CAM101 Foundations of Medicine 1 2007

Course Study Notes
compiled by Hayden Farquhar June 2007

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These course note have been compiled by Hayden Farquhar for the unit CAM101: Foundations of Medicine 1 in the degree Bachelor of Medicine and Bachelor of Surgery. They are not intended to be in the hands of anyone but the person who compiled it. Hayden Farquhar is not claiming this to be his own work; indeed this information is not correctly cited and it is never the intention of the compiler for this work to be reproduced, sold, or used by anyone other than himself, as a reference study aid. This note set omits the course components of cell biology and immunology.

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H I S T O L O G Y ................................................................................................................................................................................ 1 EPITHELIA......................................................................................................................................................................................... 1 Characteristics of epithelium:..................................................................................................................................................... 1 Classification of epithelium: ....................................................................................................................................................... 2 EPITHELIAL CELL SPECIALIZATION ................................................................................................................................................... 4 GLANDS AND GLANDULAR TISSUE .................................................................................................................................................... 8 CONNECTIVE TISSUE ...................................................................................................................................................................... 16 What is connective tissue? ........................................................................................................................................................ 16 Cells of connective tissue: ......................................................................................................................................................... 16 Fibroblasts ................................................................................................................................................................................ 16 Extracellular Matrix ................................................................................................................................................................. 17 Embryonic connective tissue: ................................................................................................................................................... 21 Loose connective tissue: ........................................................................................................................................................... 22 MUSCLE TISSUE.............................................................................................................................................................................. 30 Types of muscle tissue:.............................................................................................................................................................. 30 Cardiac muscle: ........................................................................................................................................................................ 33 NERVOUS TISSUE............................................................................................................................................................................ 36 Synapses:................................................................................................................................................................................... 38 Myelination of axons: ............................................................................................................................................................... 38 HISTOLOGY OF THE INTEGUMENTARY SYSTEM ............................................................................................................................... 42 Structures of the skin ................................................................................................................................................................. 42 Layers and tissues of the skin.................................................................................................................................................... 42 Epidermis .................................................................................................................................................................................. 42 Epidermis and dermis ............................................................................................................................................................... 44 Appendages of the skin.............................................................................................................................................................. 46 B I O C H E M I S T R Y – N U T R I T I O N A N D M E T A B O L I S M ................................................................................... 49 AMINO ACIDS, PROTEINS AND ENZYMES ......................................................................................................................................... 49 Structure of amino acids and proteins ...................................................................................................................................... 49 Proteins ..................................................................................................................................................................................... 50 Protein and amino acid turnover in the body ........................................................................................................................... 51 Imbalances in protein and amino acid turnover ....................................................................................................................... 53 Enzymes..................................................................................................................................................................................... 54 Proteins and enzymes in diagnosis ........................................................................................................................................... 56 CARBOHYDRATES ........................................................................................................................................................................... 57 Structure of common carbohydrates in the body and in foods ................................................................................................. 57 Functions of glucose and glycogen ........................................................................................................................................... 58 Glucose and glycogen turnover in the body ............................................................................................................................. 59 Starches ..................................................................................................................................................................................... 60 Dietary fibre .............................................................................................................................................................................. 60 Glycosaminoglycans ................................................................................................................................................................. 60 Carbohydrates in diagnosis ...................................................................................................................................................... 60 Is carbohydrate essential in our diet? ...................................................................................................................................... 61 LIPID (FATS) ................................................................................................................................................................................... 61 Structure of fatty acids and triglycerides .................................................................................................................................. 61 Functions of fatty acids and triglycerides ................................................................................................................................. 62 Triglyceride and fatty acid turnover in the body ...................................................................................................................... 63 Specialised lipids ...................................................................................................................................................................... 64 Lipids in diagnosis and therapy ................................................................................................................................................ 65 Summary of fatty acid usage ..................................................................................................................................................... 65 MICRONUTRIENTS .......................................................................................................................................................................... 66 Some roles of vitamins .............................................................................................................................................................. 66 Roles of some minerals and trace elements .............................................................................................................................. 67 A HEALTHY DIET ........................................................................................................................................................................... 68 Nutrient Reference Values (NRV) ............................................................................................................................................. 68 Dietary guidelines for Australians, 2003 .................................................................................................................................. 68 METABOLISM – BASIC PRINCIPLES .................................................................................................................................................. 70 Key metabolic situations ........................................................................................................................................................... 70 Key ‘energy’ molecules: ATP, ADP and AMP ......................................................................................................................... 72 CATABOLIC AND ANABOLIC PROCESSES ......................................................................................................................................... 74 Key fuels used ........................................................................................................................................................................... 74 Fundamental process in catabolism – oxidation ...................................................................................................................... 74 Aerobic catabolism ................................................................................................................................................................... 75 Fuel catabolism under anaerobic conditions is much less efficient ......................................................................................... 76 Page 3

Anaerobic processes (introduction and examples) ................................................................................................................... 76 B I O C H E M I S T R Y – D N A C O M P O N E N T ................................................................................................................... 78 DNA STRUCTURE AND THE GENETIC CODE .................................................................................................................................... 78 Structure of DNA....................................................................................................................................................................... 78 DNA replication ........................................................................................................................................................................ 80 DNA REPLICATION AND REPAIR ..................................................................................................................................................... 82 G E N E R A L P A T H O L O G Y ................................................................................................................................................... 93 CELLULAR RESPONSES TO STRESS AND NOXIOUS STIMULI.............................................................................................................. 93 Common harmful cellular alterations ....................................................................................................................................... 94 Organelles involved in the cellular response to injury ............................................................................................................. 95 Cellular adaptations – intracellular accumulations ................................................................................................................. 96 Tissue changes .......................................................................................................................................................................... 96 APOPTOSIS AND NECROSIS ............................................................................................................................................................. 97 Apoptosis ................................................................................................................................................................................... 98 Necrosis................................................................................................................................................................................... 101 INFLAMMATION ............................................................................................................................................................................ 104 Acute inflammation ................................................................................................................................................................. 104 The five cardinal signs of inflammation.................................................................................................................................. 108 TISSUE REPAIR, FIBROSIS AND REGENERATION ............................................................................................................................. 108 Outcomes of acute inflammation ............................................................................................................................................ 109 Healing of skin ........................................................................................................................................................................ 110 Factors impairing the ability of tissue to repair ..................................................................................................................... 111 Factors that promote repair.................................................................................................................................................... 111 Summary diagram ................................................................................................................................................................... 112 CHRONIC INFLAMMATION............................................................................................................................................................. 112 Chronic inflammation arises in the following circumstances: ............................................................................................... 113 Morphologic features of chronic inflammation ...................................................................................................................... 113 Mononuclear (macrophage) cell infiltration .......................................................................................................................... 113 Other cells involved in chronic inflammation ......................................................................................................................... 114 Granulomatous inflammation ................................................................................................................................................. 115 N E O P L A S I A ............................................................................................................................................................................. 117 GENERAL FACTS ABOUT NEOPLASIA ............................................................................................................................................. 117 NOMENCLATURE AND GENERAL PRINCIPLES ................................................................................................................................ 117 Benign tumours of mesenchymal (non-epithelial) origin........................................................................................................ 117 Benign tumours of epithelial origin ........................................................................................................................................ 117 Malignant tumours of mesenchymal (non-epithelial) origin .................................................................................................. 118 Malignant tumours of epithelial origin ................................................................................................................................... 118 TUMOUR MORPHOLOGY, BEHAVIOUR AND GROWTH .................................................................................................................... 119 General features...................................................................................................................................................................... 119 Tumour history ........................................................................................................................................................................ 119 Differentiation and anaplasia ................................................................................................................................................. 120 Dysplasia................................................................................................................................................................................. 121 Neoplastic angiogenesis.......................................................................................................................................................... 122 Local Invasion ......................................................................................................................................................................... 123 Metastasis ............................................................................................................................................................................... 124 Aetiology ................................................................................................................................................................................. 126 THE MOLECULAR BASIS OF CANCER ............................................................................................................................................ 127 Five fundamental principles underlie the molecular basis of cancer ..................................................................................... 127 The essential molecular alterations for malignant transformation ........................................................................................ 128 SKIN TUMOURS ............................................................................................................................................................................. 129 Naevi ....................................................................................................................................................................................... 129 Dysplastic naevus.................................................................................................................................................................... 130 Lentigo maligna ...................................................................................................................................................................... 131 Malignant melanoma .............................................................................................................................................................. 131 Squamous cell carcinoma ....................................................................................................................................................... 133 Basal cell carcinoma............................................................................................................................................................... 134 Benign skin neoplasms ............................................................................................................................................................ 135 H O M E O S T A S I S ...................................................................................................................................................................... 138 BODY FLUID COMPARTMENTS ...................................................................................................................................................... 138 Thirst mechanism .................................................................................................................................................................... 140 Regulation of water output...................................................................................................................................................... 141 Starling's Law of the Capillaries ............................................................................................................................................ 141 Page 4

Burns ....................................................................................................................................................................................... 143 HOMEOSTATIC FUNCTION OF THE SKIN ......................................................................................................................................... 145 Body heat ................................................................................................................................................................................ 145 Effect of variation of ambient temperature on skin temperatures .......................................................................................... 145 Functional relationships within the skin vasculature ............................................................................................................. 151 I N T R O D U C T I O N T O P H A R M A C O L O G Y .............................................................................................................. 155 PHARMACOLOGICAL PRINCIPLES.................................................................................................................................................. 155 What is a drug? ....................................................................................................................................................................... 155 Some basic pharmacological terminology .............................................................................................................................. 156 Potential drug targets: membrane bound enzymes ................................................................................................................. 156 Potential drug targets: G-protein coupled receptors ............................................................................................................. 156 Potential drug targets: ion channel receptor proteins ........................................................................................................... 156 Potential drug targets: transport proteins .............................................................................................................................. 156 More terminology.................................................................................................................................................................... 156 Polypharmacy ......................................................................................................................................................................... 157 DRUG DELIVERY ........................................................................................................................................................................... 157 Why are there different routes of drug delivery? .................................................................................................................... 157 Different routes of drug administration .................................................................................................................................. 158 Different forms of medication ................................................................................................................................................. 158 How drugs get to their site of action ....................................................................................................................................... 158 Oral administration ................................................................................................................................................................ 159 Parenteral administration ....................................................................................................................................................... 159 Drug prescription quantities ................................................................................................................................................... 159 Some common abbreviations and symbols used in pharmacology ......................................................................................... 160 BASIC PRINCIPLES OF RECEPTORS, LIGANDS, AND SECOND MESSENGER SYSTEMS........................................................................ 160 Drug action at the receptor can vary depending on the drug-target interaction ................................................................... 160 The four receptor superfamilies .............................................................................................................................................. 161 Exam question examples (from all Dr Foa’s lectures) ........................................................................................................... 165 THE DYNAMIC AND EXCITABLE CELL MEMBRANE ........................................................................................................................ 166 Why are membranes dynamic and excitable? ......................................................................................................................... 166 What is a membrane potential? .............................................................................................................................................. 166 The proteins responsible for maintaining the membrane potential ........................................................................................ 166 The resting membrane potential ............................................................................................................................................. 167 Electrical activity within neurons ........................................................................................................................................... 168 Why cells need a membrane potential .................................................................................................................................... 169

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Histology
Histology is the study of tissues, and cells in tissue formation. This course covers the histology of the four basic adult tissue types (epithelia, connective tissue, muscle and nervous tissue) as well as covering the structure and function of the integumentary system, concurrent with the integrated learning topics on the skin throughout the unit.

Epithelia
General facts about epithelium:
• • •

Epithelium covers the body surfaces, both internally and externally (i.e., a surface = covered by an epithelium. Two exceptions - joint cartilage and brain) Epithelium acts as a boundary or interface between different compartments within the body, or between the body and the external environment. I.e., compartmentalisation These boundaries and interfaces can facilitate: • protection (skin) - resist abrasion, stop things getting in and out • absorption (small intestine) - increase surface area • trans-cellular transport of molecules - associated with absorption (e.g. lungs) (requires epithelium to be very thin for efficient exchange of gases - remember structure versus function) • selective permeability (because of the abilities of the plasma membrane) • filtration (kidney) - get rid of wastes and water • excretion (kidney, intestines, skin) (this is eliminating metabolic waste products) • secretion (glands, small intestine, kidney) (this is releasing chemicals from a cell) Epithelium forms all of the glands of the body (both endocrine (onto ducts; they have maintained their epithelial connection) and exocrine (into the bloodstream; they have not maintained their epithelial connection))

Characteristics of epithelium:
• • • • • • • • •

Cellularity - there is very little extracellular space between cells of epithelia, highly cellular Specialised contact between cells - cell junctions (these occur elsewhere of course) Polarity - apical, basal and lateral domains (and specialisations of cell junctions associated with these) Basal lamina (at the EM level) - (basement membrane at the LM level) - ALL epithelia sit on a basement membrane (anchorage) Supported by underlying connective tissue (lamina propria - general term) - both mechanical and nutritional support Avascular - doesn't have a vascular supply, so must receive nutrients from the underlying connective tissue, via diffusion or other methods Innervated - responds to stimuli (touch, pain, heat, etc.) Regeneration - can repair itself (e.g. in the skin, the stratum basale is highly mitotically active) Cell membrane specialisations - cilia, microvilli (not flagella because sperm is not epithelium!)

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Classification of epithelium:

First classification - number of cell layers • Simple - one cell layer thick • Stratified - more than one cell layer thick • Pseudostratified - one cell layer with all cells touching the basemement membrane that gives the appearance of stratified. Not all cells touch the surface. Second classification - shape of cells (always on the apical or luminal surface - this is where we get the classification) 1. Squamous - wider than they are tall (flattened) 2. Cuboidal - as wide as they are tall (box!) 3. Columnar - taller than they are wide Note in some epithelia the shape of cells will differ from the apical to the basal portions. We always take the classification from the apical cells. Example - skin, where the apical cells are squamous, and the cells in the stratum basale can often be cuboidal to low columnar.

Common types of epithelium:
• • • • • • •

Simple squamous Simple cuboidal Simple columnar Stratified squamous Stratified cuboidal Pseudostratified ciliated columnar with goblet cells Urinary or transitional

Simple squamous epithelium:
This type of epithelium will always form the surface where passive transport of gases or fluids will occur. This makes sense because a simple squamous epithelium is very thin, and will therefore more easily facilitate passive transport, especially where it is required to move quite quickly. Examples include the alveoli of the lungs, the glomerulus of the kidneys and capillaries (endothelium). Simple squamous epithelial cell have flattened, long nuclei, reflecting the shape of the whole cell, and contain little cytoplasm (again think structure versus function - less cytoplasm, faster transport). The cells are much, much wider than they are tall. Simple squamous epithelium covers the external surface of many of the organs - this is known as MESOTHELIUM It also lines the lumen of heart chambers, blood vessels and lymphatic vessels - this is known as ENDOTHELIUM In these cases, this type of epithelium also provides a smooth, frictionless surface.
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Simple cuboidal epithelium:
The cells of this type of epithelium have a round, centrally-placed nucleus (again, reflecting the shape of the cell). The nuclei will appear to form a single row. The cells appear square in a histological section. All cells contact the basement membrane. This type of epithelium lines many ducts. They may have excretory, secretory or absorptive functions depending on their location.

Simple columnar epithelium:
This epithelium features elongate, sausage-shaped nuclei, usually found in a basal position, forming a singlerow of basophilia under histological section. This reflects the shape of the overall cells, which are taller than they are wide. Because the epithelium is simple, all cells touch the basement membrane. This type of epithelium lines parts of the gastrointestinal system, the reproductive system and very large ducts. They play an absorptive and secretory role. If they are ciliated, they may also play a role in the transport of extracellular fluids and structures by creating a current, like in the uterine tube with the transport of the oocyte.

Pseudostratified ciliated columnar epithelium:
The appearance of this epithelium may be confusing, because it can look stratified, but in fact all cells touch the basement membrane. However, not all cells reach the surface. This epithelium is mainly found in the respiratory tract (and can be known as respiratory epithelium), in association with goblet cells. The non-ciliated type can be found in some of the ducts of the male reproductive system. Functions of this epithelium include movement of surface fluids, absorption and secretion.

