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Bioscience, Biotechnology, and Biochemistry

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Effects of Compressed Hydrocarbon Gases on the
Growth Activity of Saccharomyces cerevisiae

Satoshi KAWACHI, Yoshio HARA, Toshiaki ARAO, Yoshihisa SUZUKI &
Katsuhiro TAMURA

To cite this article: Satoshi KAWACHI, Yoshio HARA, Toshiaki ARAO, Yoshihisa SUZUKI &
Katsuhiro TAMURA (2010) Effects of Compressed Hydrocarbon Gases on the Growth Activity of
Saccharomyces cerevisiae, Bioscience, Biotechnology, and Biochemistry, 74:10, 1991-1996, DOI:

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Published online: 22 May 2014.

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and liquefied elucidate the mechanism of inhibitory action of petroleum gas (LPG). pressure (MIP). 2010. Fax: +81-88-655-7025. coli. it is difficult to evaluate the hydrophobic regions of the cell membrane. we have now inves- tigated the toxic effects of hydrocarbon gases on yeast. 0.5) n-Hexane and n-heptane resulted in growth lorimetry. are poorly suggesting that hydrocarbon gases interact with some soluble in water. and 10 g/l of yeast on humans. we report morpho- will be used increasingly. in this study because its cellular structure resembles that of higher Thus there is an increasing risk of human exposure to eukaryotic organisms. at 660 nm. scanning concentration of the gases is thus increased. including oxygen. Tsukuba.D. are widely used for domestic and industrial substances from damaged cells caused by hydrocarbon purposes. The diluted cultures were grown at 30  C for 6 h to obtain properties of liquid hydrocarbons have been studied sufficient log-phase cells (1  107 cells/ml. methane is the logical changes in yeast cells exposed to compressed most abundant organic gas in the atmosphere. Yoshio H ARA. MIP. would also not be able to metabolize the gaseous n-alkanes tested.10) Using this method. and then diluted 20-fold with fresh YPD toxicological effects of exposure to hydrocarbon gases medium (20 g/l of glucose.10) At normal pressure. Institute of Technology and Science. gases increased in the order methane  ethane < and other inert gases. and continues to increase. The toxicity of these effects on yeast growth. which the toxicity of hydrocarbon gases. nitrogen. Human exposure able to metabolize liquid n-alkanes (8 < n < 15) while some Candida to hydrocarbon gases can occur via accidental leaks from strains metabolize their chemicals.2) The hydrocarbon gases as observed by scanning electron atmospheric concentration of methane has increased microscopy (SEM). Key words: hydrocarbon gases. dramatically over the last century. found in gaseous hydrocarbons. we investigated the inhibitory pressure. There are. are by microcalorimetry. this Saccharomyces species is not hydrocarbon gases in the atmosphere. In particular. high In the present study. the release of UV absorbing 2010 [ Abbreviations: IP50 .10. Accepted July 20. 50% inhibitory pressure. however. and hydrocarbons. Therefore. future global warming has the Materials and Methods potential to destabilize methane clathrates. 74 (10). minimum inhibitory pressure . toxicity. which are Cladosporium resinae. animals. It was used enormous amounts of methane. Japan Received March 4. their toxic electron microscopy indicated that morphological effects on microorganisms become more apparent and changes occurred in these cells. calorimetry effects of compressed hydrocarbon gases with one to four aliphatic carbons on yeast growth quantitatively Large amounts of hydrocarbons are used and. We determined the 50% inhib- released into the atmosphere from many sources. y To whom correspondence should be addressed. gases hydrocarbon gases correlated to their hydrophobicity. In contrast. we found that UV absorbing materials at media. Additionally.3) Moreover. and Katsuhiro T AMURAy Department of Life System. coal mines. such as oxygen.9). Japan) is a common model organism. Online Publication. possibly resulting in the release of Technology. Toshiaki A RAO. yeast. yeast cells taken from a frozen stock culture were ing plants. Before each test.4–9) Toluene is reported to cause partial gases on the growth of the yeast Saccharomyces dissolution of the plasma membrane in Escherichia cerevisiae was investigated quantitatively by microca. 2-1 Minamijosanjima-cho. 20 g/l of peptone. October 7. were determined from growth thermograms. few data on the incubated at 30  C for 40 h. which can be regarded as indices of bile exhaust.tokushima-u. Based on Recently. and automo. 1991–1996. Both the 50% inhibitory pressure (IP50 ) and inhibition and loss of potassium ions and proteins in the minimum inhibitory pressure (MIP). 2010.Biosci. itory pressure (IP50 ) and the minimum inhibitory including oil extraction plants. When the compressed hydrocarbon gases. through observation of their propane < i-butane < n-butane. final O. and microbes.11) Further. we pressurize 260 nm were released from yeast cells exposed to yeast suspensions directly with the gases. 2010 Effects of Compressed Hydrocarbon Gases on the Growth Activity of Saccharomyces cerevisiae Satoshi KAWACHI. we developed a new method to quantify the these values. composed mostly of propane and gaseous hydrocarbons. The University of Tokushima.4) We assumed that this yeast species gas cylinders or by spills from hydrocarbon manufactur. nitrogen. In order to solve this problem. In support effects of these gases on microbial growth in aqueous of this. Biotechnol. the toxic extract). the inhibitory potency of the hydrocarbon toxicity of various gases.. Biochem. measurable.1) It is expected that these hydrocarbon gases gases was examined.7) Similar toxic effects might be regarded as indices of the toxicity of hydrocarbon gases.100156] The inhibitory action of compressed hydrocarbon intensively. Additionally. E-mail: tamura@chem. which are Yeast strain and cultivation. The yeast Saccharomyces cerevisiae found in permafrost regions and in continental slope IFO10149 (National Institute of Advanced Industrial Science and sediments worldwide.1) Liquefied natural gas (LNG). Tokushima 770-8506. In an attempt to mainly contains methane and ethane. Yoshihisa S UZUKI.

