB alleles. DUDWELLER LANDSTR. GERMANY . H. 3/4-difucosylated) blood group antigens on human gastric epithelial cells. When H. babA2. One of the most common epitopes on the surface of gastro-intestinal epithelium and expressed in soluble glycol-conjugates in saliva and tears of 80% of the population is the histo-blood group antigen Lewis b. pylori colonizing the mucus. Based on the conditions of the host tissue.

CagA localizes to the inner surface of plasma membrane and undergoes tyrosine phosphorylation by several members of the SRC family of kinases (SFK) such as SRC. Clinically.Several pathogenic factors contribute to the outcome of disease. It is presumed that cagA gene was most likely incorporated into the H. and express a functional vacuolating cytoxin A. H.Dhingra. Tyrosine phosphorylation of EPIYA motif of CagA by SFK occurs in the absence of any stimuli. FYN. but the most important ones are the virulence factors produced by any given strain. and these genes are associated with chronic gastritis. a 40 kb DNA segment containing 31 putative genes. pylori virulence factors . 70]. a 120–145-kDa protein with a carboxyterminal variable region [68. The cagA gene is localized at one end of the cag pathogenicity island (cag PAI). On the basis of the cagA and vacA genotypes. pylori have been defined.Glu-Pro-Ile-Tyr-Alu (EPIYA) motif that is present in multiple numbers in the carboxy-terminal variable region of the protein [74]. The cag PAI -DNA segment is comparable to encoding components of a bacterial type IV secretion system. 69] is the most important pathogenic factor of H. pylori strains are associated with higher grades of gastric inflammation and severe atrophic gastritis and has been suggested to play an important role in the development of gastric carcinoma [71]. infection with the cagA-positive H. two types of H. 73]. (a) Cytotoxin-associated gene A (cagA) . et al. vacA. and do not produce functional VacA cytotoxin. Once injected into the gastric epithelial cells by the type IV secretion system. cagA.CagA. 2011 Current Topics in Biotechnology & Microbiology further enhanced adhesion [67]. pylori that was first described as a virulence factor associated with peptic ulcers [68. Tyrosine phosphorylation of CagA occurs at a unique 5-amino-acid sequence. Type II strains do not contain the cagA gene. Type I strains contain cytotoxin-associated gene A. the resulting SFK are constitutively activated in gastric epithelial cells. LYN and YES [72. KG. On the basis of 252 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Type II strains are generally not associated with gastric pathology [37]. contributing to cell transformation. Depending on the presence of virulence genes of the type IV secretion system. which is a molecular syringe through which macromolecules are delivered from inside of the bacteria to the eukaryotic cells. peptic ulcers and gastric carcinoma. DUDWELLER LANDSTR. GERMANY . pylori genome by a process of horizontal transfer. The above interaction indicates that the CagA might disrupt key signaltransduction pathways. Such precise and programmed adaptations increase the “phenotypic fitness” of H. (ii) H. pylori towards successful persistent infection. translocation of CagA into host cells occurs.

SHP-2 also dephosphorylates a cellular substrate and decreases the level of tyrosine phosphorylated cortactine. (b) Vacuolating cytotoxin A (vacA) . which is involved in the induction of hummingbird phenotype that is associated with elevated cell mortality. followed by the EPIYA-C site (‘A-B-C’ type EPIYA). The EPIYA-D site of East-Asian CagA represents the major tyrosine phosphorylation site.e. The EPIYA-C segment. CagA proteins of H. et al. each contains a single EPIYA motif which surrounds the amino-acid sequence. 253 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. which is placed within a 32. EPIYA motif is part of four distinct EPIYA sites i. a 34 amino acid stretch is mostly repeated one to three times in different Western CagA species as a result of homozygous recombination or misaligned replication of a 102 bp cagA gene segment. 73]. EPIYA–B and EPIYA-D site (‘A-B-D’ type EPIYA) and lack repeatable EPIYA-C segment. possess EPIYA-A and EPIYA-B sites. KG. 40 and 34 amino acids respectively. interference with cytoskeleton dependent cell function. This protein plays an important role in the pathogenesis of both peptic ulceration and gastric cancer [10].. The CagA proteins from H. pylori and causes cytoplasmic vacuolization in gastric epithelial cells. The effect of the toxin was first observed on monolayer cell culture of eukaryote when vacuolization was induced by a cell-free supernatant of H. pylori strains isolated in East-Asian countries such as Japan. North America and Australia. effects on integrine receptor-induced cell signalling. EPIYA-A. DUDWELLER LANDSTR. The physical complex formed between SHP-2 and CagA is detectable in cell infected with CagA positive H. pylori in vitro as well as in vivo. Korea and China are known as ‘EastAsian CagA’. CagA acquires the ability to bind specially to cytoplasmic protein (SRC homology 2) SHP-2 domain containing protein tyrosine phosphatase (PTP) called SHP-2. The activities of VacA toxin include membrane channel formation.Dhingra. The CagA-activated SHP-2 potentiates ERK MAP kinase activity and regulates both cell morphology and cell growth. 2011 Current Topics in Biotechnology & Microbiology flanking sequence of EPIYA motif. B C and D. H. VacA is a major virulence factor of H. SHP-2 has two tandemly repeated SH2 domain termed as N-SH2 and C-SH2 protein tyrosine phosphatase domain. GERMANY . Once tyrosine phosphorylation by SFK takes place. disruption of endosomal and lysosomal activity.Vacuolating cytotoxin. and possess the EPIYA-A. pylori strains of Western countries (Western CagA) such as Europe. pylori culture. whereas those present within the EPIYA-A and EPIYA-B segment are also phosphorylated at tyrosine residues but less frequently than EPIYA-C segment [74. The tyrosine residue of EPIYA-C segment is the major site of tyrosine phosphorylation by SFK in the gastric epithelial cells in Western CagA.

H. 2011 Current Topics in Biotechnology & Microbiology induction of apoptosis and immune modulation [10]. and occurs as either an s1 or s2 type. These toxin clusters are transferred to the host cell surface and exert their toxic action when bacteria contact with host cells. Vacuolating activity is high in s1/m1 genotype than s1/m2 genotype and there is no activity in s2/m2 genotype. whereas the C terminal known as m contains the p58 cell binding domain and exists as an m1 or m2 type. KG. the intermediate (i) region. Using the same technique several researchers have subsequently substantiated a high level of diversity in a number of isolates world wide [6]. GERMANY . The involvement of specific receptors that mediate the bacterium-cell contact [77] has been suggested by this contact–dependent direct delivery mechanism. In the mature toxin. The VacA protein is produced as a 140 kDa protoxin that is cleaved into the 88-kDa mature form when secreted into the extracellular space as soluble protein. VacA has a role in membrane channel formation in epithelial cell and is required for VacA-induced cell vacuolation thereby inducing the release of urea and anion from the host cells. DUDWELLER LANDSTR. there is considerable variation in vacuolating activities among strains. but can also remain localized on the surface of H. pylori secrete VacA toxin. The N terminal known as s of the gene encodes the signal peptide. pylori and is not released into the environment [77]. 6. 22 and 26) [75] are contained in this region that is characteristic of transmembrane dimerization sequences. Recently. A chromosomal gene of H. pylori encodes the VacA responsible for the above effects. Transcellular permeability is also increased which leads to the release of nutrients and cation. pylori genome. pylori [75]. 18. This is due to the sequence variability within the signal region (s) and the middle region (m) of vacA gene [49]. The variability between strains was initially reported by the restriction endonuclease digest analysis of genomic DNA. a significant part of toxin remains associated with the outer membrane of H. Three tandem GXXXG motifs (defined by glycine residues at positions 14. Although nearly all H. A 254 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. the p33 domain near the N-terminus of VacA contains only one strongly hydrophobic region. Interestingly. et al. displays an unusual degree of diversity.Dhingra. a new vacA polymorphic site. Due to high activity in s1/m1 genotype it is more frequently associated with peptic ulceration and gastric cancer. has been reported where vacuolation assays showed that i-type determined vacuolating activity among s1/m2 strains [76]. DIVERSITY OF GENOME Compared to other bacterial species H.

uptake of DNA by natural transformation or silent mutations. H. 2011 Current Topics in Biotechnology & Microbiology high degree of diversity between strains of the H. Till date no studies have been made for monitoring the evolution of a H. base composition and phenotype appear to be conserved. Data obtained have clearly indicated the apparent colonization by their own particular genomic variant of H. This occurred between and after two different treatment regimens. CONTRIBUTION OF HOST GENETICS It has been reported that not only the characteristics of the pathogen and environmental factors but also host genetics play an important role in determining susceptibility and severity of H.Dhingra. GERMANY . The stresses associated with colonization and adaptation to a new environment may be responsible for diversity [78]. A high rate of genetic variation due to mutation such as ‘silent’ point mutation may be the mechanism that enables persistent infection of H. However. It is speculated that the most relevant candidate genes would be ones whose products were involved in handling the H pylori infection and resulting inflammation. pylori in most individuals. pylori for many years in a hostile environment. pylori strain over a period of several years in situ in the gastric mucosa. The degree of change observed within almost every part of the genome so far analysed cannot be attributed to a single factor and yet the genome size. Laboratory maintained strains show evidence on the stability of the genome even on being subjected to the stress of lyophilization and frequent sub-culturing under laboratory conditions. DUDWELLER LANDSTR. These candidate genes would be 255 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 7.and anti-inflammatory cytokines regulate this inflammation. Prominent among these are profiling of rRNA gene [6. pylori strains remain unexplained. pylori has also been confirmed by analyses of the genome by a variety of other molecular techniques. It has been demonstrated that strains undergo genome rearrangements after they infect a new human host however. which is combined with a high rate of recombination events. et al. It is known that an array of pro. pylori infection. the reasons for the genetic diversity among H. KG. Genetic polymorphisms directly influence inter individual variation in the magnitude of cytokine response. A number of factors may be involved in this variation such as the movement of short repetitive DNA sequences. overall inter-strain DNA sequence homologies. 52]. evidence suggests that the same genotype was maintained in one strain in an individual who had been subjected to endoscopy on four separate occasions over a period of 9 month. fingerprinting based on polymerase chain reaction (PCR) and macro restriction digest profiling by pulsed field gel electrophoresis [78]. the expression levels of gene products by generation or deletion of transcription factor sites or by affecting RNA splicing and subsequent translation.

pylori is not directly affected by these mutations. et al. This phenotypic subject has high antral inflammation.” and is the most serious phenotype which is characterized by a multifocal gastric atrophy. GERMANY . Interleukin 1B (IL-1ß) fits this profile perfectly because it is not only most important proinflammatory cytokines in the context of H pylori infection but also the most powerful acid inhibitor so far known [81]. 256 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. DUDWELLER LANDSTR. an endogenous agent is up regulated and has a profound proinflammatory effect and is also acting as an acid inhibitor.The expression levels of gene products directly affect genetic polymorphisms by addition or deletion of transcription factor sites or by affecting RNA splicing and subsequent translation. gastric acid secretion. is known as “simple or benign gastritis” phenotype and characterized by mild pangastritis with little disruption of gastric acid secretion [79]. (i) Role of IL-1 gene cluster polymorphisms in H. Secondly the effects of mutations in genes encoding antioxidative proteins such as glutathione S-transferase (GST) have also been reported [83]. This phenotype is observed in subjects who have no symptoms (asymptomatic) and they develop no serious gastrointestinal (GI) disease. The second phenotype is duodenal ulcer and this phenotype is characterized by an antral-predominant pattern of gastritis with relatively limited acid producing corpus mucosa.Dhingra. Thus. H. pylori infection [84]. However. which affects the metabolism of protein pump inhibitors (PPIs) and antibiotics thus affecting the efficacy of eradication treatment of H. the initial search focuses on genes that are most relevant to physiology of gastric and. pylori-induced gastric damage . colonization of H. Previous studies laid stress on the role of certain mutations in the gene that encodes cytochrome P450. pylori. in particular. The metabolism of certain compounds is directly influenced or the expression of immune mediators downstream of the gene is affected indirectly with specific polymorphism. The complex interplay of proinflammatory and anti-inflammatory mediators control and maintain the chronic active inflammation which is responsible for the pathogenic effects of H. corpus-predominant pattern of gastritis and achlorhydria or hypochlorhydria [81]. KG. and each is associated with a set of pathophysiologic abnormalities. The most common phenotype by far. very high acid secretion and relatively healthy corpus mucosa. all these pathophysiologic changes lead to development of peptic ulcer [80]. 2011 Current Topics in Biotechnology & Microbiology prohibitively extensive. Interesting part of above observation is that subjects who develop duodenal ulcers are protected from gastric cancer development. Three main gastric phenotypes have been observed. high gastrin. it is clear that in the presence of H pylori. suggesting that the 2 outcomes are mutually exclusive [82]. The third phenotype is the “gastric cancer phenotype.