Stratified squamous epithelium:
This epithelium has many layers of nuclei, and will show a progressive change in nuclear shape from basal to apical regions. In some cases, the nuclei will not be present in cells of the apical surface. The epithelium may be keratinised or non-keratinised. Because it is stratified, not all cells touch the basement membrane. Found in areas the require an abrasion-resistant, protective surface (skin, mouth, vagina, anal canal).

Stratified cuboidal epithelium:
This epithelium consists of 2-3 layers of cuboidal/low columnar cells. They are found lining larger excretory ducts of exocrine glands (e.g. the salivary glands).

Transitional epithelium:
Found only in the excretory passages of the urinary system, that is, the renal pelvis, ureters and bladder.
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Also called urinary epithelium, or urothelium. Stratified epithelium with domed cuboidal cells on the surface of the relaxed epithelium, and irregularlyshaped nuclei. The surface cells show cell membrane specialisations to allow for expansion of the organ (like plaques).

Epithelial cell specialization
Basement membrane (and basal lamina etc.):
Always remember that every epithelium sits on a basement membrane. The basement membrane is sandwiched between the epithelium and the underlying loose connective tissue. Both layers - the epithelium and the connective tissue - form and contribute to the basement membrane. "Basement membrane" is a light-microscopical term. "Basal lamina" is an electron-microscopical term. The basement membrane is normally resolved after special staining of tissue and is not normally visible in normal staining. At the EM level, there are two layers which define the basement membrane: 1. Basal lamina - formed by the cells of the above epithelium 2. Reticular lamina - formed by the cells of the underlying connective tissue Properties of the basement membrane include:  it can be visualised using special stain, for example PAS stain (periodic acid Schiff's reagent stain)  the basal lamina can be subdivided into the  lamina densa - 50-100 nm  lamina rara (or lamina lucida) The basement membrane is composed of: • Type 4 collagen - a product of EPITHELIAL CELLS • Proteoglycans (heparin sulphate and condroitin sulphate) - a product of EPITHELIAL CELLS • laminin (a glycoprotein) - a product of EPITHELIAL CELLS • entactin and fibronectin (glycoproteins) • anchoring filaments (type 7 collagen) - a product of FIBROCYTES • reticular fibres of type 3 collagen - a product of FIBROCYTES Functions of basal laminae: 1. Structural attachment 2. Compartmentalisation 3. Filtration 4. Polarity induction 5. Tissue scaffolding Muscle and nervous tissue also have a surrounding basal lamina (known as the external lamina).

Cell Membrane Junctions (lateral and basal cell membrane modifications):
There are five types of cell junctions. Occluding junctions, or tight junctions:
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zonula occludens Adhering junctions zonula adherens macula adherens, or, desmosomes hemidesmosomes Communicating junctions gap junction or nexus

Occluding junctions - remember - OCCLUDIN and CLAUDIN

These junctions help to define the polarity of the cell. Their principal function is to control the passage of substances between adjacent, connecting cells. Occcluding junctions, called zonula occludens, have a beltlike distribution like a ribbon, internally bracing the cells. These junctions are particular evident between epithelial cells that have secretory or absorptive functions. This makes sense because once a cell secretes a product, it doesn't want the product seeping back next to the cell itself. The actual occluding part of the zonula occludens are intramembranous proteins, occludin and claudin, that "stitch" the membrane of adjacent cells together. This prevents substances passing through this junction. This means that if substances need to get through the epithelial layer, it must do so via the transcellular pathway.

Zonula adherens - remember - ACTIN and CADHERINS

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The zoluna adherens is also called the belt desmosome. It has a belt-like distribution. It is associated with actin filaments. In the diagram, it is the junction on the left (note the belt-like distribution). These cell junctions are for strength, and robustly hold cells together, but they are very leaky, and require the presence of occluding junctions to stop substances getting past the cells. Important - plasma membrane plaques are used as anchorage points by the actin filaments of the cytoplasmic skeleton. The cadherins (desmocollins and desmogleins) are transmembrane proteins.

Macula adherens or spot desmosome:

This in an anchorage junction with a spot-like distribution. They have identical structure to zonula adherens except they are spots rather than a belt. Also, their plaques are associated with intracellular intermediate filaments, not actin filaments like in zonula adherens. There may be many macula adherens per cell.

Hemidesmosomes:

Hemidesmosomes are the cell junctions that link the basal domain of an epithelial cell to the basal lamina of the basement membrane. On the cellular side, intermediate filaments called tonofilaments are associated with intracellular plaques, which attach to anchoring filaments (laminin 5) in the basal lamina.
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Communicating or gap junctions/nexus:

These cell junctions essentially connect the cytoplasm of one cell to another. A gap junction is formed by connexons, which are a group of 6 channel-like proteins called connexins (notice the difference) that permit the passage of small molecules between adjacent cytoplasms. That is, six connexins = one connexon. This allows for coordinated action of multiple cells. Remember that electrochemical gradients can instigate intracellular responses? Well these gradients can be facilitated by gap junctions. This means that not every cell needs to be innervated. A key example of the use of the gap junction is the intercalated disks of the cardiac muscle cell.

Microvilli:

Microvilli (singular microvillus) are an apical surface modification. Microvilli are formed from a core of microfilaments (actin) that are extensions of the cytoplasm that contain a bundle of actin. Because they are extensions of the cytoplasm of the cell, they have the ability to greatly increase the surface area of the cell. In this manner, the microvilli can be very short on non-absorptive cells, to very long on highly absorptive cells (think of the small intestine). The benefits of increasing surface area depend on the function of the cell. It could increase the amount of plasma membrane proteins (like digestive enzymes or ion channels) or allow more room for endocytotic processes. At the LM level, because there are a large number of glycoproteins present at the end of long microvilli, in certain areas the microvilli form a brush border. Remember, lots of glycoproteins on the surface of cells all together are known as a glycocalyx.
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Microvilli are immotile. They do NOT move anything along the surface.

Stereocilia are elongated, immotile microvilli, and are thus poorly named. They are only found in two places: the epididymis of the male reproductive system, and the sensory cells of the ear. For the epididymis, they increase surface area for absorption, and in the ear they act as sensory receptors.

Cilia are much longer and thicker than microvilli. They have a core of microtubules (known here as an axoneme), which arise from an area just deep to the projection of the cilia from the plasma membrane, called the basal body. The axoneme is an arrangement of microtubules we term the 9+2 pattern: a ring of 9 pairs of microtubules with a pair in the middle. Each pair interacts with its neighbour via protein extensions called dynein arms. The dynein arms function as motor elements that enable the cilia to move. Cilia produce a whipping-like motion. Therefore they are responsible for the transport of surface materials. They can be found in the respiratory and female reproductive systems.

Glands and glandular tissue
Glands:
Glands are still epithelium, and therefore always have a basement membrane and associated loose connective tissue.
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All glands form as downgrowths of epithelia. That is, the epitheliem grows downwards during the gland's development, and some cells differentiate into the secretory portion of the gland, and some cells differentiate into the ductal portion of the gland. If the gland retains its connection to the original surface epithelium, it is known as an exocrine gland. If the gland does not retain its connection to the original surface epithelium, it is known as an endocrine gland, and they release their secretions into the bloodstream.

Classification of Exocrine glands:
Exocrine glands can be classified in a number of ways. The first of which is how many cells make up the gland: Unicellular glands The mucous-secreting goblet cell found in the digestive and respiratory system is an example of this type of gland. They are single cells that secrete their products onto the epithelial surface of a lumen. They are components of that epithelium such that the epithelium is described as containing goblet cells (e.g. pseudostratified ciliated columnar epithelium with goblet cells for the respiratory tract). Multicellular glands These are further subdivided based upon the structure of their DUCT. They can either be: Simple (with only a single duct) Compound (with a brancing duct system) Compound glands are again further subdivided based upon the structure of their secretory portion. They can be either: Tubular Alveolar (or acinar) Tubuloalveolar. So, breaking it down: Exocrine glands Unicellular Multicellular Simple duct system Compound duct system Tubular secretory portion Alveolar secretory portion Tubuloalveolar secretory portion

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Structure of compound exocrine glands:
All compound exocrine glands contain epithelial components (secretory acini and ducts), which we call the parenchyma, and the supporting tissue associated with it (which includes blood vessels and nerves), called the stroma. Often, a gland will be surrounded by a connective tissue capsule, which branches itself inside the gland, forming septa that subdivides the septa into pieces. Larger compound glands may have lobes, which are divided by interlobar septa, and the smaller lobules, divided by interlobular septa.

Within the lobules of alveolar and tubuloalveolar glands the secretory cells are arranged into groups called acini (acinus singular). Each acinus may be surrounded by several contractile cells called myoepithelial cells. These are modified epithelial cells whose cytoplasm have contractile properties that help the secretion move up the duct system. The duct from each acinus joins to form intercalated ducts (unlike the diagram above), which are lined by simple squamous epithelium. In salivary glands, intercalated ducts join into striated ducts. These ducts are lined by a simple cuboidal to low columnar epithelium, and have many basal infoldings that give them a striped appearance under histological section. These then join to form intralobular ducts, which join with others to form interlobular ducts, which are lined by pseudostratified cuboidal to low columnar epithelium. Interlobular ducts join to form intralobar ducts and intralobar ducts join to form lobar ducts, which are lined by stratified cuboidal to columnar epithelium. To clarify:
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Acinus secretes into Intercalated ducts--------------simple squamous epithelium--------------------many of these join into (Striated ducts---------salivary glands only-----------basal infoldings-------------simple cuboidal to low columnar--------many of these join into) Intralobular ducts-------------------------------------------------------------------------many of these join into Interlobular ducts---------------------------------pseudostratified cuboidal to columnar epithelium----------many of these join into Intralobar ducts---------------------------------------------------------------------------many of these join into Lobar ducts-------------------------------lined by stratified cuboidal to columnar epithelium.

Methods of secretion from a cell:
There are three main methods by which cells release their secretion. These are: • Merocrine secretion • Holocrine secretion • Apocrine secretion

Merocrine secretion is when cells release their secretions by the normal process of exocytosis. The secretory material of each secretory glandular cell is held inside membrane-bound vesicles inside the cell until the cell is stimulated to release them. When they are stimulated to release their secretion, the vesicles bind to the plasma membrane, the secretion is released into the excretory duct. The membranes of the secretory vesicles then become a part of the plasma membrane, and are not lost. There is no alteration or damage to the cell in the process of merocrine secretion. Examples of merocrine secretion are the sweat glands, salivary glands, and the exocrine pancreas.

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Holocrine secretion is where the glandular cells themselves become part of the secretion. The secretory cells accumulate the main component of the secretion. The cells then die (whether by the overwhelming accumulation and disruption of metabolic process or a process of apoptosis), and rupture. The secretory product and the cell's cytoplasm is released as part of the secretion. More basally located cells replace the lost cells. Sebaceous glands of the skin use this method of secretion. A way to remember this is holocrine = whole cell dies, like holocaust = whole race dies.

Apocrine secretion involves large parts of the cytoplasm of the glandular cell pinching off and becoming the secretory product, a bit like holocrine secretion, but not the whole cell. The secretory product is stored in the glandular cell near the luminal surface. When stimulated to secrete, that region of the cytoplasm is isolated by the plasma membrane and released into the duct system, much like a giant vesicle. Classification of apocrine secretion is somewhat controversial. Many glands previously identified as apocrine are now, with developments of EM, classified as merocrine. The important example is the mammary glands, that produce milk, and in particular, the fats involved in milk. This makes sense because it most likely involves a fat-based secretory product that is not membrane bound, and therefore cannot pass through the plasma membrane in normal exocytosis.

Type of secretion from an exocrine gland cell:
There are two main types of secretions that come from an exocrine gland (note we are no longer talking about method, we are talking about the secretion itself). The salivary glands are a really good example of showing their difference. There are, of course, many more types of secretion than will be mentioned here (for example, think of the cells of the gastric glands of the stomach that secrete hydrochloric acid), but for first semester, these are probably the only ones you will encounter. The salivary glands (exocrine glands found within the oral cavity) secrete either a mucous secretion (and is therefore a mucous gland), or a serous secretion (and is therefore a serous gland), or a mucoserous secretion (a combination of both, and is therefore a mucoserous gland).

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The three major salivary glands are: Parotid gland - entirely SEROUS (100%) Submandibular gland - mostly SEROUS, some MUCOUS (80/20%) Sublingual gland - mostly MUCOUS, some SEROUS (80/20%)

A mucous-secreting cell (which you should not confuse with a mucous secreting goblet cell) has a flattened, peripherally-located nucleus and mucin granules that stain very lightly under H&E. This sort of secretion is viscous, sticky, and high in carbohydrates.

A serous-secreting cell has a rounder, centrally-located nucleus, and a cytoplasm that contains typical protein-synthesising organelles. They therefore stain well under H&E. The secretion is thin, watery, and high in proteins (usually enzymes). When a gland is mucoserous, the arrangement is usually of note. Under histological preparation, certain things happen that push the serous-secreting cells to the edge of the acinus, resulting in what we call serous demilunes.

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Examples of exocrine glands:
Simple unbranched tubular gland Simple coiled tubular gland Simple branched tubular gland Simple branched acinar gland Compound tubular gland Compound acinar gland Compound tubuloacinar gland Intestinal glands (crypts of Lieberkuhn) Eccrine sweat glands Gastric glands of the stomach Sebaceous glands of pilosebaceous apparatus Brunner's glands of the duodenum Mammary glands; exocrine portion of pancreas Salivary glands

Endocrine glands:
Endocrine glands do not have ducts. Their secretions diffuse directly into the surrounding connective tissue and bloodstream within. Their secretory products are usually hormones, whose action is usually effective far away from its point of secretion. Example: the pituitary gland is located inferior to the hypothalamus and produces hormones that affect many parts of the body far from its location.

Pancreas:
The pancreas is interesting because it is a mixed gland, which means it is both endocrine and exocrine. In this case, the exocrine and endocrine secreting cells are different cells that are found in different compartments within the gland. A group of exocrine cells secreting into a common duct, is, as we know, an acinus. The exocrine portion of the pancreas is considered either a compound acinar or compound tubuloacinar gland. The main secretions of the pancreas leave the pancreas by one or two major excretory ducts and are released into the duodenum of the small intestine. The endocrine portion of the pancreas form small island clusters of cells, known as the Islets of Langerhans. They have many capillaries associated within them so that their hormones (e.g. insulin, glucagon) can enter the bloodstream.

Some additional notes to remember from the practical:
1. Remember that slide 78 is of the trachea and oesophagus of a rat, and the oesophagus is lined by a stratified squamous keratinised epithelium. This is not the same as humans. Rats have a different diet to us and require a more abrasion-resistant epithelium in theor oesophagus. Humans have a stratified squamous non-keratinised epithelium lining our oesophagus. Also, a moist environment will not harbour a keratinised epithelium.
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2. You should also review the diagrams depicting the types of DUCTS for exocrine glands. A good one is in Kierszenbaum p59 (2nd edition)

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Connective Tissue

What is connective tissue?
Connective tissue is one of the four basic adult tissue types. Embryonically, it is of mesodermal origin (the middle plate). Functionally, it provides structural and metabolic support for other tissues, and has an an enormous range of functions. Connective tissue consists of two components: • Cells (relatively, few) • Extracellular matric (relatively, connective tissue is mostly extracellular matrix, by volume) (Recall that the reticular lamina from the basement membrane comes from connective tissue). When talking about the terms parenchyma and stroma, connective tissue is represented by the stroma. If you recall the functional lobules of the structure of the liver, the actual lobules are the parenchyma, and the connective tissue stroma is the tissue in between each lobule, providing support (both structurally and functionally) for the lobules. Recall: ectoderm - "external" epithelia mesoderm - all connective tissue, most muscle endoderm - epithelium of internal organs NOTE: the above is NOT a comprehensive list and is intended only to remind you where connective tissue comes from.