as employed in this study. makes it possible experiment. Hydrocarbon gases clearly decreased the growth rate the absorbance of the supernatant from 220 to 300 nm was measured of yeast and increased the incubation time required for with a double beam spectrophotometer (U-2001. measure OD. All gases for the experiments measuring turbidity (data not shown).1 M phosphate buffer. 1). Moreover. When the visible among the observed growth signals when an value of the specific growth activity was zero. ers. pressure gases. the loss of specific growth activity caused by gas detailed quantification of gas toxicity is possible. changes are was plotted against each gas pressure (Fig. For each gas. and dried in a freeze dryer (ES-2030. air. the specific growth activity monitoring microbial growth. Osaka. gases at 30  C for 24 h.10) these inhibitory effects can pressed hydrocarbon gases were collected. cerevisiae. This metabolic heat can be inhibition is available. 2). Hitachi). The yeast cells were fixed in 2% lated from each growth thermogram: the growth rate glutaraldehyde for 2 h at 4  C and 2% w/v osmium tetroxide for 2 h at constant  and the growth duration time t. growth was completely inhibited.10–13.1 M phosphate buffer inhibition. the cell suspensions were centrifuged at 1. They agree with the were applied above normal atmospheric pressure. 1). reached a peak. Hence calorimetry is a useful tool for for each hydrocarbon gas. More units. but 10. Besides. gamma rays. yeast trations. were constant between different gas pressures that do not induce high levels of growth inhibition. For quantification of UV absorbing substances. decreased with increasing gas pressure. we Each time-course of changes in metabolic heat introduced both the 50% inhibitory pressure (IP50 ).9% pure. The specific growth activity at pressure Results P MPa is given by p =m or t ð0Þ=t ðPÞ.2. produced during yeast incubation was recorded as a which reduces the growth of yeast by 50%.. In the case Calorimetry. activity of 1. of living cells.0561 MPa in the case of n-butane. pH 7. washed. The specific growth activity at a particular pressure. inhibitory pressure curve based on the hypothesis that Since measurement is continuous and fully automated. Measure- ments were done in duplicate so that four pressures could be tested in indicate that all the hydrocarbon gases tested had an one experiment. 1. Scanning electron microscopy. both specific growth activities.10) In short.1992 S.2. they were obtained from Takachiho Chemical Industrial Co. Hitachi. These results Instruments. 2A–E were then fitted to the minimum decompression (to count cells. We pressure P (1  p =m or 1  t ð0Þ=t ðPÞ) is propor- applied this method to analyze the effects of compressed tional to the m-th power of this gas pressure. essentially as described previously effects of chemical substances. argon.1 M phosphate buffer to a final concentration of 2:5  107 cells/ml. where p is the growth rate constant at pressure P MPa. p =m and to measure these changes in growth signals among t ð0Þ=t ðPÞ. Nippon Medical and Chemical incubation time with increasing pressure.10) employed as an index to evaluate the growth activities Based on the growth thermograms depicted in Fig. Eight high-pressure containers containing yeast of each gas. the calorimetric signals increased due to connected via high-pressure lines to gas cylinders. and When yeast cultures are incubated in the calorimetric t ð0Þ is this time when no extra pressure is applied. By contrast.15) nitrogen. Japan)10–15) and maintained at 30  C. Tokyo). After fixation. and krypton. For all the hydrocarbon gases tested in this lorimetry. After treatment with hydrocarbon effect on yeast growth than methane. (Tokyo). The area under the curves made as slowly as possible. The experimental set-up was as described growth thermogram.10) As indices of the toxicity of hydrocarbon gases. specific duration time from the parameters  and t respectively. The various compressed cultures without the need for gas data in Fig. Growth thermograms were ob- previously. and finally returned hydrocarbon gases were injected at the selected pressure. etc. the specimens were dehydrated with a series of quantitative description of the inhibitory effects of solutions of increasing ethanol concentrations. KAWACHI et al. Pressure treatment. gases t ðPÞ is the incubation retardation time under P MPa. and were better than 99. which dried cells were coated with platinum using an ion sputter (E-1010.10–15) Microca. washed in 0. can be determined as specific growth rate or as the scope (FE-SEM S-4700. Hitachi) and observed by a field-emission scanning electron micro. and resuspended in 0. Tokyo). Hitachi. yeast cultures or suspensions were aseptically tained that represent the incubation of yeast cultures added to sterile stainless steel (SUS630) 17-ml high pressure contain- under various compressed hydrocarbon gases (Fig. indicating that n-butane has a more inhibitory pH 7. from which growth of the yeast.). The air in the headspace of each container was displaced by the experimental gas.600  g for 5 min. and the absence of growth curves determined by colony counts and by extra pressure is indicated as 0 MPa. and resuspended in be quantitatively expressed using two parameters calcu- 0. substituted with t-butyl hydrocarbon gases on yeast growth is given by the alcohol. This hydrocarbon gases on the growth activity of baker’s hypothesis is applied to analyze the growth inhibitory yeast. m is the Growth thermograms under compressed hydrocarbon growth rate constant when no extra pressure is applied. and high- for the measurement of the effects of oxygen. A more 4  C. all pressures given in MPa corresponds to total growth activity. the apparatus detects the metabolic heat produced detailed information on the quantification of growth during yeast growth. In this study. the cultures to reach a certain activity level (Fig. a 50% reduction in peak height was achieved at 0. which lasted as indicated or until the calorimetric antimicrobial effect over the range of pressures applied. yeast inhibitor is added to yeast cultures at various concen. signals of all samples returned to baseline. Log-phase cells treated with com- As has been described. Ltd. Gas to baseline due to nutrient depletion and the accumu- (de)compression at the begining (and end) of each measurement was lation of toxic metabolites. Calorimetry is used to study the interaction growth was unaffected in the case of a specific growth between microorganisms and inhibitors. the initial slope of the growth thermograms culture were placed in the calorimetric units of the Biothermo decreased and the peak shifted towards a longer Analyzer (Biothermo Analyzer H-201.0 MPa of Release of cellular material. and the . S. The vessels were sealed with stainless steel caps Generally. and the supernatant and cell fractions were IP50 and MIP of the hydrocarbon gases collected separately. Exponentially growing yeast cells methane was needed to cause the same degree of growth were harvested by centrifugation.