2011 Current Topics in Biotechnology & Microbiology Independent studies conducted in recent years have shown the role of H. and IL-1RN 2/2. the IL-10 gene haplotypes affect the expression of the anti-inflammatory cytokine IL-10. Thus the association of TNF-A*308A genotype with H. Overall. a master regulator of pro-inflammatory cytokines and anti-apoptotic signalling molecules.89].89]. and increased risk of gastric cancer [85. DUDWELLER LANDSTR. Higher expression level of IL-10 is related to the GCC haplotype and hence gives rise to antiinflammatory response. This eventually contributes to decline in acid production. 89]. KG. whereas increased risk of gastric cancer is related to the ATA haplotype [86. 86. such as IL-1B31C/C. A gene cluster which contains the polymorphic IL-1B (encoding the IL-1β cytokine) and IL-RN (encoding the IL-1 receptor antagonist) genes encode the IL-1 cytokine. Several independent studies have established that vulnerability to development of gastric cancer is increased only two-to three fold by single polymorphisms while the presence of multiple proinflammatory genotypes increases it substantially [86. and TNF-A gene shows several polymorphisms. Eventually the disease outcome is jointly contributed to factors like the interaction between the different pro257 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. NFκB. which is related to corpus-predominant colonization by H. pylori. (ii) Other cytokines . pylori strains.89]. 81. TNF-β is a proinflammatory cytokine. as much depends on the vulnerability to H. GERMANY .Dhingra. is one of the most well-studied transcription factors activated by H. IL-1B-511T/T. The most powerful known inhibitor of acid secretion is IL-1 β which is also a powerful proinflammatory cytokine. the increased risk of development of gastric cancer cannot be attributed to a specific polymorphism.Increased TNF-α production. pylori infection [90]. Polymorphisms in other inflammation-associated genes e.g.. pylori gives rise to infection and increases the risk of gastric cancer [86. et al. pylori in gastric disorders and the genetic polymorphisms that affect the expression levels of these inflammatory mediators. The GCC haplotype favours colonization with more-virulent H. influences the production of gastrin and consequentially affects acid production by gastric parietal cells [88]. Thus it can be concluded that IL-1 gene is related to increased risk of developing gastric cancer which is caused by proinflammatory genetic polymorphisms. pylori. resulting the formation of atrophic gastritis. 87]. High-level expression of IL-1 β is enabled by the IL-1 gene cluster that contains several polymorphisms. the genes encoding tumour necrosis factor alpha (TNF-α) and IL-10 show similar effects. Likewise. is associated with the TNF-A* 308A genotype which along with IL-1. whereas decreased level of IL-10 is related to the ATA haplotype which favours a shift toward a proinflammatory response [86. pangastritis. H.

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Regardless of the prevalent use of AgNPs. et al. more and more apprehensions have been raised regarding their potential toxicity and jeopardy to the environment.. 10-9 m or one-billionth of a meter) size range.3 Madhu Yashpal. cosmetics etc.mail: madhuyashpal@gmail. 2Brigesh Shahare. Assessing the risks of AgNPs in human health as well as environment requires an understanding of their mobility. detergents or antimicrobial coatings. DUDWELLER LANDSTR. therapeutics. H. relatively few studies have been carried out to determine the detrimental consequences of AgNPs exposure.2. toxicity INTRODUCTION Nanotechnology. are being used increasingly as vehicles in drug delivery systems. With the strong growth in commercial applications. KG. use or disposal and thus. E. biosensors. characterization and application of molecules in the nanoscale (i. envisaging the whole of the Encyclopedia Britannica written on the head of a pin and foreseeing increasing ability to examine and control the matter at nanoscale. wound dressing. ecotoxicity and persistency. 268 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. is the science involved in the design.. Banaras Hindu University. Varanasi Corresponding Author: Madhu Yashpal. it is most likely that AgNPs enter the environment in the course of their production. are rapidly becoming a part of our daily life in different forms. and impacts of AgNPs on the environment in order to generate information appropriate to attempt a risk assessment. The conceptual keystone of nanotechnology arose in 1959 by the physicist Richard Feynman [2] in his lecture. exposure. synthesis. [1].e. 2011 Current Topics in Biotechnology & Microbiology Chapter XIII Toxicity of Silver Nanoparticles: The Flip Side of the Coin 1 1. Department of Anatomy. silver.com ABSTRACT: Silver nanoparticles. 3Gajendra Singh Electron Microscope Facility. Keywords: Nanoparticles. He explored the likelihood of maneuvering material at the scale of individual atoms and molecules. GERMANY . Institute of Medical Sciences. reactivity. food packaging. which have recently been found to be established antimicrobial agent.Dhingra. “There’s plenty of room at the bottom”. The chapter reflects on the sources.

magnetics. a naturally occurring precious metal. With the escalating charisma of NPs in a broad array of commercial products. mechanical. by definition. et al. 2011 Current Topics in Biotechnology & Microbiology The dramatic expansion of this emerging multidisciplinary science has led to the potential application of nanopartilces (NPs) in various fields e.g. Owing to the specific physiochemical properties of NPs. Because little is known about the toxicity profile of NPs.16].5 billion and because of their widespread application. clothes. Also. known to be used in a wide variety of applications. Compared to their bulk materials. the use of NPs raise concerns about possible detrimental effects. magnetic. building materials and as a disinfectant for water and human wounds [10]. NPs are proven to be the quintessence of the nanoscience. nanoelectronics. NPs. ornaments. it is estimated that of all the NPs in consumer products. more and more apprehensions have been raised regarding their potential toxicity and jeopardy to the environment [7]. Silver. bone cement or wound dressings [13. 269 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. including elevated reactivity due to a large surface to volume ratio [8]. money (coins). silver nanoparticles (AgNPs) have received substantial recognition due to their attractive broad continuum of antibacterial activity [11]. DUDWELLER LANDSTR. fabrics. electrical. KG. catalysis. Moreover. neurosurgical catheters. especially in biological and medical applications. are the structures that have dimension in the 1-100nm range [4].14]. At present. Ancient civilizations used this precious metal in medicine. the estimated global market was worth US $ 10. pharmaceutics. aerospace engineering. no benchmarks or ‘‘safe’’ levels have been set for the concern of human health. GERMANY . has some special properties like high electrical and thermal conductivity [9]. In 2006. photonics. H. Over the last decade. inadequate availability of toxicological information on NPs renders people to endure a high risk of using these novel materials. cosmetics. AgNPs applications have the highest degree of commercialization [12]. due to their special chemical.Dhingra. central venous catheters. home appliances to water treatment [15. the characteristic plasmon-resonance optical scattering properties of AgNPs make them available to be used in bio-sensing [17] and imaging applications [18]. AgNPs are deployed in a wide range of medical and consumer products ranging from cardiovascular implants. the commercial nanotechnology industry is expected to increase significantly to $3 trillion by 2015 [6]. eating utensils. environmental remediation and medicines [3].. and optical properties [5].

the skin and the female genital tract [13]. shape. inhalation into the respiratory tract. comprehensive biological and toxicological information is lacking. exposure to AgNPs is becoming more and more complex. surface 270 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. In this chapter. immunological system. the precise toxicity mechanisms are still ambiguous. exhibit the greatest toxic effects [31-34]. Ji et al. concentration. we endeavor to emphasize on the adverse effects of the AgNPs. it is well known that these versatile particles cause detrimental effects by their oxidative and inflammatory [26] nature. Further escape of these NPs into the effluent pollute the aquatic and soil environment results in direct exposure to humans via skin contact. EXPOSURE ROUTES OF SILVER NANOPARTICLES In this booming era of nanotechnology. H. 2011 Current Topics in Biotechnology & Microbiology Despite the upcoming prevalent use of AgNPs. GERMANY .Dhingra. in general.21].22-25]. accidental or involuntary contact of AgNPs is most likely to occur via the lungs. and blood vessel walls [20. liver. subsequently inducing genotoxic [27] and cytotoxic [28] outcomes. et al. In addition.g. KG. consequential in an escalating entrée to tissues. mammalian. exposure and allied jeopardy to human and environmental health have not been explored systematically. inhalation of aerosols. olfactory bulb. kidney. DUDWELLER LANDSTR. MECHANISM OF SILVER NANOPARTICLE INDUCED TOXICITY Copious research studies have examined the toxicity of AgNPs on an array of organisms. and human cells [17. However. The inhaled AgNPs can reach systemic circulation in the body and thus can be disseminated to a number of different target organs including brain. [20] proposed three feasible routes of their translocation to the blood – from tracheobronchial region by a mucociliary escalator. into lymph nodes & lymphatics and via alveolar epithelial cells to the circulatory system. cells and biological molecules within the body through various portals e. the gastrointestinal tract (GIT). contaminated drinking water or indirect exposure from ingestion of sea food such as fish. Even though. In addition. The aim is to give an overview and scrutinize the various toxic effects of AgNPs. with subsequent ingestion in to the alimentary tract. Toxic effects of AgNP possibly stems from their small size [29] as the tiny size of the NPs makes them highly motile both in the body and the environment [30] and smaller particles. It has been revealed that AgNPs used in our household and industrial merchandises find its way through the waste disposal routes into the wastewater treatment facilities and end up in wastewater sludge [19].

stimulation of reactive oxygen species (ROS) via a reaction with oxygen [46.25.. can educe a spectrum of impacts on tissues and the novel properties. in a variety of cells. Inside a cell.47] that damages cellular constituents causing disruption of cell functions and interaction with specific proteins and/or enzymes inhibiting their activities [48. these NPs can get metabolized and/or altered and go through a series of processes like binding and reacting with proteins. deposited. there is an inadequate understanding into the prejudicial upshots of AgNP exposure. H. GERMANY . most likely through decreasing cell viability and/or increasing apoptosis. KG. oxidative.Dhingra.63]. cytotoxic and genotoxic responses. cleared or translocated ensuing inflammatory. IMPACTS OF SILVER NANOPARTICLES ON BIOLOGICAL SYSTEMS AgNPs. MLO-Y4 osteocytic [63].49]. et al. In addition. NPs are distinguished by a higher surface reactivity and can produce comparatively more ROS than larger particles [51. and cytotoxic effects.52]. In contrast to their micro-sized counterparts. mouse peritoneal macrophage RAW264. 53-60]. could prove to be deleterious and contribute to their toxicological profile. and stimulating inflammation. Cytotoxicity Studies reveal that AgNPs exert perilous cytotoxic effects. However. 2011 Current Topics in Biotechnology & Microbiology chemistry. whether inside or outside of the cell has been considered as a key factor resulting in toxicity induced by NPs [50].7 [60]. human lung carcinoma cell line. including peripheral blood mononuclear cells [61] and human cervical cancer HeLa [62. when in contact with biological systems. which make them so attractive in numerous applications. eventually culminating in cell death [36. Augmentation of ROS generation triggers the apoptotic cascade by activating oxidant sensitive signaling pathways. other imperative factors accountable for AgNP toxicity include association of AgNPs with cell membranes that might cause physical damage (e. A549 [64] and Drosophila melanogaster [65] cell lines. genotoxic. human acute monocytic leukemia THP-1 [64]. capping agent and propensity to aggregate are also important factors that may influence the AgNP induced toxicity [35-37].38-40]. 271 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. get phagocytosed. as they move into the cells leading to the production of superoxide radicals [41] and disruption in the ion efflux system [42]. pitting) or the subsequent penetration resulting in cell malfunction including cell wall destruction [43-45]. Another possible means for the AgNPs to cause toxicity is the production of silver ions (Ag+) [24. The production of ROS.g.37. DUDWELLER LANDSTR.