Cells of connective tissue:
There are four major cell types that are principally classified as connective tissue. They are: 1. 2. 3. 4. Fibroblast - the true connective cell. Produces the extracellular matrix Macrophage - an antigen-presenting phagocytic cell (part of the immune response) Mast cell (originated from elsewhere) Plasma cell (originated from elsewhere)

Fibroblasts

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Fibroblasts have different names that depend on the location and type of tissue they are present in. For example, • a fibrocyte is present in fibrous tissue (like loose connective tissue), • an osteocyte is a mature cell present in bone, • a chondrocyte is the main cell in cartilage, and • an adipocyte is a fat cell. We also need to make clear the difference between -blast and -cyte cells. The suffix "blast" usually refers to an immature cell that is actively producing the connective tissue component (that is, the extracellular matrix), whereas the "cyte" is referring to the mature cell that is just maintaining the tissue. For example, a fibroBLAST is acively producing and secreting colagen fibres and ground substance, and an osteoBLAST is actively synthesising and secreting the bone matric (osteoid). An osteoCYTE will be the mature form of the osteoBLAST, will be less active, and will be maintaining the secretion. The cells of connective tissue are widely scattered, separated from each other by extracellular matrix. Few, if any, cell junctions exist between the cells of connective tissue. Very important to remember: The cells produce the extracellular matrix! Also very important: many other cell types may be foound in connective tissue, that is, they make use of the connective tissue properties to "do their own thing".

Extracellular Matrix
Extracellular matrix (ECM) is composed of (amorphous) ground substance and fibres. Amorphous ground substance consists of: GAGs (glycosaminoglycans) Hyaluronic acid Proteoglycans These substances form a hydrated gel in which the cells and fibres are found. Fibres include: Collagen fibres (including reticular fibres) Elastic fibres Always remember: ground substance and fibres are secretions of the connective tissue cells (fibroblasts, chondroblasts, osteoblasts, adipoblasts).

Glycosaminoglycans:
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There are four groups of glycosaminoglycans. Glycosaminoglycan 1. Hyaluronic acid Sulation? no Protein linked? no Distribution synovial fluid, skin, support tissue cartilage, bone, skin, support tissue skin, blood vessels, heart BM, lung arteries lung, liver, skin, mast cell granules cartilage, cornea, vertebral disc

2. Condroitin sulphate 2. Dermatan sulphate 3. Heparan sulphate 3. Heparin

yes yes yes yes

yes yes yes yes

4. Keratan sulphate

yes

yes

The sulphation is important. Sulphation causes the groups to be highly negatively charged, which contributes to their ability to retain sodium ions and water. All of the glycosaminoglycans are bound to a protein to form a proteoglycan (except hyaluronic acid). This aggregate is described on p119 of Kierszenbaum, for clarification.

Glycosaminoglycans remain expanded and therefore occupy an enormous volume for a small mass. This forms a flexible gel through which metabolites can diffuse easily.

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Fibres:
Collagen

Collagen is the principle fibre in the ECM. It is the most abundant fibre found in the body. Approximately 19-21 different types of collagen have been described, and up to 27 identified. Each type of collagen has a different 3-dimensional structure which confers different properties. (Recall that with proteins, it is the three-dimensional structure that defines its function). Type I: Type II: Type III: Type IV: Type VII: The most common form of collagen Cartilage Reticulin (more often referred to as reticular fibres) Basal lamina Anchoring filaments in BM

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Each collagen molecule is three proteins woven together. This gives collagen its great tensile strength. Under electron microscopy, they are arranged as 67-68 nm-thick bands, and can sometimes be confused with skeletal muscle because of its striated appearance. You would rarely, if ever, be asked to differentiate between skeletal muscle and dense regular connective tissue, and if you are, it would be stained in a particular way to make it quite obvious. The above picture contains both collagen and elastic fibres - make sure you learn which are which because it's a good prac exam question. Reticular fibres (type III collagen) These fibres are found at the boundary between epithelium and its underlying loose connective tissue. Reticular fibres also surround adipocytes, small blood vessels, nerves, and muscle. See below section of the liver, stained particularly to highlight the presence of reticular fibres.

Reticulin is an early form of connective tissue and is often later replaced by type I collagen. It forms scar tissue in wound healing, but because type I has greater tensile strength, is usually replaces reticular fibres. Reticular fibres also function as supporting stroma for haemopoietic (blood-forming) tissue and lymphatic tissue. It is produced by reticular cells. Elastic fibres A good exam question is to identify an organ or structure based on the presence of a big black band of something. You can pretty much straight away guess that this black band is the presence of elastic fibres! But ONLY under a certain stain called ORCIEN STAIN. These are the second type of fibres found in ECM. They are made of the protein elastin, and require the presence of a fibril-forming glycoprotein called fibrillin. Characteristically, elastic fibres are useful for places that require stretch, because they do not lose their structure when stretched. Additionally, elastic fibres are able to provide a tissue with a feature called elastic recoil, which is where the tissue will passively return to normal structure after stretching.
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In tissues, elastic fibres can be present both as individual fibres (both straight and branching),or as sheets of unidirectional elastic fibres called elastic lamina. The key example of an elastic lamina is the aorta, which requires elastic recoil after being forcefully filled with blood from the left ventricle of the heart. See below picture of a muscular artery, note the presence of both an internal elastic lamina (close to the lumen of the vessel), and an external elastic lamina (after the middle muscular component).

You will always find some collagen associated with elastin.

Classification of Connective Tissue:
The following is also a really good exam question (it was asked in CHG113 last year) so make sure you know it! 1. Embryonic connective tissue, known as mesenchyme. (Note this is NOT the same as mesoderm, which is a layer of the embryonic plate much earlier in development). 2. Ordinary Connective Tissue (connective tissue proper) A. Loose connective tissue (general function: movement of metabolites). It is only sometimes divided into: 1. Areolar connective tissue (what we usually know as loose c.t.) 2. Reticular connective tissue (lymphatic organs, spleen etc.) B. Dense connective tissue (general function: strength) 1. Dense regular (only in tendons and ligaments; unidirectional fibres) 2. Dense irregular (many locations e.g. dermis; fibres in many directions) 3. Special connective tissue (this is a category by itself mainly because these tissues are not described accurately by any of the categories above) A. Bone and cartilage B. Adipose tissue C. Blood (and haemopoietic tissue = bone marrow)

Embryonic connective tissue:
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Structurally, it resembles loose connective tissue. It has a semi-fluid ground substance. It develops from mesoderm (the middle embryonic disc). It is the precursor for virtually all connective tissues.

Loose connective tissue:

Notice in the above picture, the loose connective tissue underlying the epithelium. (Yes this section is a bit funny, don't worry about it). Loose, or areolar, connective tissue is the most common form of connective tissue. It is found beneath every epithelium. The reason for this is that epithelium is avascular, and requires nutrients, and loose connective tissue's properties provide for the diffusion of nutrients to overlying epithelium.

Fixed cells of loose connective tissue:
A common practical exam question is to provide a slide of connective tissue, and you must name a few cells that should be present. Fixed cells of loose connective tissue include: 1. Fibrocytes and fibroblasts 2. Macrophages (immune-response cells; in blood, these cells are known as monocytes and undergo conformational changes when they migrate into tissue to become macrophages). 3. Adipocytes (these are very easy to identify; they have a spongy appearance)
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4. Mast cells (these secrete histamine as a part of the immune response) 5. Undifferentiated mesenchymal cells (these are basically stem cells; they could differentiate into some of the above cells.

Wandering/transient population of loose connective tissue:
These could include: 1. Lymphocytes (immune cells) 2. Plasma cells (differentiated B cells; produce antibodies) 3. Neutrophils (also known as polymorphonuclear leukocytes) 4. Eosinophils (granulated white blood cells, obviously eosinophilic) 5. Basophils (highly basophilic white blood cells) 6. Monocytes (the precursor to macrophages, see above) Also will include all blood-based cells that are able to exit the blood. You could also find smooth muscle cells, nerves, blood and lymphatic vessels, sensory receptors. Hopefully you are understanding that loose connective tissue can basically function as a nutritious place for many other cells to reside in or use temporarily. Essentially, if you see loose connective tissue, it means that: there are scattered fibroblasts (or fibrocytes), separated by ground substance that is very thin and watery, with a mixture of fibre types (collagen and elastic), which do not form bundles (like in dense connective tissues), and have a random arrangement of fibres (which are always irregular), and allows easy passage through the tissue of fluids and dissolved substances. Loose connective tissue may be very cellular, but it also may not. It also may have a population of wandering accessory cells, or it may not. This will depend on the location and activity of the loose connective tissue. It will also change when under the influence of disease. Always remember! Every epithelium has a layer of loose connective tissue directly below its basement membrane!

Differentiating dense connective tissue from loose connective tissue:
Remember, in dense connective tissue, the fixed cells do not change. Most of the cells present are inactive fibro-what? Fibrycytes (recall they are the mature, inactive ones), rather than fibroblasts. Most of the defining characteristics are in the extracellular matrix. (Recall that it is the ECM that defines the classification of connective tissue). These characteristics include: 1. more fibres 2. fibres may be in bundles, not individual 3. one fibre type may be favoured, depending on function 4. fibres may be orientated: regular or irregular 5. less ground substance 6. overall, there may be more ECM because of the tissues dense fibre content 7. generally, fewer wandering cells, and will appear less cellular Dense connective tissue has a range of functions, and these functions are different to loose connective tissue.
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Dense irregular connective tissue:

Dense irregular connective tissue is characterised by collagen bundles arranged in many different directions. The primary example of this tissue is the reticular layer of the dermis. The nature of the collagen bundle arrangement allows the tissue to withstand moderate forces from many different directions. This is its primary function.

Dense regular connective tissue:

Dense regular connective tissue is characterised by bundles of collaged arranged in a parallel fashion. It is found only in tendons, ligaments and aponeuroses (membranes separating muscle from each other). This parallel arrangement provides the tissue with the ability to withstand and transmit (i.e. to bone) great unidirectional forces. Smooth muscle tends to look similar to dense regular connective tissue in a histological section. To tell them apart, remember that smooth muscle would never appear by itself (i.e. it would appear in association with something like a visceral organ or a blood vessel), where as dense regular connective tissue could appear as a tendon or ligament by itself (in almost all cases). Also, the nuclei of myocytes in smooth muscle tissue tends to look like a Twistie, whereas the fibrocytes of dense irregular connective tissue will look elongated and flattened, and parallel to the direction of the fibres. Very little ground substance exists.

Dense elastic connective tissue:
This is basically just aggregations of elastic tissue. The only really important example is the elastic lamina mentioned earlier (because elastic fibres present in other areas, like the skin, aren't really classified as dense elastic connective tissue). Elastic lamina are sheets of elastic fibres. Recall that elastin has the ability to be stretched greatly and return to its original shape with no energy
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requirement. Elastin can be broken down over time (like in the skin, where it is accelerated by prolonged exposure to UV light, hence the appearance of wrinkles). Always remember that where there is an elastic lamina, it would also include collagen fibres and bundles. Elastic fibres are stained black under Orcien stain. Elastic connective tissue is an important component of the heart's ability to maintain pressure, even when the ventricle is relaxed. That is, the pressure inside the aorta during diastole is usually about 80 mmHg, even though the ventricle is relaxed. It is the effect of the elastic lamina that encourages recoil of the aortic wall.

Adipose tissue:

Adipose tissue is a form of special connective tissue. Make sure you do not confuse adipose tissue for mucous acini. The nuclei of adipocytes will be much smaller than mucous secreting cells, and mucous secreting cells will have some serous secreting cells associated with them too (which are easier to identify).

White adipose tissue:
White adipose tissue, the tissue in both pictures above, is also called unilocular adipose tissue, because it has only one lipid droplet in its cytoplasm. The droplet pushes the nucleus and the rest of the cytoplasm to the periphery of the cell. Adipocytes in white adipose tissue are packed tightly together and will be associated closely with blood vessels. It is often found in areas of both loose CT and dense irregular CT. The hypodermis (deepest layer of the skin) contains large amounts of white adipose tissue. Its functions: a support tissue for example, in the hypodermis for example, as shock absorption in the soles of feet, buttocks, around the kidneys, and in the orbit of the eye an energy source as the producer of proteins that regulate metabolism for example, leptin It has a rich capillary network that is innervated by the Autonomic Nervous System.

Brown adipose tissue:
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Also known as multilocular adipose tissue. It has many small lipid droplets in its cytoplasm instead of one large one. It contains many mitochondria which give it an eosinophilic appearance under histological section. It will also appear foamy. Its main function is to metabolise fat to produce heat for neonatal babies. It usually is only present in babies, but it may be present in adults at selected sites (although this is disputed).

Cartilage:

Cartilage is also a special connective tissue. Why? • It has a supporting and protective role as part of the musculoskeletal system. • Cells are widely spaced and separated by extracellular matrix (ground substance and fibres!) • Variations in the extracellular matrix indicates different types of cartilage tissue • Embryological origin from mesenchyme (and therefore mesoderm) • Cartilage formation is a precursor to bone formation in both endochondral and membranous ossification. Some key terms to know when talking about cartilage: Cells of cartilage: chondroblasts and chondrocytes Lacuna: this is the space within which the condroblasts and chondrocytes sit. "Lacuna" means "little lake". Matrix: the extracellular matrix of the cartilage. This makes up 95% of cartilage by volume. It contains lots of GAGs. Perichondrium: the connective tissue covering of cartilage. Remember that almost all cartilage is avascular, so it needs a connective tissue coating for its nutrition.

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There are three types of cartilage Hyaline cartilage: • E.g. trachea, lining the surface of joints, no perichondrium • Predominance of Type II collagen • Is the skeleton in the fetus Elastic cartilage: 1. E.g. epiglottis, parts of the ear, the auditory tube, a small amount in the nose. 2. Dead giveaway when stained by Orcien Fibrocartilage: • This is an intermediary between dense regular connective tissue and cartilage. You might like to think of it as tough, tendinous cartilage. • Exists between the vertibrae, where you don't want much movement. • Has great strength Cartilage is covered in greater detail in CAM102.

Bone:

Bone is also a special connective tissue, because it has cells (osteoblasts and osteocytes) embedded in an extracellular matrix, secreted by the cells themselves. The major difference is that the ECM has become mineralised with calcium salts. There are two forms of bone in the adult human: Compact bone Cancellous (or trabecular or spongy bone) Bone is covered in greater detail in CAM102.
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Red and white blood cells:
The practical includes blood smears, so we should go through these quickly. Blood is a specialised connective tissue that consists of a variety of cells suspended in a fluid medium called plasma. When you look at a blood smear under histological section, you notice two overlying types of cells: erythrocytes (red blood cells) and leukocytes (white blood cells). Erythrocytes

Red blood cells lack a nuclei. They appear slightly eosinophilic (because of the protein content of their cytoplasm). Leukocytes White blood cells. There are five types, divided into "granular" or "agranular". Lymphocyte

An agranular leukocyte. Most lymphocytes are small and not much larger than a RBC. They have a round, densely stained nucleus and only a little amount of pale, basophilic, agranular cytoplasm. Larger lymphocyts have both more cytoplasm and a larger, paler nucleus. You'll remember from Dr Woods' lectures that there are B and T lymphocytes, and that some lymphocytes differentiate into plasma cells. If this happens, you would expect their cytoplasm to grow to accommodate the enlargement of organelles that are required for rapid protein (immunoglobulin) synthesis. Monocytes

These are the largest of the leukocytes. Monocytes are agranular, and they are the cells that differentiate into macrophages once they leave the bloodstream and enter the surrounding tissues. They have a large nucleus, stained less intensely than other leukocytes, and is usually indented (horseshoe shaped). Neutrophil

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Granular leukocyte. Called neutrophils because they are neither heavily basophilic or heavily eosinophilic. Also known as a polymorphonuclear leukocytes because their nucleus is composed of several lobes connected to each other by fine strands. The cytoplasm is packed with many fine, "peppery" granules. The balance of staining will make the colour and visability of the granules differ a lot in different slides. Eosinophils

These are granular. They are the most easily identifiable leukocytes because of their large, (usually) orangered cytoplasmic granules. The nucleus may be obscured by the granules but are usually bi-lobar. Basophils

These granular leukocytes are the hardest to find. The large, deeply-staining blue-black cytoplasmic granules may obscure the simple or bi-lobed nucleus. The granules are water soluble and may therefore dissolve during histological processing.

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Muscle Tissue
Muscle tissue is one of the four adult tissue types. There are three types of muscle: cardiac, skeletal, and smooth. All three types of muscle are composed of elongated cells that have many names that all mean the one thing: myocyte, myofibres, muscle fibres. In all three types of mucle, energy from the splitting of ATP is transferred into mechanical energy that results in movement. We should note here that there are other types of contractile cells in the body: • Myoepithelial cells - you will recall that these are associated with exocrine glands, and they contract to help propel the secretion into and through the duct. A good example is the eccrine sweat gland. They are contractile cells of epithelial tissue. • Myofibroblast cells - these cells are normally associated with repairing tissue, but can also be associated with other tissues such as in the interstitial tissue of the testis. They are the contractile cells of connective tissue. • Pericytes - these cells surround endothelial (inner lining of blood vessels) cells beneath the basal lamina. They are sometimes considered myofibroblast. Their contraction causes a decrease in the size of the lumen of capillaries.