for instance. C. A. cerevisiae cells. Ethane. .000 cells revealed that in the case of 5 to 10% of with compressed ethane and n-butane at various pres. 4) Examination spectra of the supernatants of yeast suspensions treated of about 1. either hydrocarbon gases varied with the length and shape of nucleic acids or fragments of these materials or amino the carbon chain. Methane. i-Butane.5 in the fitted compressed n-butane at 0. Effects of Compressed Hydrocarbon Gases on Yeast 1993 A B C D E Fig. under pressure of hydrocarbon gases might be affected due to these compressed gases. which was also observed after treatment with growth-activity related effects would not be measured. The 260 nm activity of zero in the fitted curve. 1.and about The cell morphology of S. defined as the gas pressure that yields a specific growth most cells survived the 24 h period. This increase might reflect the amount pressure. since. corresponds to the gas pressure that of dying cells. Propane. greater the growth inhibitory effects of the gas. The order of the inhibitory activity of hydrocarbon gases on SEM observation of yeast cells treated with com- yeast was methane  ethane < propane < i-butane < pressed hydrocarbon gases n-butane ( and < representing steps of 10. was dead. exposure to n-butane caused major cells resuspended in phosphate buffer were used so that intrusions. n-butane compressed cells. and hence these results indicate that summarized in Table 1. E. at which yeast pressures resulted in an increase in the absorbance at growth is completely inhibited. B. IP50 . the heat output (in mV) as measured over the time of incubation (in h) is represented by the indicated curve. sures for 24 h. the acids were released from the S. respectively). exponentially growing As shown in Fig. The inhibitory potency of the upon exposure to compressed hydrocarbon gases. In this experiment. Effects of Various Compressed Hydrocarbon Gases on the Growth of Yeast Saccharomyces cerevisiae. after treatment with results in a specific growth activity of 0. The lower the IP50 and MIP values. The minimum inhibitory pressure. For each applied pressure (MPa) of the tested gas. when yeast is We tested to determine whether cellular material is inhibited only by high-pressure gas) under 0. The 50% inhibitory 260 nm (Fig. 4B. 3). the cell shape was deformed. The IP50 and MIP absorbance peak can be attributed to compounds derived values for the various hydrocarbon gases tested are from nucleic acids. Hence we analyzed cells Leakage of cellular UV-absorbing substances grown for 15 h (during the log phase. and hydrocarbon gases by determining the UV absorption compared them to untreated cells (Fig. minimum inhibitory pressure (MIP). cerevisiae cells growing 3-fold reduction. MIP.030 MPa released when yeast cells are exposed to compressed n-butane by scanning electron microscopy (SEM). whereas when no extra gas pressure was applied. n-Butane. the other hydrocarbon gases at pressures that inhibited The application of these hydrocarbon gases at increasing growth activity by at least 15% (data not shown). D. the most cells were curve.07 MPa.