[68] reported that AgNPs induce cytotoxicity in mouse’s fibroblast cells (L929) with manifestation of severe morphological deformities. and increased gene expression of matrix metalloproteinases (MMP-3. MMP-11. et al. [60] reported cytotoxicity of AgNPs to mouse peritoneal macrophages derived RAW264. increased NO secretion. H. increased TNF-α in protein and gene levels. a tissue explants culture model and mouse expurgated wound model [66]. DUDWELLER LANDSTR. Hackenberg et al. and MMP-19). human hepatoma derived cell line HepG2 [23] and in mouse embryonic stem cells and fibroblasts [12].7 cell lines. More recently. Wei et al. the cytotoxic effects of AgNPs are reported to pilot the impairment of the developmental potential of embryo. Moreover. Using mouse blastocysts as the assay model. [70] reported AgNPs induced inhibitory effect on tissue regeneration by suppressing stem cell migration in mesenchymal stem cells derived from human adipose tissue at high exposure concentrations. More recently. few studies as well revealed that AgNPs treatment induced DNA damaging effects on aquatic [59.71. AgNPs decreased intracellular glutathione level. In addition. Recent studies have reported that the AgNPs result in DNA damages such as double strand breaks and disrepair of strand breaks in DNA. resulting in chromosome reorganization and chromosomal aberrations thus disturbing genomic stability and integrity. These NPs were found to induce genotoxic effects in human glioblastoma and fibroblast cells [27]. disturbed metaphases and multiple chromosome breaks. Despite increasing use of NPs. very little has been done with respect to examine the genotoxicological safety of AgNPs.72] and plant [73. GERMANY . genotoxicity being an imperative endpoint to be tested. 272 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. AgNPs were also found to be notably cytotoxic on different cultures such as in a monolayer cell culture. They described that AgNPs were ionized in the cells to cause cytotoxicity by a Trojan-horse type mechanism by increasing sub G1 fraction indicating cellular apoptosis. Li et al. [67] reported that AgNPs repress embryonic cell proliferation during the blastocyst stage predominantly via inducing apoptosis in the inner cell mass and trophectoderm.74] cells with impairment of cell-division and causing different effects such as chromatin bridges. Park et al. AgNPs are reported to exhibit its genotoxic activity mainly via induction of somatic recombination in the wing imaginal disk cells of Drosophila melanogaster [69]. Genotoxicity Health risk assessment of NPs is a sprouting field. 2011 Current Topics in Biotechnology & Microbiology Moreover. stickiness. KG.Dhingra.

et al.68].33.Dhingra. The key event in NP induced cytotoxicity is the mitochondrial perturbation that has significant biological effects together with the commencement of apoptosis and diminished ATP production [92. Pulmonary Toxicity 273 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.78]. H. However. DUDWELLER LANDSTR.101] and thus apoptosis. which in turn cause DNA damage [27. which may pose adverse effects [83. AgNPs induced ROS generation cause release of cytochrome c into the cytosol of mitochondria [33. Mitochondrial toxicity: The foremost toxicological apprehension is the fact that some manufactured NPs are transported across cell membranes. followed by axonal translocation to CNS structures.91].99].31. as it has been reported that NPs exposure can induce impairments of normal neurons [85].76] and pass through biological membranes. NPs could progress inside the brain and induce detrimental effects [3.97] consequential in an uncoupling effect on the oxidative phosphorylation system [98]. Several reports have demonstrated a clear deleterious effect of AgNPs on mitochondrial function in the liver.95].94. In addition. Another possibility is that these NPs gain access to mitochondria because of their small sizes or ROS generated at its exterior and might then release redox cycling chemicals that damage the inner membrane [91] or by disrupting the mitochondrial respiratory chain.77-79]. ultimately resulting in the production of ROS and disruption of ATP synthesis. AgNPs can induce alterations in mitochondrial membrane permeability [96. NPs can also access the brain by sensory nerve endings embedded either in airway epithelia [80] or in olfactory bulb [3.89]. 2011 Current Topics in Biotechnology & Microbiology Neurotoxicity Owing to their ability to cross the blood brain barrier (BBB) via systemic distribution [10. AgNPs were found to be toxic by ensuing cell shrinkage and irregular membrane borders and reducing the level of dopamine in a neuro-endocrine cell line (PC-12 cells) [88. how exactly AgNPs gain access to and induce mitochondrial damage is unknown. Recently.84].93]. AgNPs entering via the BBB can accrue in different regions of the brain [82].75. lung. microglia [86] and even aggravate the process of brain pathology [87]. especially into mitochondria [90. KG. GERMANY . NPs may also be taken up directly into the brain by trans-synaptic transport [81]. which commences a cascade of signaling mechanisms that leads to the activation of caspase 3 and caspase 9 [100. nervous system and kidney [12.

following pulmonary exposure. AgNPs are reported to pervade through 274 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Dermal toxicity: The potential of NPs to breach the skin and to diffuse into underlying structures raises a considerable health and safety issue for their topical use. AgNP can induce toxicity to germ line stem cells via reduction in mitochondrial function and induction of membrane leakage and apoptosis [113]. et al. they are quickly taken up by alveolar cells and are likely to induce oxidative stress. and translocate to sensitive organs via the blood or lymph [52]. DUDWELLER LANDSTR. be taken up by cells. [113] reported that AgNPs can induce a significant decline in mouse spermatogonial stem cells proliferation. Braydich-Stolle et al. which necessitates their transfer into blood or transport within neurons [36]. Scarcely available studies on inhalation toxicity of AgNPs demonstrate that these particles may translocate from their site of exposure. Both inhalation and instillation experiments have revealed that AgNPs are taken up by alveolar macrophages and persevere there at least for up to 7 days [21]. spleen. and brain.105.106]. Moreover. 2011 Current Topics in Biotechnology & Microbiology The NPs are allied with adverse pulmonary and cardiovascular effects following inhalation in humans [102. and the underlying mechanisms causing pulmonary toxicity [104]. and get deposited and bioaccumulated in gonads [108-111]. AgNPs can also induce DNA damage response in embryonic stem cells and embryonic fibroblasts [12]. GERMANY . Review of literature has shown that NPs can translocate systemically from primary sites of access across the blood testes barrier. including the liver. Prolonged exposure to AgNPs is reported to elicit an inflammatory response within the alveoli and induced alterations in lung function [20. In aquatic species it has been shown that NPs can translocate in the testes after gill absorption of contaminated water [112].Dhingra. Once NPs enter the interstitial air spaces of the lungs. there is a lack of information regarding the adverse consequences associated with pulmonary exposure to AgNPs. Unfortunately. Reproductive toxicity: Despite the fact that the NPs might pose potential reproductive harm. Recently. KG. H.103]. Airborne NPs can deposit in all regions of the respiratory tract. so that they have the potential to accumulate within a number of secondary targets. there has been little substantive evidence to support or refute these concerns. These aggregated AgNPs are reported to be cytotoxic to alveolar macrophage cells as well as epithelial lung cells [107]. DNA damage and inflammation leading to fibrosis and pneumoconiosis.

vascular system and reproductive organs. The preliminary results necessitate further comprehensive studies to clearly 275 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Arora et al. because these are employed in widely peddled products for water disinfection and food stabilisation [123] and “health maintainers” or “immuno-boosters”. it has been demonstrated that AgNPs could enter the fibroblasts and liver cells and cause DNA damage and apoptosis [122]. oxidative stress. CONCLUSION AgNPs have emerged as noteworthy class of NPs for a wide range of industrial and medical applications. KG. including eosinophils [94] and other hepatoxic consequences including lymphocytic infiltration and expression of genes related to apoptosis and inflammation [32].116] have substantially increased the potential for human skin exposure. Despite the promising applications of AgNPs. Commercial wound dressings containing AgNPs are cytotoxic to keratinocytes and fibroblasts [118-120]. fabric quality. Probable mechanisms of AgNPs induced toxicity include generation of ROS. These are reported to induce toxicity in a range of organs including the lung. silver could be released from antibacterial fabric products into “sweat” [117].Dhingra.125] or through the intestinal lymphatic tissue [126]. H. Review of literature reveals that depending on the quantity of silver coating. but it has been demonstrated that the uptake can take place trans-cellularly via normal enterocytes and through paracellular pathways [124. The applications of AgNPs in cosmetics. surgical prosthesis and anti-microbially active “smart textiles" [15. et al. GERMANY . These initial findings assure that AgNPs do pose a certain degree of potential hazards to environmental and human health. DNA damage and apoptosis. there are still doubts regarding their safety. [121] found that AgNP induced cell death and oxidative stress in human fibrosarcoma and skin carcinoma cells. bile duct hyperplasia with infiltration of inflammatory cells. brain. 2011 Current Topics in Biotechnology & Microbiology the intact skin and appreciably through the damaged skin [114] and the intradermal NPs could gain access to systemic distribution through subcutaneous lymphatics [115]. liver. In subsequent studies. topical products. After ingestion. gastrointestinal ingestion is the most common voluntary route of exposure for AgNPs. the kinetic mechanism of AgNP translocation is unclear. Gastrointestinal tract toxicity Probably. DUDWELLER LANDSTR. Oral exposure of AgNPs can also cause noteworthy changes in serum alkaline phosphatase and cholesterol concentration. pH and sweat formulation.

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Ji. S. J.. Schlager.. M. Z. 3. Zhu. T. 105. Wu. Distribution of nanoparticles in the Seethrough Medaka (Oryzia latipes).. B. 2011 Current Topics in Biotechnology & Microbiology 103. Han. J.K. 1697-1702. Toxicological sciences.C. Sodium chloride modified silica nanoparticles as a nonviral vector with a high efficiency of DNA transfer into cells. Kim. Ryu.B. Uptake of PMMA nanoparticles from the gastrointestinal tract after oral administration to rats: Modification of the body distribution after suspension in surfactant solutions and in oil vehicles.. Long... (2006). 1118-1125.J. B. 338-347. Tang. & Yu.. K. B.U. 114. Schrand. H. Hyun.. Cho. 182. GERMANY .114. L. 351-358.. M. M. 109. J.H. Acta Biomaterialia. Toxicological Sciences.Y.. B. L. K. American Journal of Respiratory and Critical Care Medicine.. Lee.. S. Heinrich. Environmental Health Perspectives.. I. H. 452-461. Lee..J. Kim. Y. Soto.. R.... Xia.H. M. Garza. & Heyder.. F. J.. Subchronic inhalation toxicity of silver nanoparticles. Y. J... (2008).Dhingra.. (2006).. L. J.E. 89. Lee. 50. Yoon. & Kreuter J. J.. Jeong. K. K. D.S. 577-589. Park. H. J. Xue. Lee. (2009).R. 273-279. K.. Lucas.H. 155. L. Han. Hussain. Kim.S. 2323-2327. T.. 104. Chen. D..S. Kim. K. Sheppard.. J. Xue. Z. (2010). Yu. Li. H. 24-28. Xia.H. & Val Vallyathan. Yoon. Environmental Health Perspectives. T. et al. D. KG.. & Hofmann. Chung. Z. Sung.J.. S. Liang. S.S.S. H..C. Kashiwada S. Gwinn. Toxicology Letters. 112. M... Lobenberg. Araujo. H. J. 110.H. Effects of repeated silver nanoparticles exposure on the histological structure and mucins of nasal respiratory mucosa in rats. 108. Zheng. Q. Peters. (2006). Jeon.. A. 107.H. J. A. J. Braydich-Stolle. J.. Long. Toxicity and tissue distribution of magnetic nanoparticles in mice. Tuch. Yu. Lui. 209-224.. Y.. Internal Journal of Pharmaceutics.M.. Park.. Cai.K.. B. Murdock.W. & Zia. K. B. 116.. Chinese Science Bulletin. 111.. V. J. DUDWELLER LANDSTR. & Murr..J.S. & Cho. Silver nanoparticles disrupt GDNF/Fyn kinase signaling in spermatogonial stem cells. 3. Dai. M.D. (1999). J. Wichmann.E.N.. Park. (1997). Cytotoxic effects of aggregated nanomaterials. 106.. Current Gene Therapy. Kelman. Zhao. Chung. Lee. (2005). K. (2003). Toxicological sciences. Sung.. I. X.. Pan. Chang.Y.H.H. T. J. 113..J... Y. Silica nanoparticle is a possible safe carrier for gene therapy..M. Liu.. (2007).K..G. 108.H.. 176.H.. Respiratory effects are associated with the number of ultrafine particles. Song. 285 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.J. Z. 1376-1383. Nanoparticles: Health Effects—Pros and Cons. R. & Xia.