Types of muscle tissue:

The three types of muscle can be easily classified by a couple of distinguishing features: Microscopic appearance: Striated Skeletal and cardiac Non-striated Smooth Innervation: Voluntary Skeletal
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Involuntary

Cardiac and smooth

Skeletal muscle:
Skeletal muscle develops from cells called myoblasts, which fuse to form large multinucleated cell. That is, myoblast + myoblast + myoblast = 1 large, multinucleated myocyte. (A muscle cell). It is for this reason that skeletal muscle is referred to as a true syncitium. A key part of distinguishing skeletal muscle from smooth and cardiac muscle is that the nuclei are located at the periphery of the cell in skeletal muscle. So if you see a cross-section of muscle, and the nuclei are at the edge of each cell, you know it is skeletal muscle. There is a lot of annoying, complicated terminology used for skeletal muscle tissue, and it is just best to sit down and learn it now. To make it easier, you can try and visualise the terms from getting bigger to smaller. A muscle, e.g. your biceps brachii, is composed of bundles of muscle cells called fascicles. Each fascicle is composed of many muscle cells that span the length of the muscle. Intracellularly, there are many groups of cytoskeletal contractile proteins called myofibrils. These are bundles cytoskeletal microfilaments named myofilaments. They are arranged in a particular pattern to give a striated appearance to skeletal muscle. There are two types of myofilaments: thick and thin. Thick are composed primarily of myosin, and thin are composed primarily of actin. (To remember this, just remember actin has less letters and is therefore thinner than myosin). Aside from these terms related specifically to muscle tissue, there are terms about the surrounding connective tissue of a muscle cell that we need to learn. The connective tissue surrounding a muscle is what enables the muscle to transfer its force to bone so that movement can occur. There are three connective tissue capsules: epimysium, perimysium, and endomysium. They are primarily composed of a dense arrangement of collagen fibres. Within these connective tissue components lies blood vessels, nerve fibres and lymphatics. Epimysium surrounds the entire muscle, for example, it would surround your entire biceps brachii. Perimysium exists around, and in between, fascicles. Endomysium lies around each muscle cell (which, remember, spans the entire length of the muscle). Actin and myosin are also important to understand on a microscopic scale. They are both proteins, and form components of thin and thick filaments.

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Thin filaments are: 7nm in diameter and 1 micron in length Each thin filaments is 2 strands of filamentous actin arranged in a double helix formation Also contain the proteins troponin, tropomyosin and nebulin. Thick filaments are: 15nm in diameter and 1.5 microns in length Arranged like two golf clubs with intertwined shafts, with the golf club heads pointing in opposite directions. Each myosin head (the golf club head) has an actin-binding-site, and an ATP-ase, that is an enzyme that breaks down ATP. Thick filaments also contain titin (easy to remember because it is a big, big protein). The thick and thin filaments are arranged together in a particular formation that allows contraction by a mechanism called the sliding-filament mechanism. This will be covered next semester so we won't worry about it here. But, we need to know that the functional unit of skeletal (and cardiac) muscle is the sarcomere, which is a portion of the muscle that looks like this next picture.

A muscle cell will have thousands of these sarcomeres running along its cytoplasm. When they are stimulated to contract, the thin and thick filaments slide over each other and they basically get smaller, reducing the length of the whole cell and creating contraction. Don't worry about the mechanism too much, just know what it looks like and be able to identify it if it comes up in the practical exam.
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Also within a skeletal muscle myocyte, you should be aware of the sarcoplasmic reticulum. It is a reticulum much like smooth endoplasmic reticulum, and holds lots of things, but especially calcium, which is essential for the contraction mechanism. To recap this section: Actin and myosin - are cytoskeletal microfilamentous proteins that form components of thin and thick filaments Thin and thick filaments

The above picture is skeletal muscle. Note the peripherally-located nuclei in each muscle cell, the endomysium connective tissue surrounding each muscle cell, the perimysium connective tissue surrounding and dividing up the fascicle. The epimysium cannot be seen, because as you know, it surrounds the whole muscle. This next picture puts it into a little more perspective. Test yourself and see if you can name what sort of tissue each component is. (I didn't mention this before, but the plasma membrane of a muscle cell is also referred to as the sarcolemma).

Cardiac muscle:

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Cardiac muscle, like all other muscle, is derived embryonically from mesoderm. It is found only in the heart, and in portions of the large arteries closer in the heart. A cardiac cell, known as a cardiocyte, has a diameter of 15 microns and a length of 80 microns. (Note that what is says in Kierszenbaum is incorrect here). Like skeletal muscle, it is striated. But unlike skeletal muscle, cardiocytes are branching. See the diagram back at the start of the section on muscle, and the one directly above. Each cardiocyte junctions with a couple of other cardiocytes. Other important features of cardiac muscle include: • cardiocytes have a centrally placed nucleus (useful for distinguishing between cardiac muscle and skeletal muscle) • cells joined by specialised junctions called intercalated discs • innervated by the autonomic nervous system • inherent rhythmicity (that is, will contract without nervous system stimulation) • mitochondria make up 50% of the volume of the cell (hence the eosinophilia of cardiocytes in histological section) • calcium is supplied from outside the cell (important for contraction)

Intercalated discs are exclusive to cardiac muscle. They are junctions located between cardiocytes. They are a step-like arrangement that include vertical and horizontal components. The transverse component is located at the z-line of the sarcomere in adjacent cardiocytes (A in the diagram). Here, both fascia adherens and desmosomes hold the cells together (B in the diagram). Also associated closely are gap (communicating) junctions (C in the diagram). The gap junctions in cardiac muscle are extremely important and it is really important to understand why cardiocytes are referred to as a functional syncitium. Unlike skeletal muscle, which is a true syncitium and composed of aggregates of precursor cells in one big cell, each cardiocyte is a cell by itself. Because of the gap junctions, they are able to very quickly transmit a chemical signal, first originating in a cell that has a connection to a nerve, to thousands of other cardiocytes by the passage of ions. This allows the cardiocytes to contract at the same time. Hence, they are not a structural syncitium, because they are separate cells, but they are a functional syncitium.

Smooth Muscle:

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Smooth muscle is found as part of most organs that have a hollow component (the gastrointestinal tract, the bladder, the ureters, for example) and in blood vessels. It is innervated by the autonomic nervous system, like cardiac muscle, and is therefore involuntary. Cells are attached to each other via gap junctions and a basal lamina. Unlike sketal muscle, which has highly organised arrangement of actin and myosin microfilaments, smooth muscle has a random arrangement of actin to densities on the cell membrane called dense bodies or focal densities. See picture below. The cells communicate to each other via gap junctions.

At the EM level, numerous pinocytotic vesicles are characteristic of the cell membrane. They also have a centrally-located nucleus, like cardiocytes, and unlike skeletal muscle fibres.

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Nervous Tissue
Organisation of nervous tissue:
This semester is only a very, very brief taste of the nervous system. We'll cover it in a lot more detail in other units. It's my favourite system so I hope they do it justice. Nervous tissue can be organised broadly into one of two locations: the Central Nervous System (CNS) and the Peripheral Nervous System (PNS). The CNS is quite simply the brain and the spinal cord. The PNS is composed of cranial nerves (twelve of them), spinal nerves, and ganglia. The PNS has two components: • Sensory afferent (afferent = towards) • Motor efferent (efferent = away from) The MOTOR EFFERENT component is further divided into: • The Somatic Nervous System (SNS) • The Autonomic Nervous System That is, both the SNS and ANS are motor systems. The ANS is further divided into: 1. Sympathetic Nervous System (the system that controls the "fight, flight and frolic" responses in most cases) 2. Parasympathetic Nervous System (the system that controls the "rest and digest" responses in most cases)

Characteristics of nervous tissue:
Cells fall into one of two categories: • Neurons • Glia, aka neuroglia, (supporting cells) - these cells differe between the CNS and the PNS In most cases of nervous tissue, neurons are widely scattered between areas filled with the processes of neurons and the processes of glia. The processes of neurons and glia we call neuropil.

Neurons have the special ability to transmit a signal (known as an action potential) along a cell surface by reversing the polarity of the cell membrane. Highly specialised contact points known as synapses exist between neurons and allow this signal to pass from cell to cell.
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Cells of nervous tissue:
1. Neurons 2. Glial cells 1. oligodendrocytes (CNS) 2. astrocytes (CNS) 3. microglia (CNS) 4. ependymal cells (CNS) 5. Schwann cells (PNS) 6. satellite cells (PNS) Don't stress about what they are for now, just know which ones appear in which nervous system.

Neurons:

Some key properties of a neuron: 1. Cell body, also called the soma or perikaryon 2. Nissl bodies in cytoplasm - large accumulations of rough endoplasmic reticulum and lots of free ribosomes 3. Axon hillock - the point at which the axon and the soma are connected; where the action potential begins 4. Dendrites - branch-like process extending from the cell body 5. Axon - a bigger, thicker dendrite 6. Myelin sheath - wrappings of the plasma membrane of glial cells around the axon 7. Nodes of Ranvier - the gaps between the wrappings of plasma membranes of glial cells around the axon 8. Axon terminals - the end of the axon, where the synapse occurs 9. Synaptic end-bulbs - branches of the axon that bulb at the end to carry an action potential to multiple cells 10. Synapses - where the action potential is transferred to another cell; can be from axon to axon (axoaxonic), from axon to soma (axo-somatic) or axon to dendrite (axo-dendritic). Neurons are put into broad categories based on their structure: • Pseudounipolar neurons
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Bipolar neurons, and • multipolar neurons. There are other classifications. See diagram.

Synapses:

Synapses are communication points between neurons and other neurons, or, the effector cells such as muscle cells in the neuromuscular junction. Synapses have several important features from a histological viewpoint: 1. Presynaptic end bulb (terminating bouton) containing: 1. Synaptic vesicles containing neurotransmitter 2. Presynaptic plasma membrane thickening 2. Synaptic cleft (basically a space) 1. Postsynaptic plasma membrane thickening 2. Ligand-gated (neurotransmitter receptors) ion channels (not visible in histological section)

Myelination of axons:

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Oligodendrocytes produce myelin in the CNS. Schwann cells produce myelin in the PNS. Myelin is a wrapping of portions of the axon by the cell membrane of a glial cell (Schwann or oligodendrocyte).

As the myelin sheath is composed of plasma membranes, they will appear differently under light and electron microscopes. Under the LM, lipids are usually removed through processing and so myelinated axons in cross-section appear as pink rings of predominantly white empty spaces with a small eosinophilic dot in the centre (representing the axon). This is important! It featured in our formative practical exam and will probably come up again in our next one! Under EM, the myelin sheath will appear as concentric electron-dense rings (big and black!) around axons. There are critical differences in the myelination by Schwann cells and oligodendrocytes. • Oligodendrocytes send out long processes that wrap around the axons of neurons to form the myelin sheaths, and so each oligodendrocyte can myelinate a number of axons, or more than one site on a single axon • Schwann cells are closely associated with the axons they myelinate, and a Schwann cell only myelinate one part of a single axon. So an axon that can be up to two metres long (suppling your toe for example) may have thousands of Schwann cells associated with it. Other key histological features differ between the CNS and the PNS: • Nodes of ranvier (the spaces between adjacent myelin sheaths) in the CNS are covered by the
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processes of a type of CNS glial cell called the astrocyte. In the PNS, the nodes are covered by extensions of the Schwann cell cytoplasm. The Schwann cell also has an external lamina external to it; this is not present in the CNS.

Not every axon has myelination! In the CNS, non-myelinated axons are structurally supported by surrounding non-oligodendrocyte cells (mostly astrocytes). In the PNS, however, non-myelinated axons are still closely associated with Schwann cells, and become embedded in the cytoplasm of the Schwann cell, without being myelinated by it. See the bottom-right part of the diagram above (the part that says 5). Several axons may associate with one Schwann cell in this non-myelinated manner.

Fast versus slow conduction:
Some nerve impulses are required to be faster than others. There are a few principles that determine fast versus slow conduction of action potentials. • Myelination. If the axon is myelinated, the action potential will be very quick. This is due to the principle of saltatory conduction (saltatory = "jumping). Because the plasma membrane of glial cells are associated so closely to the axon's plasma membrane, no transfer of ions can take place, and the action potential skips to the next location of possible ionic transfer, which happens to be the nodes of Ranvier. • Diameter. Generally, the larger the diameter of the axon, the faster the conduction of the action potential is.

Peripheral Nerves:
So, what actually makes up a peripheral nerve that we are going to come across in our practicals? Peripheral nerves contain: 5. Axons (both myelinated and unmyelinated) 6. Schwann cells (associated with nerve fibres) 7. Fibroblasts/cytes (associated with the following connective tissue layers) 8. Endoneurium (loose connective tissue surrounding individual axons and the Schwann cells) 9. Perineurium (denser connective tissue surrounding bundles of axons known as fascicles) 10. Epineurium (dense connective tissue surrounding the entire peripheral nerve) 11. Jamie forgets to mention here that there will be blood vessels and lymphatics in the nerve also Do those connective tissue layers sound familiar? They should. Remember the connective tissue layers of skeletal muscles? Kierszenbaum has a pretty good diagram that sets it out well:

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Notice that in the peripheral nerve there are no nuclei of neurons. If you're looking at a peripheral nerve and see nuclei, you're probably seeing a Schwann cell or a fibroblast (or another cell associated with the connective tissue component, although this is less likely). Don't make the mistake of saying there are cell bodies of neurons! This would only occur in ganglia.

Ganglia:
A ganglion is a collection of neuronal cell bodies (somas) located outside the CNS. All ganglia are part of either the sensory (afferent) nervous system, or the ANS. They could be either: • Dorsal root ganglia (sensory) • Found just outside the spinal cord; contains ONLY sensory neurons • Sympathetic ganglia (motor) • Found predominantly in a long chain running parallel to the vertebral column but also in several sites associated with the major blood vessels of the abdominal organs • Parasympathetic ganglia (motor) • Found intramurally (within the wall of the organ/tissue they supply) This will all be covered in much more depth, including how to find a ganglion in a histological slide, in CAM102.

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Histology of the integumentary system
Integumentary system – skin • Skin and its various accessory structures (hair, nails, glands, muscles and nerves) make up the integumentary system. • The tissues are arranged into organs (epidermis, dermis, hair follicles, sweat glands, nails etc) which together form the system.

Structures of the skin
Epidermis – epithelium Dermis – connective tissue Hypodermis – connective tissue

Accessory structures of the skin
• • • • • • Hairs (derives from the epidermis) Arrector pili muscles (responsible for goosebumps) Sebaceous glands Sweat glands Sensory receptors Blood vessels

Layers and tissues of the skin
• • Epidermis o Keratinised stratified squamous epithelium Dermis o Papillary layer  loose connective tissue (because it underlies an epithelium) o Reticular layer  dense irregular connective tissue (collagen bundles in many different directions) Hypodermis o Adipose tissue o Dense irregular connective tissue (anchors overlying skin with underlying tissue)

Epidermis
An avascular layer of stratified keratinised epithelium sitting on a basement membrane. (Recall that a basement membrane is made up of a basal lamina and a reticular lamina) The epithelium can be subdivided into 4-5 layers from the base to the free surface: 1. stratum basale (stem cells for skin) (1 layer) 2. stratum spinosum (spiny cells because of lots and lots of desmosomes and shrinkage during histological processing) (4-7 layers) 3. stratum granulosum (lots of granules) (2-3 layers) 4. stratum lucidum (only identifiable in thick skin) (translucent) (a couple of layers) 5. stratum corneum (flattened cells like scales on the surface) (many layers)

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Keratinocytes
• The cells that constitute the layers of the epidermis are called keratinocytes. They appear by division of the cells in stratum basale and move (are pushed) towards the surface by further division of the stem cells in the stratum basale. At the surface of the epithelium, the cells are removed by abrasion. Keratinocytes at the surface are little bags of plasma membrane and keratin. The shape of the cells change as they progress through the layers towards the surface from cuboidal, to irregular spiny, flattened with granules, and then squamous.