cerevisiae. B.132 0.80 Propane 0. The inhibitory effect of physiological properties of the dissolved gases. n-Butane. the inhibitory potency of the gases conclude that growth inhibition under compressed increased in the following order: methane  ethane < hydrocarbon gases is caused mainly by chemical or propane < i-butane < n-butane. and the minimum inhibitory pressure (MIP). Based on gases evaluated in this study (Table 1).776 1. we obtained the 50% at a value of about 50 MPa. Methane.16) which far exceeds any of inhibitory pressure (IP50 ) and the minimum inhibitory the MIP values (19 to 0. KAWACHI et al. In order to deter.0874 0. Propane. . i-Butane. The specific growth activity was determined from the growth rate constant (open circle) and the growth duration time (solid triangle).101 0.07 MPa) for the hydrocarbon pressure (MIP) of various hydrocarbon gases.0420 0.856 1. C. Effects of Various Compressed Hydrocarbon Gases on the Specific Growth Activity of the Yeast Saccharomyces cerevisiae.265 0.0723 The 50% inhibitory pressure (IP50 ).50 19. Ethane. A B C D E Fig. provide means of comparing the toxicities of different gases. 3.6 10.1994 S. 2. E. at which yeast growth is completely inhibited. A. we IP50 and MIP values. Ethane. Effects of Two Compressed Hydrocarbon Gases on the Release of UV-Absorbing Materials from the Yeast S.450 i-Butane 0. Yeast quantitatively by microcalorimetry. Therefore. espe- compressed hydrocarbon gas on yeast growth results cially in the case of n-alkanes with n > 2.275 0. B.3 18. and the dotted curve for the growth duration time.161 n-Butane 0. which reduces the growth of yeast by 50%. The solid curve was drawn to fit plots for the growth rate constant. Table 1. n-Butane.0481 0.75 0. growth is completely inhibited by hydrostatic pressure mine comparative toxicities. Fig. D.6 Ethane 0.0687 0. the inhibitory effects of compressed hydrocarbon gases on yeast growth were determined from both gas toxicity and hydrostatic pressure.430 0. Discussion A. In this study. IP50 and MIP Values of Various Hydrocarbon Gases A B Butane Growth rate constant Growth duration time Gas IP50 (MPa) MIP (MPa) IP50 (MPa) MIP (MPa) Methane 8.