R...D. Srisung. (2004). Journal of Pharmacy and Pharmacology. & Burd. J.. G. A. 119. A Study on the bio-safety for nano-silver as anti-bacterial materials. Bovenzia.133-43. Agostin..93-100. & Xu. S. V. Langridge. 125. 31. K. C. K. 179. C. D..T. 61. C. & Liew. Jain. 2011 Current Topics in Biotechnology & Microbiology 114. 821-826. P. Particle and Fibre Toxicology. Human skin penetration of silver nanoparticles through intact and damaged skin. H. (2010). Nanoparticle uptake by the rat gastrointestinal mucosa: quantitation and particle size dependency. W. V. & Paknikar. J. K. Rajwade. W. 7. F.V. Webb. G. Lee. Renzic.. P. Plastic & Reconstructive Surgery. & Howard. Zhang. 121. British Journal of Biomedical Sciences. (2009). Toxicology Letters. et al. Humbert.J. 30... Toxicology and Applied Pharmacology..K. J.S. 110S-118S. In vitro cytotoxity of silver: implication for clinical wound care... Yu.F. (2004)..E.M.. GERMANY . Jani. Chinese Journal of Medical Instrumentation. C.C. 42. Lam... P. Michel. 122. J.M. F. X. J. Arora.. Toxicological Sciences.J. M. (2007). N. 140-147. J. A.M. 124. (1987). Paddle-Ledinek. Cellular responses induced by silver nanoparticles: in vitro studies. Cozart.J. R. (2008). 255. 123. 118.. Toxicology.Dhingra.H.M.8. Jain J. Larese. (2007). 286 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. K.. Migration of intradermally injected quantum dots to sentinel organs in mice. & Maniratanachote. Kangwansupamonkon.K..N. Siitonen. Osgood. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. 33-37 115. Chan. C. Z.L. Ho. & Damge. G. M. Halbert. M. 120.M.. & Paknikar.D. Effect of different wound dressings on cell viability and proliferation. A. W. Kulthong. Burns. Gopee. Roberts. L.. 1. & Mainad. Poon. Arora S. Adamib. In vitro cytotoxicity testing of a nanocrystalline silver dressing (acticoat) on cultured keratinocytes. & Cleland. H.. ACS Nano.. Boonpavanitchakul.W. N. 98. Determination of silver nanoparticle release from antibacterial fabrics into artificial sweat.P. N. 249-257. Rajwade. Browning. In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. 236. Nasa. (2009). Warbritton. & Florence. (2007). 69-76. Aprahamian. J. W. 116. 117. Nallathamby. E.. K.. P.W.. P.. 61. Transmucosal passage of polyalkylcyanoacrylate nanocapsules as a new drug carrier in the small intestine. & Sun.S. 125-127. 35-38. Devissaguet. (2006).W.T. Biology of the Cell.R. S.. KG. Croserab. 310-8. DUDWELLER LANDSTR. (1990). P.. Colvin. Y. 117. Walker.J...

DUDWELLER LANDSTR. 2011 Current Topics in Biotechnology & Microbiology 126. 735-743. P. Experimental Physiology. H....G. Jenkins.. N. Selective transport of microparticles across Peyer’s patch follicle-associated M cells from mice and rats. M. GERMANY . (1995).W. D. et al.W. & Porta. 80. C. Thomas. KG. Cremaschi.Dhingra. N. 287 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Smith.G. Miller.

human genome sequence has been completed and there has been upsurge in newer diagnostic tools. its applications in immunobiology and benefits of the technology are discussed as compared to other analytical tools. Kapre Research and Development Department. Enzyme-linked immunosorbant assays (ELISAs). GERMANY . cytokine profiles.Dhingra.com * ABSTRACT: Analytical assays are one of the important part of biomedical research. PhD In-Charge. The basics of technology. KG. repeatable and cost effective. time consuming and laborious which have been mostly replaced by advanced and better assay systems e. diagnostics and prophylactics. fluorescent antibody assays. Immunobiology and Molecular Research Laboratory Email: manoj. A useful assay should be simple. Pune. Various qualitative and quantitative assays have been developed till date having respective advantages and disadvantages. Keeping in view the huge increase in the demand of diagnostics and the new vaccine development. MVSc. DUDWELLER LANDSTR. there has been major advances in the field of the biomedical science especially biotechnology and microbiology. H. serum antibody titers. the analytical methods have improved significantly during this time. fast. In recent times there is a focus on multiplexing assays due to an extensive increase in the number of analytes being required to be tested from the same sample e. et al.kumar@seruminstitute.g. detection of pathogens.411028 INDIA Corresponding author: Manoj Kumar. INTRODUCTION: During the last couple of decades. The present chapter focuses on suspension array technology for multiple analyte profiling in a single assay with a major focus on immunodiagnostics. various diseases have been targeted for prophylaxis and control. Serum Institute of India Research Foundation 212/2. The conventional assays had disadvantages of being less sensitive.g. gene expressions and single nucleotide polymorphism etc. radio 288 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 2011 Current Topics in Biotechnology & Microbiology Chapter XIV SUSPENSION ARRAY TECHNOLOGY: A MULTIPLEXING APPROACH IN IMMUNODIAGNOSTICS *1 1 Manoj Kumar and 1Subhash V. specific. sensitive. Hadapsar. Maharashtra .

digital signal processing and traditional chemistry that have been combined in a unique way. GERMANY . flow cytometry. specific antibody and secondary antibody or other combination of reactants. The instrument is equipped with two types of lasers. Various chemistries have been used for coupling of the analytes to the carboxylated microspheres [2-8]. each of which is coated with a particular analyte (antigen/antibody/Nucleic acid fragment etc. DUDWELLER LANDSTR. A 635 nm red diode laser excites the two fluorochromes contained within the microspheres 289 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. real time quantitative Polymerase chain reactions (qPCR) assays. Samples from microplate wells are aspirated by a sampling probe and transported to the flow cytometer through a flexible sampling line. In this technique Luminex 100/200 or FLEXMAP-3D system (or similar) controlled by a software is used in conjunction with the Luminex microspheres of 5. An auto sampler moves the probe from well to well while a continuously running peristaltic pump causes the alternating uptake of fluid samples and air.. 2). A rotating cell suspension system periodically rotates microplate to keep sample particles (microspheres) in suspension during any required pre-analysis incubation steps. KG. The technology has several advantages over the conventional and competitor techniques and is described in the following sections. et al.6 or 6.. the most common being an EDC (1-ethyl-3-(3After reaction with the dimethylaminopropyl)-carbodiimide hydrochloride) based reaction. Figure 3). Luminex x-MAP (Suspension array) technology is built on proven existing technologies viz. Various manufacturers have come up with multiplexed bead based flowcytometric assays for applications in diagnostics. The technique started as FlowMetrix System [1] and several advancements have been seen in the technology over several years. A suspension array multiple analyte profiling (MAP) technology developed by Luminex Corporation is one of the emerging technology in this field. red laser and green laser (Figure 4). 2011 Current Topics in Biotechnology & Microbiology immunoassays.Dhingra. H. With the advent of technologies. microspheres which are composed of 100 (500 in case of FLEXMAP-3D) different combinations of 2-3 different fluorochromes (Figure 1. microspheres. biopharmaceutical industry and medical research. lasers.5 micron diameter. these microspheres are made to flow through the needle of the instrument. The technique has been highly useful in dealing with the issues related with conventional assays in terms of speed. economy and sensitivity. several multiplexing assays have been developed. The microspheres flow through the fluidics system to the cytometer where the laser falls on the beads and the emitted fluorescence is measured by the detector. The basic mechanism used is. one of which are microsphere based flowcytometric assays.

GERMANY . Phycoerythrin (PE) emits different wavelengths so there is no chance of overlapping of fluorescence. H. Then the beads are subjected to green laser beam which gives the Median Fluorescence Intensity (MFI) when the emitted florescence from the reporter dye attached to the secondary antibody/primer tag falls on the detector. et al. 2011 Current Topics in Biotechnology & Microbiology and a 532-nm. or Cy3) bound to the microsphere surface. MFI is proportional to concentration of analyte in the sample. High-speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface. Microspheres size. determined by 90-degree light scatter. DUDWELLER LANDSTR. Alexa 532. The red LED (635 nm) excites 290 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. The dyes on the microspheres and reporter dye e.Dhingra. The sample is aspirated into the instrument.g. yttrium aluminum garnet (YAG) laser excites the reporter fluorochrome (Rphycoerythrin. When the red laser falls on the beads it emits a fluorescence which is detected by the detector system and helps to segregate different types of beads. magnetic beads enter magnetic chamber to form a monolayer for imaging. is used to eliminate microsphere aggregates from the analysis. KG. Figure 1: Luminex microspheres (With permission from Luminex Corporation) Latest addition to the series of Luminex instruments is MagPix instrument which provides LED/Image-Based Analysis. The software controls every activities of the hardware [9-12].

In addition. microspheres and computer hardware and software. green LED (525 nm) excites detection fluorophores and measures the expression. Figure 2: Typical Luminex 200 Bead map: Bead Region Classifications showing 100 different identities (With permission from Luminex Corporation) Luminex x-MAP microspheres: The Luminex 100/200 (Luminex Corporation) or similar system performs multiplexed analysis of up to 100 (500 in case of FLEXMAP 3D) different reactions simultaneously by using a flow cytometer and digital signal processor to perform realtime analysis of multiple microsphere-based assays. which is more than sufficient for assay setup and analysis. and also provides near291 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.Dhingra. whereas. GERMANY . The fluorescence is identified and quantified with CCD imager. DUDWELLER LANDSTR. The three major components of the system are a bench-top flow cytometer. Microspheres of this size provide sufficient surface area for covalent coupling of 1–2 x106 target molecules per microsphere. KG. Microspheres serve as the vehicles for molecular reactions. The microspheres are 5. H. the small size allows the microspheres to remain in suspension for several hours.5 µm polystyrene microspheres that bear carboxyl functional groups on the surface. et al. 2011 Current Topics in Biotechnology & Microbiology internal bead dyes and determines which analyte is being measured.6 or 6.

The ability of an assay to measure multiple analytes at the same time has become a major consideration in the analyses where one need to quantify multiple analytes from the same sample e. 292 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. KG.com/applications). GERMANY . The microspheres are available in 100 (500 in case of FLEXMAP 3D) distinct sets that are classified by the flow cytometer by virtue of the unique orange/red emission profile of each set. xTAG Microspheres. A rapid and cost effective assay is always a preferred choice for the diagnostic facility. The carboxylated microspheres can be covalently coupled to virtually any amine-containing “target” molecule through surface carboxyl groups (Figure 3). Figure3: Coupling of an oligonucleotide to Luminex Microspheres (With permission from Luminex Corporation) Advantages of x-MAP technology: In order to be a useful assay. an analytical method should have maximum of the following characteristics. They are MicroPlex microspheres. the less will be the chances to go wrong which is important for clinical diagnostic laboratories. et al. Specificity is very important parameter in most protein-based assays. 2011 Current Topics in Biotechnology & Microbiology fluid-phase reaction kinetics. Various types of microspheres are available based on the application and type of analyte required to be coupled to the microsphere. Assays that eliminate the need for washing steps are particularly advantageous.Dhingra. H. MagPlex Microspheres.g. SeroMAP Microspheres and LumAvidin Microspheres (http://www. The simpler the assay.luminexcorp. Higher sensitivity facilitates earlier diagnosis and helps in reducing the sample requirement. Reliability and reproducibility are of significant importance. DUDWELLER LANDSTR. cytokine estimations.