• •

Dermal-epidermal junction
• • Keratinocytes are separated from the underlying connective tissue of the dermis by a basement membrane (remember only stained by PAS) Structural integrity of the dermal-epidermal junction is maintained by: o tethering fibres o highly folded membranes o ‘rete ridges’ or ‘epidermal pegs’  particularly found in thick skin

Basement membrane of epidermis
• Basement membrane at dermo-epidermal junction consists of o lamina lucida (electron lucent) on epidermal side o lamina densa (electron dense) in the middle o fibroreticular lamina (ill-defined) on the dermal side Lots of hemidesmosomes

Life cycle of a keratinocyte
During their life span, the keratinocytes undergo a process called keratinisation. This gives rise to cells that have a great resistance to abrasion. The cells fill with the protein keratin and keratohyalin granules and in addition they secrete a glycolipid (lamellar bodies) that waterproofs the skin by coating the outside of each cell. • Provides a protective layer that resists water evaporation and things from getting in The cells of lucidum and corneum are dead cells forming plates of keratin.

Stratum basale or basal layer
• • Deepest cell layer of the epidermis o Cuboidal or low columnar in shape Cells have many junctions o Hemidesmosomes (between the basal cells and the BM) o Desmosomes (between adjacent basal cells) Cells may contain melanin granules (melanocytes in basal layer produce melanin) Cells undergo mitosis (the region for constant production of keratinocytes) Non-keratinocytes found here o Melanocytes and Merkel discs (special touch sensory cells)

• • •

Stratum spinosum or prickle cell layer
• Sit above basal layer
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• •

Cells are in constant contact with each other by a system of intercellular bridges held together by desmosomes Cells contain many tonofilaments

Stratum granulosum or granular layer
• • Produces the surface keratin and water-proofing substances Cells contain basophilic keratohyaline granules, abundant tonofilaments and lamellar bodies o keratohyaline granules and tonofilaments combine when the cell dies to form keratin o lamellar bodies are released at cell death  contain water-proofing glycophospholipid

Stratum corneum or keratin layer
• • Dead cells filled with keratin o no nuclei, no organelles Surface keratin is constantly lose due to normal wear and tear from surface friction

Lifespan of a keratinocyte
• Turnover from basal cell to keratin layer varies: o 25-30 days in areas with lots of friction (e.g. soles of feet, palms of hand) o 40-50 days in areas of less friction This turnover period is considerably shortened in some disease states (e.g. psoriasis)

Soft keratin – stratum corneum (and lucidum) Hard keratin – hair, fingernails, rhinoceros’ horn

Epidermis and dermis

Non-keratinising epidermal cells
Not all cells found in the epidermis are keratinocytes. Other cells: • Melanocytes • Merkel cells • Langerhans cells

Melanocytes
Melanocytes are dendritic cells that produce melanin and then transfer the melanin to the keratinocytes. They are found in the stratum basale and can associate with as many as 36 keratinocytes. They produce melanin from a base of the amino acid tyrosine and pass it on to the keratinocytes.

Langerhans cells
• • • • • Dendritic cells of the skin o Found in all cell layers but best identified in the stratum spinosum Form a network within the epidermis Irregularly shaped nucleus No desmosomes or melanosomes (to facilitate easy movement) Contain distinctive granules (Birbeck) visible with the electron microscope
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• •

o They look like racquet handles, only visible under EM, play a role in breaking down the antigen Function as antigen presenting cells o Important component of the immune system Paler nucleus than the surrounding keratinocytes

Merkel cells
• • • • • Located in the basal layer of the epidermis Always associated with the end of a sensory neuron (nerve cell) Part of the sensory reception mechanisms of the skin Respond to touch Granules in the cytoplasm that have a neurotransmitter-like action with the sensory nerve ending

Types of skin
Thick or glaborous (hairless) • sole of the feet and palms of the hand • also called friction skin • deep folds on the basal surface of the epidermis (fingerprints) to better attach the epidermis to the dermis Thin (hairy) • covers the rest of the body • nearly all thin skin has hairs • some hairs are not obvious to the naked eye The main difference structurally between thick and thin skin is in the stratum corneum. The other layers (the living layers) are very similar.

Comparison of thick and thin skin
• • • • • • The living layers of the epidermis are similar The dead layers (lucidum and corneum) are thicker in thin skin (functional adaptation for wear and tear) Dermal papillae are greatly developed in thick skin – allows firm attachment of epidermis to dermis Both have sweat glands No hairs (or sebaceous glands) in thick skin Thick skin often has lots of sensory receptors (touch in the fingers)

Dermis
Layer of irregular connective tissue underlying the epidermis o both loose and dense irregular connective tissue o it supports the epidermis and gives great strength and flexibility to the skin 1. It contains many of the accessory structures of the skin, such as: o hair follicles o nerve fibres and sensory receptors o sebaceous glands o sweat glands o blood and lymphatic vessels • The dermis can be divided into two region
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1. Papillary layer • immediately below the epidermis • forms the dermal papillae between the epidermal pegs • a layer of loose connective tissue (areolar connective tissue • remember every epithelium has an underlying layer of loose connective tissue 2. Reticular layer • between the papillary layer and the hypodermis • the ‘true’ dermis and is a dense irregular connective tissue

Hypodermis
Below the dermis is the hypodermis (superficial fascia) • largely a layer of adipose tissue and areolar connective tissue Often considered to be part of the integumentary system Sometimes the bottom of hair follicles can be seen extending into the hypodermis.

Sensory innervation of the skin
1. 2. 3. 4. Free nerve endings which finish in both the dermis and the epidermis Merkel cells of the epidermis (specialised) (not encapsulated) Meissner’s corpuscles (dermis) (specialised) (encapsulated) Pacinian corpuscles (dermis) (specialised) (encapsulated)

Encapsulated sensory receptors • Meissner’s corpuscles are located in the dermal papillae of the dermis. They respond to light tough • Pacinian corpuscles are large ‘onion’-like structures deep in the dermis. They respond to deep pressure and vibrations. Free nerve endings • These are not encapsulated receptors • They respond to heat, pain, cold etc. • Many work via their attachment to the hair bulb; movement or touch from the hair is recognised by the nerve endings • Some actually penetrate between the cells of the epidermis.

Appendages of the skin
1. 2. 3. 4. Hairs and hair follicles Sebaceous glands Nails Sweat glands

Hair follicles
Hair follicles are down growths of the epidermis We will see the same layers of the epidermis in the layers of the hair follicle and the hair itself.

Pilosebaceous apparatus
• Hair shaft (the part we can see – very different from the part below the level of the skin)
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• • •

o cuticle o cortex o medulla Hair bulb – the growing point Arrector pili muscle Sebaceous gland

Hair bulb
Contains a specialised area of dermis = hair papilla Lots of nerves and blood vessels Lots of actively-dividing stem cells (matrix) produce the hair shaft and internal root sheath • The hair shaft and internal root sheath lie within the external root sheath • Melanocytes are found in the hair bulb The internal root sheath contains several layers including the cuticle • Cells undergo keratinisation to produce the hair shaft • Extends from hair bulb to the level of the sebaceous gland Leaves a space for sebum to be secreted. The external root sheath contains epidermal layers – basale, spinosum and granulosum. Surrounded by a thick basement membrane called the glassy membrane.

Hair follicle
All of these layers are surrounded by a fibrocollagenous follicle sheath (FFS), including the sebaceous gland Attached to the FFS is the arrector pili muscle • Smooth muscle • Attaches at, or just below, the level of the sebaceous gland • Upper attachment at papillary dermis • Contraction causes hair to stand on end – goosebumps

Glands of the skin
• • • Sebaceous glands – holocrine secretion of sebum Sweat (eccrine) – merocrine secretion of sweat Apocrine sweat glands

Sebaceous glands 1. Sac-like gland that secretes an oily thick substance called sebum 2. Open onto the skin surface via a hair follicle 3. They are holocrine glands 4. Secretion is viscous and may become secondarily infected by bacteria – acne 5. Secretion is controlled by the ANS 6. Activity may be influenced by sex hormones 7. Important lubricant of the epidermis 8. Pyknotic nuclear appearance Sweat glands (eccrine) 1. Simple coiled tubular glands
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2. Open onto the skin surface via a duct 3. They are merocrine secreting glands 4. The secretory portion of the gland lies deep in the dermis (therefore they have a relatively long duct system) 5. Sweat is a mixture of secretory and excretory products 6. There are different forms of sweating: a. emotional (adrenergic – adrenaline – sympathetic) b. thermoregulatory (cholinergic – Ach – parasympathetic) 7. Sweating is controlled by the ANS

Histological structure of eccrine sweat glands
Secretory portion • dark cells – proteinaceous secretion • clear cells – watery secretion (many basal membrane infoldings because of Na+ and Cl- secretion) • myoepithelial cells (for contraction Duct portion • stratified cuboidal

Histological structure of apocrine sweat glands
1. Simple coiled tubular glands with wide secretory lumen (as opposed to the eccrine sweat glands) 2. Open onto the skin surface via a hair follicle (as opposed to eccrine sweat glands which are not associated with a hair follicle) 3. They secrete cells by merocrine secretion, which makes their name a misnomer 4. Secretion is often viscous and may become secondarily infected by bacteria, creating body odour 5. Glands are concentrated in the axillary and anogenital areas 6. Secretion is controlled by the AND 7. Activity may be controlled by sex hormones

Sample examination questions
• List the functions of the skin 1. protection from external environment 2. cutaneous sensation 3. body temperature regulation 4. blood reservoir 5. excretion and reabsorption 6. vitamin D synthesis (via sunlight) Explain how the structure of the skin relates to its function Explain how the structure of the skin and its organs contribute to protection from the outside environment

• •

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Biochemistry – nutrition and metabolism
Amino acids, proteins and enzymes
Amino acids are ‘monomers’ that can be linked together to form ‘polymers’ called proteins. Enzymes are a sub-group of proteins. Amino acids, proteins and enzymes all play crucial roles in body function.

Structure of amino acids and proteins
• • • There are 20 common amino acids They are organic compounds, with molecular weights in the range of 70-200 They have three key functional groups to their structure

Each amino acid has an alpha-amino (-NH2) group, and an alpha-carboxylic acid (-COOH) group. • These two portions are the same for all the amino acids. The alpha refers to the C-atom to which they are both covalently linked (bound). Each amino acid has a side chain that is unique to that amino acid. • It is also covalently bound to the alpha-C-atom. • Different amino acids have different side-chains.

The 20 common amino acids
arginine asparagine aspartate cysteine glutamate glutamine glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine valine

Functions of amino acids
1. The key role of amino acids is to act as building blocks for constructing proteins and enzymes. 2. Some aminoacids are catabolised (broken down) to release energy • in this process, their amino group is removed and converted to urea, and the rest of the molecule (called the carbon skeleton) is metabolised for energy • CO2 and H2O are released in this set of reactions • Catabolism of 1g of amino acids releases 16-17 kJ of energy 3. Some aminoacids are converted into the carbohydrate glucose • this mostly happens in ‘emergency’ situations such as fasting and starvation, to maintain adequate glucose levels in the bloodstream • (the brain is heavily dependent on the steady supply of glucose, and the rest of the body tries hard to support the brain) 4. Some amino acids are converted into other useful low-molecular weigh compounds • e.g. thyroxine (thyroid hormone), adrenaline and noradrenaline, melanin, dopamine, creatine, cartinine, purines and pyrimidines, haem.

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Amino acid supply
Some amino acids are (or can be) made in our cells, but others are essential, which means they must be part of our diet because we cannot produced them. In practice, the majority of the amino acids we use come from our food supply. Some amino acids are non-essential, that is, they can be made in our cells. • We can make these ourselves, from non-amino acid substances found in our cells, so we don’t actually need them in our diet. • These are: o ASPARTATE o ALANINE o GLUTAMATE • You must know these for the exam. The term essential means that humans and animals can’t make them at all, and must obtain them from our dietary proteins. Plants and bacteria can make them. • For reference, they are isoleucine, leucine, lysine, methionine, phenylalaline, threonine, tryptophan, valine Others are conditionally essential, which means they can be made in our cells under certain circumstances. • We can make limited amounts of them from other substances found in our cells • However, when demand is heavy (for example, in growth), we cannot make enough of these amino acids • For reference, they are arginine, asparagine, cysteine, glutamine, glycine, histidine, proline, serine, tyrosine

Proteins
Proteins (or polypeptides) are linear polymers of amino acids. • They are polymers consisting of amino acids arranged in a linear sequence (e.g. A-B-C-D) linked by covalent peptide bonds between the alpha-NH2 of one amino acid and the alpha-COOH of the next. • Their molecular weight varies from a few thousand to a few million. • All proteins contain all of the 20 common amino acids. • Each amino acid plays a unique role in protein structure • If a specific amino acid is unavailable, it cannot be replaced by another The primary sequence and 3-D structure of the polypeptide must be correct! • each protein has a unique sequence (primary structure), and hence a unique three-dimensional structure • the three-dimensional structure (usually called secondary or tertiary structure) is maintained by covalent bonds and non-covalent linkages between amino acids • Alteration of the three-dimensional structure usually causes loss of function. This can happen if: o non-covalent linkages are disrupted by an increase in temperature – DENATURING o covalent peptide bonds between amino acids are split – PROTEOLYSIS

The most common proteins and their functions
Collagen • provides structural support in connective tissue • provides strength to bone, teeth and other connective tissues • its proper function requires vitamin C during its synthesis • the most abundant protein in the body.
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Haemoglobin • a globular protein that transports oxygen • found in red blood cells Actin and myosin • responsible for the contraction of muscle tissue

Other extremely important types of proteins
Enzymes • specialised proteins that act as catalysts • in small amounts, under the mild conditions inside cells (pH usually close to 7.4, temperature usually about 37o) they catalyse the conversion of one substrate into another • each cell has many, many enzymes • they are crucial for the digestion of food, obtaining energy from our food, and in making proteins and other body components Antibodies (immunoglobulins) • help fight infections • they do this by binding to specific foreign material as part of the body’s immune respone Myoglobin • helps deliver ozygen to muscle cells • ‘downloads’ oxygen from haemoglobin, for temporary storage until it is used Insulin and glucagon • hormones that control metabolism • glucagon is made by alpha cells in the endocrine pancreas, and is secreted into the blood if blood glucose levels fall • insulin is made by beta cells in the pancreas, is secreted into the blood if blood glucose levels rise • both have crucial roles in controlling metabilism Keratin • major component of skin, hair and nails

Protein and amino acid turnover in the body
The body’s protein content: • Virtually all the amino acids in our diet are part of proteins • A 70kg human body has about 11kg of protein (including enzymes) • Most of this is in: o Muscle (~43%) o Blood (~16%) o Skin (~15%) Sources of protein in the human diet: • Major contributors to protein intake among Australian humans are: o meats (especially beef and chicken) o milk (and cheese and yoghurt) o breads, breakfast cereals, pasta and rice
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Other significant sources that have high protein content, but because we don’t eat a lot of them, aren’t major dietary sources are : o fish o eggs o nuts o legumes

Dietary proteins provide most of the amino acids for the body’s cells. Proteins cannot be absorbed intact from the gut. Instead, dietary proteins are split by digestive proteolytic enzymes in the stomach and small intestine. The amino acids are then absorbed into the blood and cells where they become available for protein synthesis and other purposes. Some amino acids are made by our cells from non-amino acid precursors; about half of the twenty amino acids can be made by cells from substrates derived from metabolism of such fuels as carbohydrates (discussed later). Amino acids are constantly being incorporated into cellular proteins. Amino acids from dietary protein and amino acids synthesized from non-amino acid precursors all become part of the amino acid pool. Proteins are also constantly being broken down into amino acids in the body. • proteins in cells or body fluids have only a short life expectancy, usually only a few days • this is because they gradually denature, that is, the non-covalent links between amino acids are disrupted, even at 37oC • denatured proteins then get broken down into amino acids by proteolytic enzymes in cells Some amino acids, instead of getting incorporated into proteins, get diverted to other purposes: • being catabolized and used for energy (in a low energy state or an amino acid surplus) • being converted to glucose (starvation or low-carbohydrate diet) • being converted into other specialised low-molecular weight compounds