20) the remnants of burst cells observed after gas decom- Furthermore.4) As discussed above. the hydrocarbon gases. Scanning electron microscopy indicated that the shape carbon gases can act on hydrophobic regions of the cell of yeast cells when treated with compressed hydro- membrane. cerevisiae cells and damage elles and that hydrostatic pressure thus gives rise to the membranes of DNA-containing organelles like mito. Exponentially growing cells untreated with n-butane.20) and decompression. in the case of n-butane even If hydrocarbon gases cause membrane damage.9) the permeability barrier of the cell membrane is disrupted and loss of proteins and potassium ions can It is known that the anti-microbial potency of alcohols be observed. Still. The MIP values were determined from the growth rate constant (open circle) and the growth duration time (solid triangle). Scanning Electron Microscopy Images of the Yeast S. of microorganisms has considerable effects on the B. is of negligible influence in the case the cell membrane. 5). with a correlation coefficient of 0.or ER. Researchers have reported that the stress caused by n-butane exposure.030 MPa n-butane inhibited yeast growth membranes might lead to loss of ribosomes and tRNA.9982.1. the outer shape of the derived compounds increased when the yeast cells were yeasts treated under hydrostatic pressure of up to treated with hydrocarbon gases. 5. the relationship between the inhib. The observed phenom- would manifest itself by the release of cellular materials. We is a result of harmful action on membrane permeability is cannot exclude the possibility that the majority of the in line with the mechanism proposed to explain toxicities cells were derived from a population that has adapted to of liquid hydrocarbons. Although alternation of membrane of a low test-pressure of 0. this at a low pressure. carbon gases was deformed. pressure of 0. Thus. since at an extra chondria or nuclei. Candida strain capable of metabolizing such alkanes. The linear relationship evaluated from the growth duration time is expressed by the formula log MIP ¼ 2:661{1:294 log Pow.030 MPa. This indicates that these hydro. Fig. A. cerevisiae Grown in YPD Medium Untreated and Treated with accumulation of liquid hydrocarbons in the membranes Compressed n-Butane.9981. The linear relationship calculated from the growth rate constant is expressed by the formula log MIP ¼ 2:703{1:318 log Pow. whereas damage to cell. of 0. Effects of Compressed Hydrocarbon Gases on Yeast 1995 Fig. by 25% (Fig.7) These effects might be due to disruption of that are structurally similar to hydrocarbons is dependent lipid-lipid and lipid-protein interactions in the plasma on their hydrophobicity. the amounts of extra-cellular nucleic-acid pression of the gas. ena might arise from hydrostatic pressure or decom- Indeed. Another study reported inhibition of the some gaseous hydrocarbons on yeast growth might glucose transport system by n-alkanes (8 < n < 12) in a therefore be related to their hydrophobicity. 3). 300 MPa was unaffected. this would not happen at the relatively low different from the appearance of holes in the cell wall or pressure used in the case of n-butane (Fig. When we examined the the smaller gaseous hydrocarbons affect glucose trans- relationship between MIP and the hydrophobicity of the port in a similar manner.19) The solid line fits plots for the growth rate constant. nearly linear (Fig. 5). it appears that hydrocarbon possible that hydrocarbon gases impair cellular organ- gases are taken up by S. Exponentially growing cells treated with compressed n-butane structural and functional properties of those mem- at 0. branes:5.030 MPa. The octanol-water partition coefficient used was as described else- where. 4. was higher cell deformity is the chemical or physiological proper- in the case of ethane than for the more hydrophobic ties of the dissolved hydrocarbon gases. . with a correlation coefficient of 0. Hydro.18) The inhibitory effects of membrane. The type of cell permeability is also known to be induced by hydrostatic damage when exposed to compressed hydrocarbons is pressure. we initially expected a larger fraction The hypothesis that the toxicity of gaseous hydrocarbons of visibly deformed cells than the observed 5–10%. deformities of the impaired cells. suggesting that com.6) phobicity for hydrocarbon gases is expressed by log That S. toxicity of the hydrocarbon gases studied here probably itory effect (log MIP) and hydrophobicity (log Pow) was stems from their capacity to affect membrane function. the pressure required for the release of pression. However.17.21) This suggests that the cause of the observed comparable levels of UV absorbing material. 2E). Comparison of Logarithm of the Minimum Inhibitory Pressure (Log MIP) and Hydrophobicity (Log Pow) for Various Hydrocarbon Gases. and the dotted line those for the growth duration time.030 MPa. which gives the distribution of a compound cannot use them as a carbon source makes it unlikely that between octanol and water. which pressed hydrocarbon gases affected the permeability of we applied slowly.7. cerevisiae does not oxidize such alkanes and Pow. It is also n-butane (Fig.

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