Favorable reaction kinetics of liquid bead array approach with high dynamic range 4. DUDWELLER LANDSTR. Flexible multiplexing in the range of 1-500 analytes 3. it has been utilized for several application successfully over several years in various fields of biomedical research including drug-discovery. planar assays PMT 532 nm 635 nm Figure 4: Optics of Luminex 200 instrument (Detection of identity of microsphere and fluorescence intensity by red and green laser beams). Smaller sample requirement due to multiplexing 5. GERMANY . (With permission from Luminex Corporation) APPLICATIONS OF x-MAP TECHNOLOGY Keeping in view the various benefits of the x-MAP technology. The details of various major applications are described in the following sections. 293 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. H. et al. Cost and labor is reduced by multiplexing 2. Table 1 shows in brief the applications of the Luminex technology. more reproducible results than with solid. KG. Liquid reaction kinetics gives faster. diagnostics and basic research [8-9.Dhingra. 13-24]. 2011 Current Topics in Biotechnology & Microbiology The x-MAP technology has several benefits over the similar techniques in terms of the following: 1.

Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology

x-MAP IN IMMUNODIAGNOSTICS Various types of immunoassays are required in the vaccine development, its evaluation, biomedical research and diagnostics. Briefly, vaccines are the antigenic preparations, which stimulate body’s immune system to produce antibody against the antigen injected. Vaccination is the administration of a vaccine to stimulate a protective immune response that will prevent occurrence of disease in the vaccinated person, if contact with the corresponding infectious agent occurs. Presently, there are number of vaccines available in the market. We can divide the major vaccine preventable disease in two parts, that are bacterial vaccine preventable disease, for example diphtheria, tetanus, pertussis, pneumonia, meningitis, cholera, influenza, and viral vaccine preventable diseases such as poliomyelitis, influenza, hepatitis-B, rabies, measles, mumps, rubella etc. Few of the vaccines for protozoal, parasitic and mycotic diseases are also developed or are under development. With the increase in the potential vaccine candidates, there is a trend towards development of multivalent vaccines e.g. Trivalent, quadruvalent, Pentavalent and hexavalent etc. Further, there are vaccines against several serotypes/strains of the same pathogen e.g. Bacterial pneumonia, rotaviruses etc. With this approach, multiplexing technique has become an essential requirement for vaccine development and evaluation. The assays can be categorized in to antibody and antigen screening assays. Screening antibodies: Efficacy studies of a vaccine are highly required to evaluate its potency in protecting from the disease. Normally with response to the different antigens/serotypes different type of IgG are formed by the immune system of the affected host. To measure the concentration of IgG, different types of antibody titration methods are available. Among them few of the conventional methods are gel diffusion method, immunoelectrophoresis etc. where diffusion lines are formed due to the antigen (Ag)-antibody (Ab) reaction [12]. RIA (Radioimmunoassay) is also a sensitive method to estimate antibody, in which the antigen labeled with radio isotopes and the precipitation formed by Ag-Ab reaction is estimated by autoradiography but it has potential health hazards. Enzyme Linked Immunosorbant Assay (ELISA) is another method for quantitative estimation of antibody, in which the microtiter plate is coated with Ag followed by reaction with antibodies in serum and a secondary Ab conjugated with an enzyme, and the substrate. The reaction in terms of optical density is then measured with an ELISA reader.


Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology

Conventional Ab titration methods have a lot of disadvantages. 1. Low sensitivity 2. Require large volume of sample 3. Huge time consumption 4. Laborious and tedious method 5. A number of critical steps 6. A number of washing steps 7. If the assay is done for multi-serotype vaccine then it is not cost effective 8. Safety issues Multiplexed immunoassays have been a focus for development and evaluation of multivalent vaccine which help get rid of the disadvantages of the conventional monoplex assays. Using x-MAP technology, multiple immunoassays can be performed simultaneously on an array of microspheres as long as the conditions of the assays (incubation times, sequence of reagent additions, washes) are the same or can be combined. A basic format of IgG estimation assay is presented in the figure 5. The technology has been applied by various authors for development of assays for simultaneous identification and quantitation of antibodies in sera, plasma, culture supernatants etc. A multiplexed indirect immune-fluorescence assay was developed for antibodies to

Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip). The multiplexed Luminex assay was compared to individual ELISAs for the same analytes. The correlations (r2) between ELISA and Luminex assay of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively (Pickering et al., 2002a). A fluorescent covalent microsphere immunoassay (FCMIA) method was

developed for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14 [4].


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Current Topics in Biotechnology & Microbiology

Table 1: Various fields of application of x-MAP technology

Sl. No.

Field of application

Targets (Examples) Detection and diagnosis of Infectious diseases (Bordetella pertussis, M. tuberculosis, Clostridium spp., Epstein Barr viruses, Influenza viruses, Rota viruses, Toxoplasma infections etc.) Vaccine testing (Vaccine efficacy surveillance, immunology research etc.) studies, immunization



Allergy testing (Alternaria, Asp f 1, Bermuda Grass, Bla g 1, Der f 1, mountain cedar, wheat, timothy grass etc.) Autoimmune diseases (Chromatin, Histone, Gliadin, SSA etc.) Human Leukocyte Antigen testing (Donor specific antibody testing, HLA class I or II testing) Newborn screening Gene expression profiling (BCL2, IFN-gamma, IL-10, NKFB1, VEGF genes etc.)


Genomic research

Genotyping (HPV genotyping, Signet genotyping panel etc.) Cystic fibrosis (Testing for various mutations associated with CF) Cytochromes Cytokines, Chemokines and growth factors (ILs, MCPs, MIPs, EGFR, IFNs, TNF, V-CAM etc) Cardiac markers (Adiponectin, Cystatin C, ILs, SGOT, VCAM-1, Fibrinogen etc.)


Protein expression Cancer markers (AFP, CAs, CEA, f-PSA etc.) profiling Isotyping (IgA and IgG isotyping) Metabolic markers (Apolipoproteins, Collagens etc.) Cellular signaling (Akt, CRK, Erk, EGFR etc.) Neurobiology Testing of bio-warfare pathogens and bio-threat agents (Bacillus anthracis, Yersinia pestis etc.)


Biodefence/ Environmental


Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology

A rapid and simple method for the simultaneous quantitation of serum IgG antibodies specific for Neisseria meningitidis serogroups A, C, Y, and W-135 was developed and evaluated. Four bead sets were generated, each conjugated with one of the meningococcal capsular polysaccharides (A, C, Y, or W-135) and serologically assessed by the use of anti-meningococcal international reference sera. Cross-reactivity studies demonstrated no inhibition between monoplex and multiplex assays, and the assay was linear over a 24-fold serum dilution range. Inhibition studies demonstrated that the assay is specific, with <25% heterologous inhibition occurring [25]. Lal et al. [26] described a rapid and simple method for the simultaneous quantitation of IgG to nine pneumococcal serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, 23F). Comparison of monoplex and nonaplex assays revealed no evidence of microsphere interference. The nonaplex assay was shown to be specific with <30% heterologous inhibition occurring and assay sensitivity was high with the Limit of detection ranging between 32.3 and 109.7 pg/ml. The assay was shown to be repeatable and conjugated microspheres were shown to remain stable over 12 months. There was a good correlation of the nonaplex assay with the ELISA. Finally, four bead sets coated with meningococcal polysaccharides (serogroups A, C, Y and W135) were added into the nonaplex assay, with no effect on the pneumococcal or meningococcal IgG levels generated. A multiplex system was developed for measuring antibodies against 9 malarial vaccine-candidate antigens, including recombinant proteins from 2 variants of merozoitesurface protein 142 (MSP-142), 2 variants of apical merozoite antigen-1 (AMA-1), erythrocyte binding antigen-175 (EBA-175), merozoite surface protein-3 (MSP-3), and peptides from the circumsporozoite protein (CSP), ring erythrocyte surface antigen

(RESA) and liver-stage antigen-1 (LSA-1). Plasma samples were screened by SAT in a mono- and multiplex format and by ELISA. Optimal amounts of protein required to perform the SAT assay were 10-100-fold less than that needed for ELISA. Excellent agreement was found between the single or multiplex formats (r2 >0.96), even when two variants of the same antigen were used. The multiplex assay was rapid, reproducible, required less than 1 l of plasma and had a good correlation with ELISA [27]. A 13-plex assay for quantitation of pneumococcal antibodies was optimized which showed satisfactory correlation with ELISA [20]. The multiplexed assay has been used for 297

Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology

screening the human sera for antibodies against 19 of the pneumococcal serotypes in the Indian population [18]. Further, the technique has been successfully utilized for screening of the culture supernatants from hybridoma cultures during development of various monoclonal antibodies [19]. Various multiplexed pneumococcal IgG quantitation kits are being explored for their use in vaccine efficacy studies [8]. In addition to antibody detection in vaccine development, IgG levels against 6 extractable nuclear antigens associated with collagen vascular diseases were measured by a multiplexed assay in various samples. The results of multiplexed assay corroborated well with those from ELISAs and the results could be used for diagnosis and prognosis of the disease [15].


Unknown Sample

Phycoerythrin Secondary Antibody

Known antibody Unknown antibody Antigen Polystyrene Bead

Figure 5: Schematic diagram for identification and quantitation of antibody in a serum sample 298

Dhingra, H. et al. 2011

Current Topics in Biotechnology & Microbiology

Screening antigens: In addition to the antibody estimation for determining efficacy of various vaccines, vaccine manufacturing process requires highly sensitive analytical tools for antigen estimation also for process development e.g. Fermenter harvest yields, purification process yields and antigen identification and quantitation in the final vaccine vials. Further, the antigen identification and titration is of significant importance in the clinical sample sent to the diagnostic laboratories. Similar to the antibody assays, the multiplexed assays impart big advantage over conventional time consuming and less sensitive assays for antigen identification and quantitation/titration.

Figure 6: Typical standard curve during identification and quantitation of antibody in a serum sample There are two most common formats of antigen estimation assays viz., sandwich assay and competitive inhibition assay. In a typical sandwich assay, an antigen is captured by the antibody on surface of microsphere and detected by a secondary antibody conjugated with a fluorochrome (Figure 7). On the other hand, in a competitive inhibition assay, an unknown antigen is pre-adsorbed with the known antibody and the reduction the signal of antibody due to pre-adsorption with homologous antigen is determined by putting the mixture in an antibody quantitation assay. Lower the fluorescence signal (MFI) indicates higher the content of antigen in a sample (Figure 8). For quantitative 299

and carcinogenesis. hematopoiesis. and not be reactive with the same epitome. The assay was found to be simple. Often microsphere-based assays can be performed with no wash steps if the background and potential interfering substances in the samples are minimal. Since each microsphere population is distinguished by size and/or spectral address. robust and repeatable as compared to conventional techniques. standard curves are optimized one analyte at a time. chemokines. Commercial antibody pairs optimal for ELISA development may not always be optimal for multiplexed microsphere immunoassays. GERMANY .Fluorosphere-based analyses would be applicable for disease surveillance. et al. the reporter intensity relates to the quantity of analyte bound and thus establishes the relative concentration of that analyte. KG. the assay data are transformed with analytical software. The fluorescent intensities of each fluorochrome are measured and. the several individual assays can be combined into the reaction volumes of one assay. inflammation. Antibodies should be tested to find the best pairs. and growth factors (hereafter referred to collectively as cytokines) play an important role in a wide range of physiological processes including immune response. A measure of viral load was developed by covalently coupling adenovirus antibodies to microspheres and incubating with DNA specific fluorophores. The MFI was measured from a standard curve of fluorescent intensity versus viral concentration. and other high throughput screening procedures [29]. with the capture antibody coupled to the microsphere and the detection antibody bound to the reporter. be matched for affinity. A multiplexed assay has been used by Health protection agency of United Kingdom for detection of 14 pneumococcal serotypes by a competitive inhibition assay format utilizing a polyclonal serum. Once optimized. vaccine development and surveillance. Various groups have reported bacterial serotyping of multiple strains of Streptococcus pneumoniae. Quantitation of Cytokines: Cytokines. Changes in levels of cytokines in the body fluids have been linked to various ailments making them important from diagnostic point of view. DUDWELLER LANDSTR. 2011 Current Topics in Biotechnology & Microbiology sandwich immunoassays. Validation of the assay was performed with a selection of serotype specific monoclonal antibodies and the assay was found to be specific [28]. if quantitative. screening of donated blood. Cytokines are 300 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. H.Dhingra. Similar assays are being developed for identification serotype specific antigens at Serum Institute of India .