Meats

Cereals

Legumes

Proteins in diet

Proteolytic enzymes

Broken down in stomach into aa

10 of 20 amino acids made by non-aa precursors

Taken up by blood

AA POOL
Incorporated into cellular proteins Synthesising new Page 52 proteins for export Diverted for other uses

Imbalances in protein and amino acid turnover
Normally, the body’s total protein content remains fairly constant. Growth: body’s protein content increases (positive nitrogen balance) • for example, childhood, adolescence, pregnancy, lactation • creating new cells • hence, total stock of protein increases • to do this, we need a bigger supply of amino acids, hence more proteins in our diet Protein deficiency: the body’s protein content decreases (negative nitrogen balance) • symptoms: levels of many proteins in cells and body fluids decrease (among the more obvious effects) o impaired growth (including brain) in growing children or young adults o anemia (not enough haemoglobin) o muscle wasting (cannot make enough of the many proteins that are in muscle) o swollen abdomen (blood levels of protein are low, fluid escapes from blood into the interstitium osmotically; most visible in the accumulation of fluids in the abdominal cavity) • causes include: o low dietary intake of protein  (or one essential amino acid, because no other amino acid can replace an inadequate supply of one, therefore proteins cannot be made in adequate amounts) o fasting or starvation  (normal glucose levels fall, body diverts amino acids to make glucose to keep the brain operating, even cannibalising body proteins if necessary) o major infection  (cells fighting infection require more glucose than usual, so the body diverts amino acids to make glucose, even cannibalising body proteins if necessary \ o blood loss Causes: • low dietary intake of protein • low intake of one essential amino acid • fasting or starvation • blood loss Symptoms: • impaired growth • anemia • muscle wasting • swollen abdomen

PROTEIN DEFICIENCY
negative nitrogenPage 53 balance

Childhood growth Pregnancy

requires

MORE PROTEIN
for the creation of new cells

Lactation

they contain protein

POSITIVE BALANCE

NITROGEN

Enzymes
Enzymes names almost always end with –ase, and often include an indication of the substrate. For example, ‘glycogen synthase’ participates in the synthesis of glycogen. Each enzyme supports a specific conversion, at its active site. • This happens at a special site in the 3-D structure, called the active site. • The active site is a cluster of amino acid side-chains, where substrates bind and are modified, and reaction products are released. • Each enzyme has a different active site, hence different specificity. The more substrate, the faster reaction: • The higher concentrations of a substrate, enzymes work faster. • The relationship between reaction velocity and substrate concentration may be the Michaelis-Menten type, or the Allosteric type Michaelis-Menten:

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Allosteric type:

Coenzymes
Coenzymes are also called cofactors or prosthetic groups. • They are essential for many enzymes • Many enzymes require coenzymes which are mostly organic • Such enzymes cannot operate at all unless the proper coenzyme is attached (covalently for some, non-covalently for others). Many coenzymes are made from water-soluble vitamins Niacin Riboflavin Thiamin Pantothenate Vitamin B6 gives gives gives gives gives NAD+ and NADH, NADP+ and NADPH FAD and FADH2, FMN and FMNH2 TPP Coenzyme A pyridoxal phosphate
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Folate

gives

several different coenzymes (e.g. 5-methyl-THF)

Some are used by enzymes that carry out oxidation and reduction reactions in cell metabolism, for example, the following: From vitamin: Niacin Niacin Riboflavin oxidised form NAD+ NADP+ FAD corresponding reduced form NADH NADPH FMNH2

That is, the portion of the vitamin that is the coenzyme will be ‘reduced’ when helping to catalyse the reaction. X + NAD+  Y + NADH

Inhibitors of enzymes
Some substances can inhibit enzymes. • Many enzymes are inhibited by particular chemical substances • The inhibitor usually binds to the enzyme reversibly, by non-covalent bonding • Some inhibitors, mainly poisons, bind covalently and irreversibly Many drugs act by inhibiting a specific enzymes. • For example: o captopril (lowers BP by inhibiting the enzyme ACE) o lovastatin (lowers serum cholesterol by inhibiting the enzyme HMG-CoA reductase) o methotrexate (slows cancer proliferation by inhibiting dihydrofolate reductase) • When an inhibitor is used as a drug, we rarely use enough to completely inhibit the enzyme. The aim is to slow itt down. Some inhibitors are competitive • They bind to the enzyme’s active site, and so directly prevent the substrate binding there • Many drugs are competitive inhibitors Other inhibitors are non-competitive • They bind elsewhere on the enzyme, and indirectly affect the ability of the active site to bind to the substrate • Many poisons are non-competitive inhibitors

Proteins and enzymes in diagnosis
Serum/plasma levels of specific enzymes and proteins can help diagnosis. • Cells contain a variety of enzymes • Some are widespread, some are found in substantial amounts in only one or two organs. • If an organ infers rapid major damage (e.g. lack of oxygen, infection), many of its cells die simultaneously and release their contents (including their enzymes) into the bloodstream. • So, measuring serum levels of enzymes (or other proteins) from an organ you suspect is damaged is useful in diagnosis. Blood, plasma or serum – which one to use in diagnosis? • For many lab tests, either plasma or serum is suitable • For enzymes, serum is normally used. Why?
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o Blood is a mizture of formed cells and plasma. While some lab tests focus on components found mainly in blood cells (e.g. haemoglobin), most focus on components found in plasma. o However, when blood is removed from the body, it clots, and this process alters the plasma to become serum (= plasma minus the clot protein fibrin). The clotting can be stopped by adding an anticoagulant to the blood immediately after the collection o The anticoagulant affects the measurement of most enzymes, so only serum is suitable for that

Carbohydrates
Summary facts about carbohydrates: • Glucose is the main carbohydrate in the blood • It is stored as the polymer glycogen in liver and muscle • The central role of glucose is to provide energy for cells (some organs, like the brain, can only use glucose for energy) • Fatty acids also have a range of other functions • Other important carbohydrates include plant cell wall components (known as dietary fibre) and the glycosaminoglycans of animal connective tissue

Structure of common carbohydrates in the body and in foods

Monosaccharides
This is the basic carbohydrate structural unit. Example: hexoses • these have 6 carbon atoms • example are glucose, galactose and fructose Example: pentoses • these have 5 carbon atoms • examples are ribose (found in RNA) and deoxyribose (found in DNA)

Disaccharides
These are two monosaccharides linked together by a covalent, glycosidic bond. Example: lactose = galactose + glucose Example: sucrose = glucose + fructose (produced by sugar cane and other plants)

Polysaccharides
These are carbohydrate polymers (many monosaccharides). They are also called glycans or complex carbohydrates. • Some are linear (that is, unbranched) polymers, others are branched polymers. • Some consist of only glucose molecules linked together, others have more than one monosaccharide component. Examples: • Glycogen (polymers of glucose; made in the liver and muscle for storage) • Amylose and amylopectin (starches) • Dietary fibre • Glycosaminoglycans
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Functions of glucose and glycogen

Glucose: a key monosaccharide.
Glucose is carried in the blood: • it is a water-soluble molecule and travels in the blood • it can enter all cells • for some cells, like muscle cells and adipocytes, its entry requires the presence of insulin • others (the brain and red blood cells, for example), it can enter freely without hormones Issues of terminology – ‘plasma glucose level’, ‘blood sugar level’ etc. • The term ‘level’ is usually used interchangeably with ‘concentration’ • Glucose is fairly evenly distributed between RBCs and plasma, so measurements of glucose levels in blood are about the same as levels in plasma • Glucose is unique for this; for many others, they are different • Most labs perform glucose measurements on plasma rather than blood as a matter of convenience • Using ‘blood sugar level’ is incorrect because this commonly refers to table sugar, which is the disaccharide sucrose, and there is no sucrose present in blood • mmol/L (i.e. millimols per litre) is the Australian standard for measurement. • 5m/mol/L is healthy, which equates to about 90mg/100mL (US) Glucose is catabolized to provide energy. • All cells are able to use glucose as an energy source • Catabolism of 1g of carbohydrates releases about 17kJ of energy, roughly the same amount of energy as 1g of amino acids.

1g carbs  17kJ energy
The brain burns a lot of glucose. • While most cells can use a variety of fuels, the brain is unusual: it must have glucose, and plenty of it. So it is important to keep blood glucose at a level that adequately supports the brain’s needs. • The brain accounts for ~20% of the energy usage in a human at rest. • It burns about 140g of glucose per day (about half of the average daily intake of carbohydrates among adult humans)

Glycogen: a key polysaccharide
Glycogen is a branched glucose polymer. • At branches, one glucose is linked to three other glucose monosaccharides: to one at C1, one at C4 and one at C6. • Liver glycogen is a backup resource for ‘topping up’ the glucose in the blood. • Surplus glucose can be converted to glycogen in the liver for storage • If plasma glucose levels fall (e.g. in short-term starvation), the liver breaks down some of its glycogen into glucose, to ‘top-up’ the plasma glucose level. In longer-term starvation, all the liver glycogen is used up and other mechanisms are used to keep plasma glucose levels in a satisfactory range. Muscle glycogen is a backup energy reserve, for use by muscle only • Surplus glucose from the bloodstream can be converted to glycogen in muscle. • However, muscle glycogen cannot be used to top up blood glucose levels. • It is there solely to provide energy within the muscle for urgent physical activity.
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Converted to GLYCOGEN in MUSCLE

Glucose (blood)

Converted to GLYCOGEN in LIVER

broken down to GLUCOSE

broken down to GLUCOSE

STAYS IN MUSCLE

Glucose and glycogen turnover in the body
The body’s content of glucose, glycogen and other carbohydrates: • Essentially all ‘free’ glucose is in the blood, about 5-6g of it, which works out to be ~5mmol/L • The body also contains about 500g of the glucose polymer glycogen, inside cells (all of it) • The amounts of other mono-, di- and polysaccharides in the blood and the cells are much smaller. • Animal cells do not contain or make starch, though they do make the related polymer glycogen. Dietary sources of carbohydrates: • Fructose (found in fruits) • Sucrose (found in table sugar, also found in sweetened cereal products, cakes, biscuits, soft drinks) • Lactose (found in milk and yoghurt, but not cheese or butter • Starches (in bread, breakfast cereals, rice, potatoes, legumes) Disaccharides and polysaccharides cannot be absorbed intact in the gut. They are first digested, by enzymes in the gut, to their constituent monosaccharides, which can then be absorbed into the blood and delivered to cells. Glycogen does not occur in foods. Glycogen in animal tissues breaks down very rapidly after death. The diet is a major source of glucose. • Dietary sources contain little glucose as such, but plenty of glucose as part of the disaccharides lactose and sucrose and part of the starches. Non-glucose monosaccharides can be converted into glucose. • Dietary sources also provide other monosaccharides (by themselves or in sucrose) and galactose (in lactose). • Fructose and galactose are mainly converted into glucose and glycogen inside cells. The liver can convert non-carbohydrate substance to glucose. • Liver cells can produce glucose from amino acids (from protein break down) and glycerol (from triglyceride breakdown); this normally occurs only in fasting/starvation • Liver cells also produce glucose from lactate (formed when muscles carry out physical activity with a limited oxygen supply)

amino acids

Liver Page 59 can convert

triglyceride breakdown

Surplus glucose can be converted to other substances for various purposes • Glycogen (in liver and muscle) o mainly happens a few hours after a meal, when blood glucose levels are high • Fatty acids and amino acids o mainly happens a few hours after a meal, mainly in the liver o inly a limited range of amino acids can be made from glucose o conversion of glucose into fatty acids and amino acids is fairly limited in humans Liver glycogen is used to top up plasma glucose levels. Normally, there is only limited fluctuations of blood glucose and cellular glycogen levels.

Starches
Amylopectin, amylose. • • • • • Both amylopectin and amylose contain glucose as their only monosaccharide component. Amylopectin is branched (similar structure to glycogen, though branch-points are less frequent) Amylose is unbranched They are found in foods of plant origin, notably cereal, grains, potatoes, and legumes (e.g. soybeans, chickpeas) Amylopectin usually comprises the majority of the starch

Dietary fibre
• • • • • • This is a collective term used for several polysaccharides found in plant cell walls Their importance for humans and animals is increasingly being recognised One major type is cellulose Our major dietary sources are cereal grains (especially wholemeal and wholegrain), fruits and vegetables. They cannot be digested by enzymes that animals and humans produce, although, the bacteria that normally live in the gut can ferment them. They prevent constipation, probably helping to contribute to gut health in other ways too.

Glycosaminoglycans
• • These are unbranched polysaccharides that contain amino-sugar monosaccharides (e.g. glucosamine), generally with more than one monosaccharide component. For example: o Condroitin sulphate – an important structural component of cartilage o Hyalouronate – an important structural component of joints

Carbohydrates in diagnosis
Plasma (or blood) levels are often measured in human patients. High levels often indicate diabetes mellitus.

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Is carbohydrate essential in our diet?
• • • Some fairly extreme diets claim we should virtually eliminate carbohydrate from out diets. Most cells use glucose as an energy source, and some (e.g. brain, RBCs) depend on having an adequate and continuous supply of it. If blood glucose levels are low, many cells switch to other fuels, and the liver can convert amino acids to glucose. However these adaptations cannot completely supply the needs of the brain and RBCs. So some carbohydrates are still essential in our diet. Given that we can convert the common mono and disaccharides into glucose, any of these can potentially satisfy our dietary glucose need. Fibre has important benefits and is essential in our diet. Because many carbohydrate-rich foods are rich in a range of important nutrients, a diet high in these foods has positive health benefits.

Lipid (fats)
• • • • • Triglycerides are the most common lipid found in the body, where they are used as an energy store. They are made of fatty acids attached to glycerol. Apart from being an energy store, fatty acids have a range of other functions. Other important lipid (present in much smaller amounts in the body) include cholesterol, phospholipids, fat-soluble vitamins and eicosanoids. Each of these have important functions.

Structure of fatty acids and triglycerides

Triglycerides are glycerol bound covalently to three fatty acids
• • • • • Molecular weight is usually several hundred. Usually, the three fatty acids on a triglyceride are non-identical Glycerol has three C-atoms Diglycerides have glycerol bound to two fatty acids Monoglycerides have glycerol bound to one fatty acid

Fatty acid structure
• • Fatty acids are organic acids They all have –COOH groups at one end, attached to a long aliphatic chain of C-atoms, linked mostly by covalent C-C bonds

The different types of fatty acids
• • • All have –COOH groups at one end The most commonly-occurring fatty acids have between 16 and 22 C-atoms The C-atom furthest away from the –COOH group is called the omega (ω) C-atom

Saturated fatty acids • Have no double C=C bonds in the aliphatic chain • Examples include: o palmitate – C16:0 o stearate – C18:0 o The 16 and 18 refer to how many Cs there are, the zeros refer to how many C=C double bonds there are in the molecule Monounsaturated fatty acids (MUFA)
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• •

Have one C=C double bond Example: o oleate – C18:1

Polyunsaturated fatty acids (PUFA) • Have more than one C=C double bond • In polyunsaturated fatty acids, double bonds are three C-atoms apart o i.e. … C=C-C-C=C-C-C=C-C-C=C-C-C=C-C-C=C-C-C… o It is important biologically to distinguish ω3 and ω6 polyunsaturated fatty acids Omega 3 (ω3) polyunsaturated fatty acids • First double bond begins with the third C-atom from the ω end. • These examples are important and must be memorised: o ALA – alpha-linoleate, C18:3, ω3 o EDA – C20:5, ω3 o DHA – C22:6, ω3 Omega 6 (ω6) polyunsaturated fatty acids • First double bond begins with the sixth C-atom from the ω end. • These examples are important and must be memorised: o linoleate – C18:2, ω6 o arachidonate – C20:4, ω6 Note – linoleate is C18:2, ω6, and alpha-linolenate is C18:3, ω3

Cis- and trans- monounsaturated and polyunsaturated fatty acids
• • • Normally, the double C bond in monounsaturated and polyunsaturated fatty acids are in cis conformation Industrial processing (e.g. margarines) can sometimes convert this to trans conformation Cis and trans conformations can have quite different biological properties

Functions of fatty acids and triglycerides
Triglycerides are primarily a storage form for fatty acids and thus a store for energy. • They are the body’s largest energy store, most of it in special storage cells called adipocytes • Because fatty acid catabolism yields water, a camel’s hump (which is mainly triglycerides) thus acts as a backup source of water. Desert and marine animals can also use their triglyceride stores as a water source (even humans to a smaller extent) All fatty acids can be catabolized to produce energy. • This is their main function • The end products of catabolism are normally CO2 and H2O • Catabolism of 1g of fatty acid releases 37g of energy

1g fatty acids  37g energy
All fatty acids play a structural role in cell membranes • Usually as part of a triglyceride and/or phospholipids Different fatty acids have different effects on specific body functions. Cardiovascular disease
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Increase the risk of cardiovascular disease: • saturated fatty acids • trans monounsaturated fatty acids • trans polyunsaturated fatty acids Decrease the risk of cardiovascular disease: • cis monounsaturated fatty acids • cis polyunsaturated fatty acids (ω3 and ω6) So, to prevent or treat cardiovascular disease, it is is helpful to modify the fatty acid composition of our diet. Nervous tissue alpha-linolenate (C18:3 ω3) polyunsaturated fatty acid is important for normal development of the retina and synapses Skin linolenat (C18:2 ω6) polyunsaturated fatty acid is important for normal development of the skin.

Triglyceride and fatty acid turnover in the body
The body’s fat content: • A lean human contains about 12kg of triglyceride (and other species are similar) • An obese human may have over 100kg of fat Sources of fats in human diets • most dietary fat is triglyceride • In Australia, major contributors to triglyceride intake in humans are: o means o fatty spreads o oils o mulk and milk products o take-away foods o cooking fats o biscuits o pastries • Other sources with high triglyceride content but low consumption o nuts o eggs o oily fish Dietary triglycerides provide a lot of the fatty acids for the body’s cells • Triglycerides cannot be absorbed intact from the gut • Instead, dietary triglycerides are sp[lit into fatty acids in the gut by a fairly complex process (discussed later) • Fatty acids from dietary triglycerides can be absorbed into the blood from cells, where they are available for triglyceride synthesis and other usage Some fatty acids are made by our cells from non-fatty acid precursors • Some fatty acids can be made by cells from substances derived from the metabolism of such fuels as carbohydrates (discussed later) Fatty acids are constantly being incorporated into storage, mainly in adipocytes • Fatty acids both from the diet and cell-synthesized Adipose stores of triglycerides are constantly being broken down to form fatty acids
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• • •

Fatty acids catabolized to release energy, Some may be catabolized to provide energy, some may be diverted to other specialised functions (such as ketone body formation) Ketone bodies are mainly formed during starvation as an auxiliary energy source for muscles

Some fatty acids can be made in our cells, others are essential • In practice, much of the fatty acids in our body come from dietary triglycerides • Saturated and cis-monounsaturated fatty acids are non-essential (we can make these from carbohydrates) o We don’t actually need these fatty acids in our diet, through in practice most of these in our cells come from our diet • Note, we cannot convert fatty acids into carbohydrates o However, plants can Two C-18 polyunsaturated fatty acids are essential in our diet and cannot be made in our cells: 1. alpha-linolenate – C18:3 ω3 2. linoleate – C18:2 ω6 Plants and bacteria can make these fatty acids. Other fatty acids are conditionally essential, that is, can be made in our cells under certain circumstances. • We can make limited amounts of them from other substances found in our cells, but when demand is heavy, we can’t make enough of them. • They include: o longer ω3 polyunsaturated fatty acids (C:20 and C:22) o longer ω6 polyunsaturated fatty acids (C:20 and C:22) For example, to make C:20 ω3 PUFA, we can convert C:18 ω3  C:20 ω3 or, to make C:22 ω6 PUFA, we can convert C:18 ω6  C:20 ω6  C:22 ω6.

Specialised lipids

Cholesterol and steroids
• • • • Cholesterol is a complex lipid that is imported into cell membranes The build-up of cholesterol in blood and blood vessel walls increases the risk of CVD Some are converted into various steroids, mostly hormones (e.g. oestrogen, testosterone, cortisol) Small amounts exist in foods, but we can produce our own, so cholesterol is not essential in our diet

Eicosanoids
• • • • Examples are thromboxanes and prostacyclins These are ‘local hormones’ made in the body from polyunsaturated fatty acids They are not present in foods Different eicosanoids affect blood clotting, blood pressure and inflammation

Phospholipids
• • • • Structurally similar to triglycerides, but also contain phosphate and organic bases (e.g. chlorine) Crucial in cell membrane, breathing, blood clotting Small amounts are in some food, but these get digested in the gut We make them in our cells, so they are not essential in our diet

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Fat soluble vitamins – A, D, E, K
• • • • These are fat-soluble organic compounds The main ones are A, D, E and K We cannot make them on our own (except for Vitamin D) so, small amounts are essential in our diet Mostly found in foods that contain fat

Lipids in diagnosis and therapy
Diagnosis: plasma levels of triglycerides and cholesterol are frequently measured in human patients. High levels are usually indicative of cardiovascular disease. Therapy: manipulating fatty acid composition in our diet is a standard part of treating common types of heart disease.

Summary of fatty acid usage
Diet Triglycerides Fatty acids

Dietary carbs Body

FA
Absorbed into enterocytes and…

FA

cells can convert carbs into FA (and not the reverse)

CIRCULATION
storage in some

Non-FA precursors converted to storage

FA
usage

Adipocytes
TG catabolized (constant)

USAGE FOR ENERGY FA

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Micronutrients
Summary: • Micronutrients are low-molecular weight components that carry out specialised infrastructure functions in the cell • Almost all substances in this category (there are a few exceptions) cannot be made in the body • They are not catabolized to produce energy (although some play a crucial role in obtaining energy from our major energy sources) • We only need small amounts of them (most: a few milligrams, some even a few micrograms or nanograms per day) • The difference between vitamins and minerals is that o Vitamins are organic o Minerals are inorganic

Some roles of vitamins
Vitamins are organic molecules • For historical reasons, the term ‘vitamin’ excludes essential amino acids and essential fatty acids • Vitamins may be referred to by their letter names, or by their chemical names (e.g. thiamine) or in some ways, both o e.g. Vitamin C – ascorbate Some are water-soluble • Examples are: o niacin o vitamin B6 o folate o vitamin B12 o riboflavin o pantothenate o vitamin C • Water-soluble vitamins may be lost from foods that are cooked with hot water Some are fat-soluble • Vitamins A, D, E and K • Fat-soluble vitamins may be lacking in diets that are very low-fat Some are crucial for releasing energy from fuels • They form co-enzymes needed for specific energy-releasing metabolic reactions, notably niacin, riboflavin, thiamine, vitamin B6 and pantothenate • Deficiency of any of these vitamins damages organs with high energy needs, such as brain and muscle Some may help prevent/manage heart disease • Via antioxidant properties (vitamin E, C and beta-carotene) • Via effects on homocysteine levels (folate, vitamin B12, vitamin B6) Some are needed for DNA synthesis • Folate, vitamin B12, vitamin B6 (hence can help prevent anemia) Folate helps prevent of neural tube defects (spina bifida). Proper vision – vitamin A (which occurs in the diet as either retinol or beta-carotene).
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Proper bone structure: • Vitamin C – for proper collagen structure • Vitamin D – for maintaining blood calcium (bone resorption)

Roles of some minerals and trace elements
• • • Minerals and trace elements are inorganic ions The difference between the two is that trace elements are required only in very small amounts At least 30 inorganic elements are important in humans

Iron (Fe) • essential for making haemoglobin • deficiency  anemia Calcium (Ca) • essential for bone and teeth structure • important for signalling inside cells and for muscle contraction • deficiency  rickets, osteoporosis Sodium (Na) • important for neuronal impulses; affects plasma volume • excess  high blood pressure (restriction of dietary Na reduces the risk of high blood pressure) Iodine (I) • essential for making thyroid hormones (thyroxine etc.) • deficiency  formation of thyroid goitre, retarded growth, cretinism Selenium (Se) • essential part of several key enzymes • deficiency  damage to heart muscle, may increase the risk of CVD and cancers • excess  toxicity

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A Healthy Diet
Summary: • The National Health and Medical Research Council is the peak health advisory body to the Australian federal and state government • NHMRC issues diet advice in two major forms: 1. Nutrient Reference Values (expressed in terms of quantitative nutrient intakes) 2. Dietary guidelines (expressed as qualitative statements about some nutrients and some food practices) • Also, the Federal Health Department has released The Australian Guide to Health Eating, which seeks to translate the above into practical statements about food choices

Nutrient Reference Values (NRV)
• • • Nutrients are substances our bodies obtain from food that are believed to play an important role in the functioning of our bodies. Nutrient Reverence values are indicators of the quantity of various nutrients healthy humans need per day Various indicators are used, all based on assessment of an individual’s average daily intake o Estimated Average Requirement (EAR) o Recommended Daily Intake (RDI) o Average Intake (AI) o (for some nutrients, AI is used instead of EAR and RDI)

EAR, RDI and AI indicate the quantity of a nutrient we need to avoid deficiency. For a given age-gender group: • EAR – estimated average requirement o is the amount that should prevent any sign of deficiency in at least 50% of people • RDI – recommended dietary intake o is the amount that should prevent any sign of deficiency in 97% of people o therefore rather higher than most individuals would ever need o it is wrong to diagnose a person as being deficient simply because their intake on a given day is a little below the RDI • AI – adequate intake o is an amount that’s ‘probably’ enough to avoid deficiency for most people o used instead of EAR and RDI where the available data for some nutrients were considered inadequate for estimating those nutrient reference values The NRVs are based on estimates of the amounts needed to avoid deficiency of a specific nutrient. Calculation of NRVs is based (where possible) on intervention studies, where volunteers are kept on a controlled diet known to be adequate for all nutrients bot one’ levels of that one nutrient are manipulated, and the volunteers are monitored of deficiency. UI – upper level of intake This value indicates the quantity that may lead to toxic side-effects. This is the amount above which there is a significant risk of adverse effects from the nutrient.

Dietary guidelines for Australians, 2003
These are important and should be memorised. 1. Enjoy a wide variety of nutritious foods
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• • • • • 2. • • • •

eat plenty of vegetables (including legumes), and fruits eat plenty of cereals (including breads, rice, pasta and noodles), preferably wholegrain include lean meat, fish, poultry and/or alternatives such as legumes and nuts include milk, yoghurt, cheeses and/or alternatives drink plenty of water Take care to limit saturated fat, and moderate total fat intake choose foods low in salt if you choose to drink, limit alcohol intake consume only moderate amounts of sugars, and foods containing added sugars

3. Prevent weight gain by being physically active and eating according to your needs 4. Care for your food and keep it safe to eat 5. Encourage and support breastfeeding

Background for the dietary guidelines
‘Eat plenty of legumes, vegetables and fruits’ • ‘Strong evidence now exists that many compounds in fruits and vegetables (including antioxidants and folate) may help to prevent the development of a number of non-infectious degenerative diseases, including: o cancer o cardiovascular disease o diabetes mellitus (type II) o hypertension o cataract and macular degeneration of the eye • Select from a wide variety of types and colours of these foods.’ ‘Eat plenty of cereals (including breads, rice, pasta and noodles)’ • Wholegrain cereals reduce the risk of developing coronary heart disease, colorectal cancer, constipation and diverticular disease, and may help prevent obesity • Reduced-salt bread is preferable to normal bread • Cereals with a low glycaemic index are preferable to those with a higher GI ‘Include lean meat, fish, poultry and/or alternatives such as legumes and nuts’ • Lean red meat helps ensure adequate iron intake (especially among females between puberty and menopause, and among athletes) • A serving of fatty fish per week helps ensure adequate ω3 polyunsaturated fatty acid intake • The visible fat of red meat, and the skin of chicken, is mainly saturated and should be restricted • Vegetarians are encouraged to eat a wide range of legumes, nuts and seeds (including wholegrain cereals) ‘Include milks, yoghurts, cheese and/or alternatives’ • Calcium is important for achieving and maintaining acceptable bone density and strength • Recommend reduced-fat milk, cheese, yoghurt, all of which are good calcium sources • Dairy fat is mainly saturated, and should be restricted • Those with lactose intolerance are advised to use low lactose milk, or yoghurt (low lactose) and cheese (no lactose) ‘Drink plenty of water’
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• • • •

Low fluid intake may cause cognitive impairment, constipation, kidney stones, possible urinary tract infections While water is the preferred drink, the report also recognises milk and fruit juice as beneficial, having both water and important nutrients Tea is a useful source of antioxidants Because of their diuretic effect, excessive intake of alcohol, coffee and ‘energy drinks’ (e.g. caffeine) can be dehydrating

‘Take care to limit saturated fats, and limit/moderate total fat intake’ • This reflects concerns about o obesity (a reduction in total fat intake can help an individual lose weight) o coronary heart disease (recommended reductions in intake of saturated and trans fatty acids, a moderate increase of ω6 polyunsaturated fatty acids, and a doubling of ω3 polyunsaturated fatty acids ‘Take care to choose foods low in salt’ • ‘The last decade has produce an international consensus that a modest sodium reduction for people with normal and raised blood pressure has a large enough effect on blood pressure to justify a guideline advocating restraint for the entire population.’ • The emphasis is on foods low in salt; salt added in cooking and at the table is only a small proportion of total sodium intake ‘If you choose to drink, take care to limit your alcohol intake’ • Men – 2 standard drinks per day • Women – 1 standard drink per day

Metabolism – basic principles
Key fuel-metabolism hormones: Insulin encourages the storage of foods. • insulin is a protein • it is secreted by beta-cells in the endocrine pancreas Glucagon releases stored fuels in starvation • glucagon is a protein • it is secreted by alpha-cells in the endocrine pancreas Adrenaline (epinephrine) releases stored fuels for urgent activity • Adrenaline is a modified amino acid • It is secreted by the adrenal medulla

Key metabolic situations

Post-prandial state
Post-prandial state (meaning ‘after a meal’) is also known as the absorptive state. It takes place during roughly the first 4-6 hours after a meal. 1. Recent feeding  high plasma levels of glucose, amino acids and triglycerides o these are derived from digestion and subsequent processing of dietary proteins, carbohydrates and fats 2. High plasma glucose levels stimulate insulin release and suppress glucagon release o therefore high insulin, low glucagon in plasma 3. Insulin promotes fuel storage
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o promotes uptake of glucose from plasma into adipose and muscle cells o stimulates the synthesis of glycogen stores (liver, muscle) and triglyceride stores (adipocytes) o stimulates protein synthesis in cells 4. Most cells are using glucose from plasma as a major fuel at this stage

Emergency: fight, fright, flight or folic
1. Brain stimulates adrenal glands to release adrenalin 2. Adrenalin  fuel release to prepare for action (glucagon and adrenalin have similar effects – adrenalin mainly keeps muscles happy, glucagon: other cells) 3. Adrenalin stimulates key enzymes that cause a. glycogen breakdown in muscle i. build-up of ATP in muscle, making it ready to contract urgently b. glycogen breakdown in liver i. release of glucose in plasma, as further fuel for muscles c. triglyceride breakdown in adipose tissue i. release of free fatty acids into plasma (as further fuel for muscles)

Short-term fasting: the early phase of the post-absorptive state
Approximately 4-24 hours after a meal, the short-term fasting phase occurs. However, transition from the post-prandial state to the short-term fasting pattern is gradual, not abrupt. 1. Low levels of plasma glucose, amino acids and triglycerides (i.e. they have been either used or stored) 2. Low plasma glucose stimulates glucagon release and suppresses insulin secretion • low insulin levels and high glucose levels in plasma 3. Glucagon promotes fuel release • it stimulates key enzymes that cause o glycogen to break down in the liver o triglycerides to break down in adipocytes  release fatty acids into plasma 4. Most tissues (except for brain and RBCs) are using fatty acids as their main fuel • glucose supplies are reserved for glucose-dependent cells (such as the brain and RBCs)

Longer-term fasting: the later phase of the post-absorptive state
This occurs more than 24-hours after a meal, but transition from the shorter-term fasting pattern is gradual, not abrupt. 1. Low plasma levels of glucose, amino acids and triglycerides 2. Low plasma levels of glucose stimulates glucagon release from the alpha cells in the endocrine pancreas • low insulin levels and high glucagon levels in plasma 3. Glucagon promotes fuel release • liver glycogen gets used up 12-24 hours after fasting • so, in more prolonged fasting, glucagon allows the body to draw on other fuels • it stimulates key enzymes that cause: a. extensive triglyceride breakdown in adipose tissue i. release of fatty acids ii. some of these fatty acids are converted to ketone bodies (a more convenient fuel) by the liver b. conversion of amino acids to glucose, a process called gluconeogenesis, in the liver (the increased demand for amino acids leads to increased muscle breakdown)
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4. Most tissues (except for the brain and RBCs) are using fatty acids and/or ketone bodies as their main fuel. • glucose supplies are reserved for glucose-dependent cells • in prolonged starvation, the brain starts to use ketone bodies as a fuel

Key ‘energy’ molecules: ATP, ADP and AMP

The basics of nucleotides:
• Nucleotides contain the following linked covalently together: o phosphate o specific nitrogenous base (adenine, guanine, cytosine, thiamine/uracil)\ o pentose (a sugar, either deoxyribose or ribose) A mononucleotide has one base + one pentose + one phosphate A polynucleotide has many mononucleotides linked together. The two main types of polynucleotides are DNA (double-stranded) and RNA (single-stranded)

• •

ATP
• • • Adenine is a nitrogenous base It is used in ATP, and in the related substances ADP and AMP These are all technically adenine molecule (mononucleotides), but they differ in the nature of their phosphate component.