IL-6. DUDWELLER LANDSTR. High levels of circulating proinflamatory cytokines which may be predictive of congestive heart failure and myocardial infarction have been associated with chronic heart failures [30]. IFN γ. IL-5. IL-10. Correlations between data sets were evaluated using Pearson’s correlation coefficient (r). IL-4. The assays setup is similar to the sandwich assay format shown in Figure 8. in contrast to IL-12 p70 and IL-13 [33]. 14]. Fluctuations in the level of one cytokine often induce changes in others. the rates of secretion of these analytes in a variety of physiological conditions are best measured simultaneously in identical reaction formats.g. smaller sample volume. Multiplexed microsphere arrays for cytokines developed by commercial vendors or independent laboratories are primarily two-site immunoassays. Thus. Relatively high-affinity matched pairs of antibodies for ELISAs for at least 50 human cytokines and related molecules are available from commercial vendors.Dhingra. A more realistic indication of the complexity of the cellular interactions would include measurements of multiple cytokines at any time point. The selection of monoclonals for multiplexed assays has been focused to get the comparable results with the conventional ELISAs [31]. 13. The advantages and protocol for multiplexed cytokine estimation in human serum and culture supernatants have been described since old [32. H. The cytokine levels were estimated in the acute wound fluid by a 27-plex assay and IL-1β was found to be the most potent inducer of signal molecule production [34]. and TNF α. Ninety-six specimens from women over the course of their respective pregnancies were evaluated for cytokine concentrations using commercially available ELISA kits and commercially available Luminex MAP® kits according to the manufacturers’ directions. Multiplexed arrays for various number of cytokines are available based on the experimental requirements e. The limit of sensitivity of these commercial antibody pairs falls in the low picogram range. Excellent correlations were demonstrated for IL-1β. 301 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Simultaneous evaluation of multiple immune mediators was performed by Luminex MAP® technology with advantages of higher throughput. KG. et al. GERMANY . Determining type of T-helper cell response (Th1 or Th2) etc. and lower cost. 2011 Current Topics in Biotechnology & Microbiology excellent candidates for multiplexed analysis because of their complex inter-relationships in immunologic and hematologic functions.

Microspheres coated with HLA class I and class II antigens have been used to test for HLA antibodies prior to transplant. RNP. DUDWELLER LANDSTR. as well as testing for antibodies to specific HLA antigens [35]. histone. SSB. and Scl-70 are included in the kit [5]. GERMANY . 2011 Current Topics in Biotechnology & Microbiology HLA Testing: Flow cytometry has significantly impacted HLA testing. Autoimmune testing: The first microsphere-based multiplexed immunoassay application that has received FDA approval was a kit for autoimmune disease testing. et al. Centromere B. H. Phycoerythrin Detection antibody Unknown Antigen Capture Antibody Polystyrene Bead Figure 7: Identification and quantitation of antigen by sandwich assay in a sample Protein phosphorylation assay: Traditional methods for measuring phosphorylated proteins include Western blot analysis with immunologic or isotopic identification of the 302 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.Dhingra. Sm. IgG antibodies to the markers SSA. This technology has recently been implemented on the Luminex platform and has the potential for DNA-based HLA typing. Jo-1. KG.

Phycoerythrin Secondary Antibody Primary antibody Free Primary Antibody + Antigen Polystyrene Bead Unknown Antigen Figure 8: Identification and quantitation of antigen by competitive inhibition assay in a sample 303 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. GERMANY . quantifiable flow cytometric procedure. KG.5]. Micro-particle based capture and detection of phosphorylated proteins is a rapid. DUDWELLER LANDSTR.Dhingra. Phosphorylation of the signaling protein ERK-2 has been identified with antibodies specific for the phosphorylated protein [36. The oligonucleotide capture probes and PCR amplification primers were designed and the probes were coupled to carboxylated microspheres followed by hybridization of coupled microspheres to oligonucleotide targets and detection of the multiplexed-PCR amplified targets by direct hybridization (Figure 9) to probe coupled microspheres [37]. Both procedures can involve several days of sample processing. Diagnosis of Cystic Fibrosis: A suspension array hybridization assay was developed for detection of 31 mutations and polymorphisms in the cystic trans-membrane conductance regulator gene using x-MAP technology. 2011 Current Topics in Biotechnology & Microbiology modified proteins. et al. H.

Its advantages include rapid data acquisition (up to thousands of data points per second). 2011 Current Topics in Biotechnology & Microbiology Antigen identification by hybridization assays: In addition to the antibody based assays. In these methods. A universal primer-multiplex PCR system was developed and applied for simultaneous detection of Escherichia coli. Salmonella enterica and Staphylococcus aureus have been detected by x-MAP technology [22]. glass slides). [17]. a fluorescent reporter molecule is enzymatically added to a capture probe to determine the SNP genotype. The ZipCode concept permits use of a defined set of optimally cZipCode-coupled microspheres for different uses of SNPs. Similarly low levels of various pathogens including E. excellent sensitivity. Flow metric analysis of fluorescent microspheres has been identified as a novel technology platform for SNP genotype determination. as well as mobile supports. high throughput. H. KG. and 3) flow cytometric analysis of the microspheres to identify both the microsphere population and the fluorescent signal associated with a genotype [41]. et al. Bacillus cereus. and cost effective. chips.Dhingra. the antigenic load has been quantitatively estimated for Salmonella and other bacterial pathogens by a hybridization of sample DNA PCR product to the microspheres coupled to the specific oligonucleotide targets [38]. Listeria monocytogenes and Salmonella spp. The xTAG respiratory viral Panel developed by Luminex corporation has been used in the characterization of pandemic influenza A (H1N1) specimens [39. such as mass spectrometry and capillary electrophoresis. coli. 2) capture of the enzymatic reaction products by hybridization of DNA sequences (ZipCodes) on the capture probe with complementary sequences on the surface of the microsphere. The three steps in the genotyping process are 1) allele discrimination by thermal cycling of PCRamplified genomic DNA in a solution-based enzymatic reaction. The same ZipCode sequence can be incorporated into the design of new capture probes as new SNPs are added to the assay. Typical SNP genotyping technologies included solid supports (gels. and multi-parametric fluorescence analysis that permits multiplexed analysis [40]. GERMANY . One approach to microsphere-based SNP genotyping is enzymatic allele discrimination by oligoligation assay (OLA) or single base chain extension (SBCE). DUDWELLER LANDSTR. Other approaches to SNP genotyping have included direct or competitive hybridization of PCR-amplified products to 304 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.24] Single Nucleotide Polymorphism (SNP) Genotyping: SNP genotyping requires methods that are accurate.

com/applications.luminexcorp. H. KG. For more information refer http://www. DUDWELLER LANDSTR. 2011 Current Topics in Biotechnology & Microbiology complementary sequences coupled to microspheres. GERMANY . et al. 305 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. excellent accuracy at a reasonable cost. TARGET AMPLIFY DENATURE HYBRIDIZE DETECT Figure 9: Schematic Microsphere based direct hybridization assay format (With permission from Luminex Corporation) Several other applications and different markers have been targeted using this technology. The advantages of microsphere-based SNP genotyping include greater flexibility than static platforms such as the chip.Dhingra. These approaches require a unique oligonucleotide-coupled microsphere population to match each SNP allele and can require an elevated temperature during the flow metric analysis [5]. and increased throughput by multiplexing.

Clinical 306 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. C.R. Pickering. Schlottmann. Greer. D..W.B. & Hill. Snawder. & Weissman D. Schroder. 872-876.. Comparison of a multiplex flow cytometric assay with Enzyme-Linked Immunosorbant Assay for quantitation of antibodies to Tetanus. H..R. J. The technique has huge potential to the biomedical research. N. Clin. 10 (5). M.W.. et al. 3. KG.M.. diagnostic assays and gene profiling studies etc. MacKenzie. J. J. As with any high-throughput method that generates data. Astil. S. R.E. McDade.L. diagnostics and vaccine industry in the years to come. TM system. H. DUDWELLER LANDSTR. Schroder. Advanced multiplexed analysis with the FlowMetrix Chemistry.. T. Hildreth. GERMANY .. Martins.A...E. M.L. R. Luminex 200. J. J. The technique has got several advantages over the conventional techniques for various applications such as immunoassays. 589-596. (2002b).. sensitivity. M.Dhingra. Pickering. REFERENCES: 1. & Hill H. C. Martins. 9(4). shifting from low throughput FlowMetrix to Luminex 100. Smith.. Striley. There has been a series of advancements in the technology over several years i. flexibility. the challenges include integrating a robust information-management system to handle the deluge of data and extract reliable information. R.B.C. A Multiplex fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharide. FLEXMAP 3D and most recently LED/CCD based MagPix system. 744750. 2011 Current Topics in Biotechnology & Microbiology CONCLUSIONS: The suspension array based x-MAP technology is one of the emerging multiplexing techniques in the field of biomedical sciences.A. 4. L. 117.W. Diphtheria.A. J. T. (1997).e. Smith.W. Clinical and Diagnostic Laboratory Immunology. L.. Clinical and Diagnostics Laboratory Immunology..C. accuracy. 43(9). 2. (2003)..P. Method of simultaneous measurement of antibodies to 23 pneumococcal capsular polysaccharides. & Kettman. Fulton.F. B..J. Biagini. Sammons. Jr. R. 1749-1756... Pathol.J. in terms of speed. R. specificity and high throughput. S...C. Litwin. Kienker. P. (2002a). and Haemophilus influenzae Type b.

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S. Sandanger. Wang. Davis K. T.. & Jacobson. P. Viruses (e-publication). D. GERMANY . et al. Methods Mol. Rev.W.194-200.. Pabbaraju. multiplexed detection of Salmonella and other pathogens by Luminex xMAP suspension array. Comparison of a singleplex real-time RT-PCR assay and multiplex respiratory viral panel assay for detection of Influenza “A” in respiratory specimens. (1998).. Otterdal. Semin. Rapid screening for 31 mutation and polymorphism in cystic fibrosis transmembrane conductance regulator gene by Luminex xMAP suspension array. Genet. Dunbar. 310 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Journal of Reproductive Immunology. Methods for genotyping single nucleotide polymorphisms. Cellular sources and inducers of cytokines present in acute wound fluid.(e-publication).. & Nelson. Fonseca.. (2011). 175-191. Bray. 114..A. Tellier..A. Grimstad. M. 34. 41. R. Louie. & deWaal M. O. B. 147-171. M. Validation and comparison of Luminex multiplex cytokine analysis kit with ELISA: Determinations of a panel of nine cytokines in clinical sample culture supernatants. N. Landegren.. P. Lee.Y. & Drews. O. 8. 66..J. 394. 1-19.Y... F. B. & Espevik.C. 40. Reading bits of genetic information: methods for single-nucleotide polymorphism analysis. H. 769-776. K. Flow cytometry in human leukocyte antigen testing. R. duPont. Methods Mol. K. Wound Repair Regen. (2001). (2010). Lund-Johansen. & Jacobsan. (2000).. & Kwok. Bishop J. Wadhwa.W. Pukstad.. Ryan. KG.L. E. 38.Dhingra. 38. Dunbar. S. K.. Res. Influenza other Respi. Med. S. S.. J. Kwok. U. Annu.K. (2001). DUDWELLER LANDSTR. 37.. K. R. Cytometry. Genomics Hum. Quantitative... 250-259. 2. 39. L. J. Nilsson. Genome.. (2007). Culhane. 39... 35. Wong. Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein phosphorylation and protein-protein interactions. Hematol.. Biol. 2011 Current Topics in Biotechnology & Microbiology 33. P. 235-258. Damass. (2005). J.A. F. 36. J. (2005).

S. In this chapter.K. 2011 Current Topics in Biotechnology & Microbiology Chapter XV CYANOTOXINS: PHARMACOLOGICAL AND ECOLOGICAL EFFECTS R. physiological and biochemical properties. rajuashwinkumar@gmail.ac. because they are involved in much of the primary production.Dhingra. and form the basis of tropic networks [1. Birla Institute of Technology and Sciences. mechanism of action of its variants and their toxicity profile.in.K. H. GERMANY . India. The majorities of cyanotoxins are of non.ribosomal origin and can be grouped into classes with conserved structural properties. rivers and estuaries and it is a constant problem for water authorities. ponds. Biochemical processes in cyanobacterial cells are of three types: basal (providing energy and raw materials for cell functions).mail: S. Ashwin Kumar. DUDWELLER LANDSTR. INTRODUCTION Cyanobacteria are photoautotrophic prokaryotes which comprise of a large species of widespread occurrence with diverse morphological.com ABSTRACT:The Cyanotoxins produced toxic cyanobacteria are usually found in eutrophicated waters such as lakes. synthetic (replication and vegetative growth) and secondary metabolites production (utilization of substrate to give a variety of products often specific for given organism). 2]. E-mail: skverma@bits-pilani. Correspondin Author: E. They play an important role in the functioning of aquatic ecosystems. Pilani. Among these processes the maximum chemical diversity is observed in the secondary metabolites such as 311 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Verma Center for Biotechnology. Biological Science Group. Rajasthan. KG. The world health organization has shown great concern to public health over prolong exposure of low concentration of cyanotoxins. et al. The biochemical aspects of Cyanotoxins have been extensively studied throughout the world during last two decades but very little information available about its regulatory factors. Verma. we discuss about the major Cyanotoxins along with its variants. their mode of actions and their potential health risk to animals and humans.

KG.Dhingra. cardio active compounds and sunscreen pigment [1-4].4% are amides. cell-differentiation promoter. TOXINS OF CYANOBACTERIA The toxins produced by planktonic species of cyanobacteria have been particularly well studied. antimalarial. Eutrophication has been a constant problem for water authorities due to its association with the formation of cyanobacterial blooms [5]. 2011 Current Topics in Biotechnology & Microbiology cyanotoxins which provide heterogeneity as well as inter-/intra species level markers for organisms. The products of cyanobacteria contain 40. Oscillatoria. They include Anabaena. H. Lyngbya. had begun to screen extracts of cyanobacteria on Nostac. because many species are able to produce toxins that threaten both animal and human health [5].2% macrolide and 9. Lyngbya. toxins. pharmaceutical active compounds and plant growth regulators. Nodularia. copper sulphate (algicides) can lyse the cyanobacterial cells to release the toxins into the surrounding water body. During 1990’s. which led to high incidence of novel biologically active compounds such as antibiotics. Numerous poisoning events that have resulted from the consumption of contaminated water through chronic ingestion. 4. 4. herbicide. 6. 312 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Cylindrospermopsis. enzyme inhibitors 8%. Cyanotoxins in drinking water can disrupt aquatic ecosystems and cause serious public health problems. 4]. Anabaena. GERMANY . et al. antiviral 4% and antifungal 18%. antimycotic. algicides. Spirulina. Nostoc. antibiotics12%. The species such as Spirulina and Nostac have been used as a source of protein and vitamins for humans and animals [2. inhalation of droplets or contact with nasal mucous membranes. antimicroalgal. these microorganisms can also be harmful. Moreover use of chemicals for purification of water like chlorine. 4].6% of pure amino acid composition. DUDWELLER LANDSTR. However. Aphanizomenon. and Nodularia for various biological activities in fresh water and marine cyanobacteria. anticancer13%. The major focus of this chapter is therefore to compile and analyze information of cyanotoxins and discuss their ecological significance. Microcystis. Lipopeptides are a potent bioactive group of compounds with approximately 85% being active of which are toxins 41%.2% of lipopeptides.2% fatty acids. The remaining activities are as tumor promoter. Scytonema and Tolypothrix [3. 5. 7]. A heavy water bloom formed under adequate light and temperature can also serve a rich source of secondary metabolites of novel chemical and molecular structure [3. Synechocystis.

6. 9S)-3-amino-9-methoxy-2. The cyanobacterial hepatotoxins include cyclic peptides (Microcystins and nodularins) and cyclic guanidine alkaloid (cylindrospermopsin). 11. The most common and potent variant is microcystin. DUDWELLER LANDSTR. and Anabaenopsis species.e. Hepatotoxins Hepatotoxins are the most common offender’s worldwide in case of waterborne disease caused by toxins of cyanobacteria [10]. intracellular or extracellular. The Adda moiety is also required for toxicity and is important in the binding of the toxins to the protein phosphatases. They contain either five (nodularins) or seven (microcystins) amino acids. 6-dienoic acid. 12]. The Adda of microcystin provides the molecule with a characteristic wavelength absorbance at 238nm [4.LR containing amino acid leucine (L) and arginine (R) at X and Z position [10. According to the world health organization. the nature of the cyanotoxin. is the most unusual structure in this group of cyanobacterial toxins with two protein amino acids (X&Z) and five non-protein amino acid.100. 2011 Current Topics in Biotechnology & Microbiology dermal contacts through bathing or recreational activities such as wading. skiing and canoeing [5.. Microcystins: Microcystins have been characterised from Microcystis. The aminoacid Adda. and effective treatment processes [9]. KG. About 75 structural variants of microcystins have been characterized so far from cyanobacteria. H. Based on the chemical structure they are mainly divided into cyclic peptides. with the two terminal amino acids of the linear peptide being condensed (joined) to form a cyclic compound [9. Cyclic peptides are comparatively large natural products with molecular weight of 800-1. alkaloids and lipopolysaccharides. To create a comprehensive cyanotoxin contingency plan. i. it is necessary to be familiar with the physical and chemical properties of the toxin. growth and diurnal cyanobacteria bloom patterns. 3S. 12]. The nomenclature of microcystin variants has denoted by the amino acids present at the X and Z position (fig1). the guide line value for total microcystin (free and intracellular) is 1 g/L [10. 8trimethyl-10-phenyldeca-4. 12].8]. et al. Anabaena. Oscillatoria. GERMANY . 8S. resulting in marked differences in toxicity and in hydrophilic and hydrophobic property.Dhingra.12]. Nostoc. The stereochemistry of the dienes in Adda group and level of 313 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. swimming. 11. Other variants differ in methyl group and amino acid in the ring. (2S. 6.

leading to massive hepatic bleeding which may lead to death or chronic intoxication by promoting hepatic tumors. with more than 97% of protein phosphates occurring at Ser/Thr residues which suggests that the toxic effects of MC results in so-called activation of protein kinases. H. The specific dephosphorylation of Ser/Thr residues plays an essential role in control of the cell. The tumour-promoting activity of microcystin is likely to arise from its ability to potently inhibit PP2A which regulates several mitogen-activated protein kinases (MAPK). These enzymatic inhibition causes morphologic damage to the tissue. GERMANY . resulting in an overall increase in phosphorylation of regulatory proteins (Fig. 2011 Current Topics in Biotechnology & Microbiology methylation in various structures of cyclic peptides influences the level of toxicity and site of action [12. Therefore. Figure 1: General Structure of Microcystin The site of action of microcystins is by disrupting the cytoskeleton of hepatocytes. The cell cycle is regulated by a delicate balance between phosphorylation and dephosphorylation of key control proteins. KG. 2) [15]. Both PP1 and PP2A covalently bind MCLR at cysteine-273 for PP1 and cysteine-266 for PP2A. DUDWELLER LANDSTR. Microcystins in the liver can induce hepatocytes to produce arachidonic acid metabolites such as prostacyclin (6-Keto F1 alpha) and thrombaxane (TXB2) by stimulation of the cyclooxygenase pathway [15].Dhingra.14]. 314 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. by inhibiting both PP1 and PP2A with a variety of molecular targets. et al.

PP2A regulates the phosphorylation of both MAPKK and MAPK so inhibition of this PP by MCLR would allow for the continual activation of both these kinases which activates transcription factors.5-biphosphate (PIP) to diacylglycerol (DAG). KG. 2011 Current Topics in Biotechnology & Microbiology Figure 2: The proposed scheme illustrated by Gehringer dipicting cellular events upon MCLR exposure. PKC was not found to play a role in MCLRinduced kinase activation in-vivo. et al.Dhingra. The MAPK pathway is activated by receptor tyrosine kinases activated through G-linked receptors bycleavage of Phosphatidyllinositol 4. however the effect on p21WAF. role of caspases and role of JNK remains to be elucidated [15]. H. IP3 triggers the release of Ca2+ from the endoplasmic reticulum (ER). GERMANY . but this does not occur for MCLR. 315 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. MCLR increases phosphorylation of p53 and formation of reactive oxygen species (ROS) which inturn triggers mitochondrial permeability. DUDWELLER LANDSTR.

It is a cyclic pentapeptide with a similar structure to microcystin. the rate of reaction is determined by the growing heptapeptide chain. Two operons. The first report of nodularins was in 1988 when bloom infested lake in Australia containing Nodularia spumigena was shown to be the cause of stock death. Peptide synthetases have a modular construction: the order and number of modules at the gene level determines the structure of product. McyE converts the polyketide to a β-amino acid in the final step of Adda biosynthesis [16. McyE. Thus. responsible for the epimerization of the L-Glu residue of microcystin and later it founded with evidence that McyF acts exclusively as an Asp racemase and D-Glu residue is provided by L-Glu racemase residing outside the mcyS gene cluster [18]. Nodularin:Nodularin is produced predominantly by brackish water cyanobacteria. consisting of Adda. 55kb (mcy S) The NRPS module of the second hybrid PKS/NRPS enzyme. 17]. KG. The individual peptide synthetase catalyzes amino/hydroxyl acid activation and thioester formation reaction. 16. The mcyF ORF was originally predicted to encode a glutamate racemase. DUDWELLER LANDSTR. elicit their toxic effect [17].3) They are transcribed in opposite direction with a central sequences of approximaterly 900bp containing the promoter and putative regulatory cis element. McyH is in fact responsible for the active transport of microcystin (ABC transporter) to. Figure 3: Microcystin synthetase of Microcystis aeruginosa. 17]. Certain gene cluster encoding microcystin synthetase has been cloned and sequenced [13. D-glutamic acid 316 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.Dhingra. H. is thought to be involved in the activation and condensation of D-Glu with Adda. 14. 2011 Current Topics in Biotechnology & Microbiology Microcystin is biosynthesized via a large multifunctional enzyme complex consisting of non-ribosomal peptide synthetase (NPKS) and polyketide synthase (PKS) modules collectively making cluster of microcystin genes (mcy S 55kb) [16]. namely mcy ABC and mcy DEFGHI form a large gene cluster for the enzyme complex (Fig. GERMANY . exchange and rearrangement of peptide synthetase modules offer the opportunity to produce novel products. The production of the fatty acid side chain of the amino acid Adda has been responsible for the function of polyketide synthase. et al.

KG. and may be synthesized by an associated cyanobacterium. The D-Glu residue is essential for toxicity of nodularin. The L-Arg residue of nodularin is replaced by L-Val in motuporin. H. 20. GERMANY . which reduces or abolishes the toxicity of the compound [16]. Motuporin has been isolated from the Papua New Guinea sponge Theonella swinhoei. nodularin-Har and motuporin. D-erythro-β-methylaspartic acid (DMeAsp). Two of these isoforms. DUDWELLER LANDSTR. blocks protein phosphatases enzyme activity by interacting with the hydrophobic groove and obstructing substrate access to the active site cleft.amino acids Adda. N-methyldehydrobutyrine (MeDhb). harveyana PCC7804. et al. The L-Val residue is responsible for additional cytotoxicity of motuporin against cancer cell lines [24]. The hydrophobic C20 β. and L-arginine (L-Arg) (Figure 4) [19.Dhingra. Nodularin-Har is produced by the strain N. substitution at position 1 has little effect on toxicity. The other two isoforms. 21]. are variable at position 2 [22]. 2011 Current Topics in Biotechnology & Microbiology (D-Glu). MeDhb binds to Cys273 of PP2A in a similar fashion to the MeDha residue in microcystin which NH 317 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Seven naturally-occurring isoforms of nodularin have been reported to date.1nm. have variations within the Adda residue. with the L-Arg. however. replaced with L-Homoarginine (L-Har) [23]. N N OH O NH O N H O COOH O NH COOH O NH H2N Figure 4: Structure of Nodularin The mechanisms of toxicity are relatively same as that of microcystin by inhibiting pp1 and pp2A with IC50 of 0. as esterification of its free carboxyl abolishes toxicity. present in both toxins.

Later it was also reported from Aphanizomenon ovalisporum.6) [30. complete the cyclic pentapeptide by adding the final amino acid residues. D-MeAsp and L-Arg. D and F) (Figure 5). GERMANY . lungs. spumigena NSOR10 [16. At the hydroxyl bridge. Two NRPS enzymes. was sequenced and characterized in 2004. L-Thr. Because of the negatively charged sulfate group and the positively charged guanido group. The acute toxicity studies with crude extract with LD50 of 200 g/kg injected intraperitonally blocks protein synthesis of cells in kidney. in Israel.Dhingra. 2011 Current Topics in Biotechnology & Microbiology binds to Cys273 of PP1 and Cys266 of PP2A. Aphanizomenon flos-aquae in Germany and Umezakis natan in Japan. there are two possible epimers. from Nodularia spumigena NSOR10. NdaA and B. The 48 kb region of the genome consists of nine ORFs (ndaA-I) transcribed from a bidirectional regulatory promoter region (Figure 5). the molecule is a zwitterion and very water soluble. Both naturally occurring epimers are equally toxic [33]. 31. The NRPS and PKS proteins require posttranslational modification by a phosphopantetheinyl transferase (PPT) protein. NdaF. The NRPS modules responsible for the activation of D-Ala and D-Leu in mcyS (McyA and B) are absent from ndaS as nodularin lacks these moieties. The PPT required for activation of the Nda proteins is not clustered with the other nda genes. DUDWELLER LANDSTR. The toxin is a stable tricyclic alkaloid containing a guanido group linked at C7 to hydroxymethyl uracil (Fig. et al. liver. Cylindrospermopsins: Cylindrospermopsins a cyclic guanidine hepatotoxic alkaloid first isolated from Cylindrospermopsis raciborski. KG. cylindrospermopsin and 7-epicylindrospermopsin. binding of the toxin does not occur covalently and may be the reason for its additional carcinogenic properties [25]. Recently. 48kb (nda S) [16] The nodularin biosynthesis gene cluster ndaS. The Adda side-chain is produced via a mixed NRPS/PKS pathway from a phenylacetate starter unit and several malonyl-CoA extensions (NdaC. H. 318 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 26]. subsequently adds D-Glu to the growing chain [16. 27 28]. The NRPS module of the hybrid NRPS/PKS. However. identified the PPT required for nodularin biosynthesis in N. Figure 5: Nodularin synthetase of Nodularia spumigena.degenerate PCR and subsequent functional enzymatic characterization. 32].

This hypothesis was confirmed in primary mouse hepatocytes where cylindrospermopsininduced DNA fragmentation was detected by the COMET assay. which does not require toxin metabolism. GERMANY . Uptake of the toxin is relatively rapid since complete and irreversible block of protein synthesis occurs after a 1 hr exposure in-vitro. but this does not lead to an increase in oxidative stress in the cell [33. Aphanizomenon. a rapid route probably through toxicity of a CYP450 oxidation product of the toxin and a slower mechanism through the welldocumented inhibition of protein synthesis. Neurotoxins Aphantoxins. neosaxitoxin are known neurotoxins reported from the strains of Anabaena. anatoxins-a. saxitoxins.34]. 35. Recent data from primary hepatocytes show two routes of toxic action. 36]. suggesting that metabolism of the toxin to an active product was required for genotoxic action. The effect pure toxin has been identified in an outbreak of acute heptoenteritis and renal damage among the aboriginal population in Australia [33. et al. O O S O O H N H OH O NH O NH HN NH H Figure 6: Structure of Cylindrospermopsins Cylindrospermopsin has also been found to be genotoxic in a number of in-vitro assay systems. cylindrospermopsin caused DNA fragmentation and loss of whole chromosomes in a human white blood cell line and in mice liver [35]. These findings could not be confirmed in CHO K1 cells. H. Glutathione synthesis is also reduced via a CYP450-dependant mechanism. Using the micronucleus assay. an effect that could be eliminated by application of CYP450 inhibitors [36]. DUDWELLER LANDSTR. KG. 34. 2011 Current Topics in Biotechnology & Microbiology adrenal glands and intestine.Dhingra. Cylindrospermum and Oscillatoria 319 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. Inhibition of protein synthesis occurs at the ribosome during the peptide chain elongation step.

Jamaicamide from Lyngbya majuscule also shares the same site of action as compared to other neurotoxins stated above [39]. which are more pronounced than in Vero cells. Exact mechanisms of anatoxin induced apoptosis are not known [38]. The enzyme acetylcholinesterase catalyzes the hydrolysis of the neurotransmitter acetylcholine and it has been an attractive target for the treatment of Alzheimer’s disease and other possible therapeutic application in the treatment of Parkinson’s disease and Myasthenic gravis [3. H. They are generally alkaloids which are well known for its neurotoxicity with low molecular weight [3. Anatoxin-a is the structural analogue of cocaine and binds to nicotinic acetylcholine receptors which leads to respiratory failure within few minutes. It can induce apoptosis in non-neuronal cells. Anatoxins and its variants inhibit transmission at the neuromuscular junction by molecular mimicry of the neurotransmitter acetylcholine and inhibition of acetyl cholinesterase activity [37]. KG. GERMANY . H N O H2 O N Structure of Anatoxins-A N N H2N N O O P O CH3 O CH3 CH3 Structure of Homoanatoxins-A Structure of Anatoxins-A (S) 320 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. These compounds are potent post–synaptic depolarizing neuromuscular blocking agent that causes hypersalivation and death within minutes to hours in animals. et al. generation of ROS and caspase activation. 37]. 4]. Homoanatoxins-a is a potent neuromuscularblocking agents that causes severe paralysis convulsion and death by respiratory arrest. Toxin-treated thymocytes showed all the typical morphological and biochemical features of apoptosis including DNA fragmentation.Dhingra. DUDWELLER LANDSTR. They are only known naturally occurring organophosphate [6]. 2011 Current Topics in Biotechnology & Microbiology respectively.

H.Dhingra. aphantoxins) have been reported to occur naturally in marine dinoflagellates. KG. et al. and the occurrence of STX producing neurotoxic cyanobacterial blooms has been increasingly reported [3. 16. STX toxins are almost exclusively associated with cyanobacteria. thereby affecting the impulse generation in animals which can lead to death. PSP toxins block resting. Saxitoxins (STX) and its analogue compounds (neosaxitoxin. 4. filamentous cyanobacteria and in certain heterotrophic bacteria. The positively charged guanidinium group of PSP toxins interacts with negatively charged carboxyl groups at the mouth of the channel [42. 42]. active or inactive Na+ channels equally well. occupied by the so-called ‘pore-blocking’toxins. PSP toxins selectively block voltage-gated Na+ channels in excitable cells. They are well known as paralytic shellfish poisoning (PSP) toxins due to their accidental consumption in contaminated seafood. STX binds to nerve membranes with high affinity (dissociation constant Kd=2 nM). 43. In freshwater environments. 44]. GERMANY . through a receptor site named as site 1. These toxins act from the extracellular side of the plasma membrane by occluding the entry of the Na+ channel pore. Br OCH3 O O O O OH O O OH OH R4 O H N NH2 N N R5 OH R2 R3 R 1N H2 N Debromoaplysiatoxins:General Structure Saxitoxins 321 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. 2011 Current Topics in Biotechnology & Microbiology Saxitoxins comprises different isoforms and with varied toxicities. DUDWELLER LANDSTR. 41.

oral and gastrointestinal inflammation. 45]. 46]. Stimulation of chemoreceptors in the stomach. Lipopolysaccarides. Endogenous mediators of emesis such as dopamine. associated with the gut mucosa (pre-absorptive response) and central. The many structural variants of LPS are generally phylogenetically conserved (similar fatty acid and sugar components). DUDWELLER LANDSTR. they are essential defences because they are the end result of the actions of sensory motor systems that operate to identify and rapidly expel hazardous substances from the upper G-I tract. usually a hexose and a lipid. are condensed products of a sugar. activation of these chemoreceptors will also initiate nausea and vomiting. as the name implies. The lipopolysaccharide (LPS) endotoxins of cyanobacteria are generally found in the outer membrane of the cell wall where they form complexes with proteins and phospholipids [4. The considerable diversity of cyanobacteria in their chemical composition is largely related to the phylogeny [4]. acetylcholine and enkephalin are reported.Dhingra. or by bacterial enterotoxins. Prostaglandins have well-known emetic actions. H. In 1970 the first LPS was reported from the cyanobacterium Anacytis nidulans. Emetic chemoreceptors are also found in the vascular system. jejunum and ileum by irritant chemicals such as hypertonic saline. It has been proved that the fatty acid component of the LPS molecule elicits allergenic response (irritant) in humans and mammals [3].negative bacteria and cyanobacterial LPS lacks any phosphate in the lipid-A core. leads to the activation of vagal sensory afferent nerves to the brain. copper sulfate or mustard. LPS of cyanobacterium are found to be 10fold less toxic than other bacterial LPS. Vagal efferent processing through the enteric nervous system stimulates enteric motor neurons to affect emesis. specifically the chemoreceptive trigger zone of the area postrema. normally a hydroxy C14-C18 fatty acid. 322 LAP LAMBERT ACADEMIC PUBLISHING AG & CO. located in the dorsal surface of the medulla oblongata (post-absorptive response). Both unlike Gram. which exhibits both pyrogenic and toxic effects. KG. GERMANY . et al. Some of the other dermatotoxins which are will reported are Debromoaplysiatoxin from Lynbya gracilis [45. 2011 Current Topics in Biotechnology & Microbiology Dermatotoxins Most of the lipopolysaccharides of cyanobacteria cause severe dermatitis. The two main sensory systems that direct the emetic response are local. Mechanisms of vomiting and its relationship to LPS activity Nausea and vomiting are normal physiological responses to the ingestion of toxic substances.

30methyloscillatoxin D. Nevertheless. a 101 amino acid protein has recently been placed on an accelerated track for clinical development for use as a virucidal agent. There is considerable evidence for involvement of other variants in toxicity 323 LAP LAMBERT ACADEMIC PUBLISHING AG & CO.4]. hormothamnin and hormothamnione from Hormothamnion enteromoyphoides. Some of the other cytotoxins reported are diarrhetic toxin {lipopeptide) from Microcystis species. CV-N was found to have potent activity against all immunodeficiency viruses HIV1. SIV (simian) and FIV (feline). genotoxicity. Dolastatin 3.Dhingra. GERMANY . KG. H. C-14 again appeared to be too toxic to become a clinical candidate. 31-neroscillatoxin and oscillatoxin from Oscillatoria species. +-α(s)-butyramidedo-ybutyrolactone. Some of sulfolipid of Lyngbya lagerheimi. CONCLUSION A number of areas of cyanotoxins toxicology require investigation before the human health risk can be fully assessed. Cyanovirin-N (CV-N) was extracted from Nostac ellipsosporum. et al. The use of CV-N. antitumor drugs [47. 12 and Serinol-derived malyngamide 1-2 from Lyngbya majuscule also show anti-HIV activity [49]. butyrolactone 3-y and hermitamides B from Lyngbya majuscule. micromide and guamamide from Symploca and tenue cyclamide from Nostoc spongiaeforme [3. kalipyrone from Tolypothrix species. Anabaena species and Nodularia species.48]. which exhibited potent cytotoxicity against human tumor cell lines and good activity against a broad spectrum of drug-sensitive and drug resistant murine and human solid tumors. 2011 Current Topics in Biotechnology & Microbiology Cytotoxins Cryptophycin 114 was first isolated from Nostac sp ATCC 53789 and Nostac sp GSV 224. G. 11. HIV-2. allowing the transfer of the viral genetic material into the host. borphycin and nostocyclophane from Nostoc ellipsosporum. The level of mechanisms of action is not yet known by which route toxic metabolites are formed to produce rapid toxicity and more importantly. DUDWELLER LANDSTR. Cyanovirin 16. Cryptophycin 114 and its analogue 815 lead to an entirely new class of antiHIV. γ-butyrolactone. which prevent the surface receptor of HIV from undergoing the conformational changes that enable the viral and cell membranes to fuse. This led to a detailed structure function study and resulted in the isolation of Cryptophycin 815 a semi synthetic analogue. which proved to have a greater therapeutic efficiency and lower toxicity.

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