ATP – (adenosine triphosphate) adenine+ribose+phosphate+phosphate+phosphate ADP – (adenosine diphosphate) adenine+ribose+phosphate+phosphate AMP – (adenosine monophosphate) adenine+ribose+phosphate ATP acts as a direct energy source for reactions in a cell; if a cell’s supply of ATP is sharply reduced, it dies. • An adequate supply of ATP is crucial for cell functioning, and cell survival • When a cell splits stored ATP into ADP (or in some situations, AMP) then this provides energy for the synthesis of important molecules (including proteins, adipose triglycerides, glycogen, DNA) and for processes that require energy (e.g. pumping molecules across cell membranes against concentration gradients). • A cell that cannot do such things rapidly, dies. Each cell must make its own ATP via the catabolism of fuels. • Dietary ATP/ADP/AMP is completely digested in the gut • ATP/ADP/AMP cannot leave cells • So, administering them orally or by injections is completely useless • Each cell has to make its own Cells engage in catabolic process, metabolising fuels to ensure that they have enough ATP • Catabolism yields energy • That energy is used to drive the following reaction:

ADP + phosphate  ATP
Each cell tries to keep its ATP supply fairly constant. • A fall in cell levels of ATP signals an energy shortage, so the cell speeds up key catabolic reactions in metabolism • A rise in cell levels of ATP has the opposite effect on these reactions
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Because each cell has a pretty constant amount of adenine, a rise in ATP levels means that ADP and AMP levels fall, and vice versa.

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Catabolic and anabolic processes
Key fuels used
The major fuels used are carbohydrates, fats and amino acids. In some species, alcohol can also contribute. Different fuels provide different amounts of energy per gram: • Carbohydrates – 16 kJ per gram • Amino acids – 17 kJ per gram • Alcohol – 29 kJ per gram • Fat – 37 kJ per gram To put that into context, a typical adult human may use 8,000-10,000 kJ per day.

Fundamental process in catabolism – oxidation
We oxidise fuel by many small steps: energy is not released in a single ‘big bang’. The later reactions in fuel catabolism are the same for all fuels, but the early reactions are different for different fuels. Most of the oxidation steps do not use oxygen directly. Instead, they use coenzymes that can exist in both oxidised and reduced states.

The overall reaction for catabolism

Fuel + oxygen  carbon dioxide + water
This consists of two linked processes: 1. an oxidation (the reduced substance ‘fuel’ is converted to the oxidised form, carbon dioxide) 2. a reduction (the oxidised substance ‘oxygen’ is converted to the reduced substance, water) A good supply of oxygen is very important. Oxygen is delivered to cells via the blood, so a blockage to blood flow means a big drop in oxygen supply. Most oxidation steps in catabolism do not directly involve oxygen, but instead involve the conversion of coenzymes to their reduced form. • Only one of the oxidation steps directly involves oxygen. The other oxidation steps use coenzymes. The process is best explained by a typical example (from the TCA or Krebs cycle – stage B of catabolism): • Malatate + NAD+  oxaloacetate + NADH • This consists of two linked processes: 1. an oxidation (the reduced substance malatate is converted into the oxidised form oxaloacetate) 2. a reduction (the oxidised coenzyme NAD+ is converted into the reduced coenzyme NADH) Reduced coenzymes formed by such steps are later converted back to their oxidised form with the help of oxygen. • This re-conversion happens in the respiratory chain stage of catabolism (that is, stage C). • Thus, the coenzymes can be recycled to allow catabolism to continue, provided that oxygen is available. The main coenzymes involved in oxidation steps during catabolism are NAD and FAD. • Oxidised: NAD+; Reduced: NADH • Oxidised: FAD; Reduced: FADH2 They cannot be interchanged; for any specific reaction, only one of them will do the job.
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Aerobic catabolism
Aerobic catabolism of all common fuels has three key stages.

Stage A: preliminary processing to create an intermediate of the TCA [Krebs] cycle – different for various fuels
For some fuels, this stage occurs partly in the cytoplasm (cytosol); for others, it occurs in the mitochondria. a. obtain fuel molecule (from body stores or blood) b. convert it to an intermediate of the TCA cycle (that enter stage B) This stage includes oxidation steps that use oxidised coenzymes and yield reduced coenzymes (that enter stage C).

Stage B: the TCA [Krebs] cycle – same for all fuels
This occurs in the mitochondria a. oxidise the intermediate provided by stage A. (The oxidation steps use oxidised coenzymes and yield reduced coenzymes that enter stage C) b. yields carbon dioxide (breathed out)

Stage C: the respirator chain and oxidative phosphorylation
These occur in the mitochondria. a. respiratory chain oxidises reduced coenzymes (from stages A and B), using oxygen, and producing water. b. oxidative phosphorylation uses the energy released by these oxidations to convert ADP to ATP. Stage A varies between fuels: The details of stage A are different for different fuels. We don’t need to know this part for the exam, but a quick outline should be understood so that we will recognise the context when the terms are used in other parts of the course.
Carbohydrate Input for stage A Usually glucose from blood, but sometimes (in muscle) intracellular glycogen is used directly Fatty acids from blood Water-soluble by-product of beta-oxidation (in liver only) if lots of FA are being catabolized Amino acids from blood Main pathway Glycolysis ( pyruvate) + PHD step (to convert pyruvate to acetyl-CoA) Beta-oxidation A few simple steps Output to feed into stage B Acetyl-CoA

Fats Ketone bodies

Acetyl-CoA Acetyl-CoA

Aminoacids

Alchol

Alcohol from blood (direct from diet)

Firs step is transamination to remove amino group (which end up in waste-product urea). The fate of the remaining part of the AA molecule varies between AA. Alcohol  acetaldehyde  acetyl-CoA

Varies between amino acids

Acetyl-CoA

Certain vitamins are essential for catabolism
As noted above NAD+/NADH (derived from niacin) and FAD/FADH2 (derived from riboflavin) are closely involved in oxidation reactions of catabolism. Hence, dietary deficiency of either of these vitamins causes illness (due to reduced capacity for catabolism). The vitamins thiamine and pantothenate, required for other types of reactions, are also essential in the diet, and deficiency causes illness.

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Catabolism is a complex process, but smoothly integrated
Sometimes on paper, these processes look long and complicated. In cells, however, the whole process occurs very rapidly, in a second or so. Each reaction is catalysed by a separate enzymes; each enzyme passes its produce swiftly on to the next enzyme.

Fuel catabolism under anaerobic conditions is much less efficient
The processes above apply when there is an adequate oxygen supply (in ‘aerobic’ conditions). Oxygen is only used directly at stage C, but the other stages crucially depend on the operation of that stage. Sometimes cell have to operate under anaerobic conditions (poor oxygen supply), e.g., heart attack. Cells cannot catabolise fats (or ketone bodies) under such conditions. They can only catabolise glucose, and they can only partially catabolise it: the produce is lactate, a by product of stage A of the TCA [Krebs] cycle. ATP production is sharply reduced, to about 10% of normal: cells with high energy needs (e.g. heart muscle, brain) can only survive a few minutes like this, and rapidly die.

Anaerobic processes (introduction and examples)
Basic summary: • Anabolism is essentially the opposite of catabolism • It is how the cell synthesizes molecules that it needs for specific purposes, e.g., DNA proteins and enzymes, energy-storage molecules (such as glycogen and triglycerides) • Unlike catabolism, these synthetic process usually involve reductions reactions only, not oxidation reactions • Anabolic reactions are normally stimulated by insulin, and use ATP • Several different vitamins are essential for particular anabolic processes.

Example 1: Glycogen synthesis in liver and muscle
• Plasma glucose can be converted to glycogen o Plasma glucose can enter muscle and liver. o There it can be converted to glycogen o Glucose  glucose-6-P  glucose-1-P  glycogen The key enzyme is glycogen synthase o The final step (glucose-1-P into glycogen) uses the key enzyme glycogen synthase. o This lengthens glycogen, adding one glucose-1-P molecule unit at a time (the P is removed and recycled) Vitamin B6 is essential for glycogen synthesis o Pyrixoxal phosphate is a coenzyme for glycogen synthase, so vitamin B6 deficiency impairs glycogen production ATP is needed to drive glycogen formation o ATP is used for the reaction: glucose  glucose-6-P o A related nucleotide, UTP, is used fro the reaction glucose-1-p  glycogen o So, the body only makes glycogen when it has energy to spare Insulin is crucial for key steps o Insulin is needed to get glucose into muscle only – not into liver o Insulin makes glycogen synthase more active in both liver and muscle Liver and muscle glycogen stores are limited in size o Total body stores of glycogen are about 500g

Example 2: Triglyceride synthesis in adipocytes
• Process 1: Plasma glucose enters cell and is converted to glycerol-3-P
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• • •

Process 2: Plasma triglyceride (carried on the particles chylomicra or VLDL) releases its fatty acids. These enter the cell, and become FA-CoA. Process 3: Finally, 3 FA-CoA combine with one glycerol-3-P to form a triglyceride molecule The key enzyme is lipoprotein lipase o This enzyme (found on blood vessel walls near adipocytes) works on both chylomicra or VLDL, to carry out the reaction TG  FA + glycerol ATP is needed for key steps o glucose  glycerol-3-P (in glycolysis) o FA  FA-CoA Insulin is crucial for key steps o Entry of glucose into adipose cell, and activation of lipoprotein lipase

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Biochemistry – DNA Component
DNA structure and the genetic code
Structure of DNA

• •

Like protein, which is a polymer of amino acids, DNA is a polymer of nucleotide subunits. A nucleotide consists of 3 distinct components: • a phosphate group (1 to 3) • a rubose sugar, and • a base. DNA is a deoxyribose nucleic acid - each nucleotide consists of a phosphate, a deoxyribose and one of four bases.

About nucleotides: • Nucleotides are linked to each other by a single phosphate group between deoxyriboses. This forms a linear, unbranched polymer. • The nucleotides have a direction. Just like a protein has an NH2 and a COOH end, DNA has a 5' end and a 3' end. • The new nucleotides are added on the the 3'-OH end during the synthesis of new DNA. • They consist of a backbone (sugar-phosphate-sugar-phosphate - the orange part on the diagram above) with bases hanging off the backbone. • There are four different bases: • Adenine - TWO RINGS - PURINE • Gunanine - TWO RINGS - PURINE • Cytosine - ONE RING - PYRIMIDINE • Thymine - ONE RING - PYRIMIDINE
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The convention is to simply write the order of bases (or nucleotides) in the 5' to 3' direction, as for protein amino acid sequence. E.g. 5' AGC TAC CTG AAA 3'.

Base pairing
Watson and Crick found that DNA strands H-bond together in pairs, so normally DNA from cells is double stranded. Its normal conformation is twisted into 2 helices - the 'double helix'. H-bond between strands is between purine and pyrimidine basis, and does not involve the ribose-P backbone. This is called base-pairing. Base-pairing combinations are restricted to just 2 different combinations: • A always binds with T • C always binds with G This sort of restriction exists because: • The distance between the strands can only accommodate a purine:pyrimidine pair. • The hydrogen bonds don't form between G and T, or A and C. • The number of hydrogen bonds formed between G and C is 3 (remember because there are three letters between C and G!) and A and T is 2. So, DNA in one strand has the complementary sequence of the second strand.

E.g. AAA TTT CAG binds to the complimentary strand: TTT AAA GTC Note that to base pair, the 2 strands actually run in opposite directions. I.e.: 5' AAA TTT CAG 3' 3' TTT AAA GTC 5' But the convention is to write all sequences in 5' to 3' orientation, i.e. 5' CTG AAA TTT 3' is the second strand, called the reverse complement.
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Rules for base pairing
4. Base pairing is automatic and self-directed. DNA effectively seeks out its complimentary sequence, because molecules in solution move around 5. Base pairing is reversible. The cell has proteins that can pull the strands apart when needed (e.g. replication the DNA for cell division). 6. Base pairing is exact. Two DNA strands MUST have complimentary base sequecnes to base-pair. Mismatched sequences results in no or only limited binding.

DNA replication

• • • • • •

All DNA in cells is double stranded, with the second strand the reverse complement of the first. The enzyme that makes the second strand is DNA polymerase. It synthesises new complementary strands, using each of the existing strands as templated. An incoming free nucleotide base-pairs opposite the existing base and is then added to the 3' end of the new DNA strand. An incorrectly base-paired nucleotide is released before it can be added to the 3' end of the new DNA strand. Both DNA strands are resynthesised before cell division, each new double-stranded DNA going to a daughter cell.

The DNA base sequence is the source of all amino acid sequences in polypeptides - the "Genetic Code"
We know that each protein has a specific, linear sequence of amino acids. DNA sequence of bases is also linear, and specific. In fact, the 2 sequences are co-linear - it is the sequence of bases in DNA that the cell "reads" or translates to specify the aminoacid sequence. The DNA sequence "codes" for the aminoacid sequence. Each aminoacid is represented by a specific 3-base sequence, called the codon. IMPORTANT THINGS TO REMEMBER ABOUT CODONS: The start codon: ATG. This is also methionine. This tells the cell where the coding sequence starts. The stop codons: TAG, TAA and TGA. This tells the cell where to end the polypeptide. (It is not translated into an amino acid). Remember redundancy. Many amino acids have more than one codon. The reason is that there are 64 possible codons (4x4x4), but only 20 amino acids. This means there is a redundancy in the code. The more commonly used amino acids have more than one codon.
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Open reading frame (ORF), also called the 'coding sequence', or CDS. This is the sequence that is translated into the amino acid sequence, it starts from ATG and ends at first 'in frame' stop codon. As the cell reads STRICTLY in codons, i.e., every 3 bases of sequence, the stop codon needs to be 'in frame' to be recognised, i.e., an exact multiple of 3 bases downstream from the start codon. E.g.: AAGGATGCAGACTAGATAGA is read as: A AGG ATG CAG ACT AGA TAG A, not A AGG ATG CAG AC TAG ATA GA.

Mutations in proteins arise from changes in DNA sequence
A mutation is a change in nucleotide (base) sequence in DNA. If this resides in a section of DNA that codes for protein, then there are going to be consequences for protein structure. This occurs for 2 main reasons: 3. damage to DNA that escapes repair, and 4. incorporation of the incorrect nucleotide into the DNA sequence during DNA replication prior to cell division. If it occurs in body cell, the worst it could result in is the death of the cell, or if in protein controlling cell growth or division, it could lead to cancer. If in a gamete (sperm, egg), it could conceivable be passed on to the next generation) inherited mutation). Spontaneous mutations affecting germ-line cells are quite rare events. Most you will encounter have been passed down the generations.

Some types of mutations
 Substitutions:  ATG GGC ACT AGT AG to  ATG GGG ACT AGT AG = no result, because both code for Gly. But if it mutates to:  ATG GCC ACG AGT AG then Gly is replaced by Ala.  Because of the redundancy in genetic code, many changes to 2nd or 3rd nucleotide of a codon either doesn't change the amino acid or it changes it to one of similar character.  Deletion or insertion:  ATG GGC ACT AGT AG = Met-Gly-Thr-Ser. With one nucleotide deleted, it becomes  ATG GGC CTA GTA G = Met Gly Arg Val.  Much larger deletions can occur, wiping out large sections of amino acid sequences.  Premature stop codon leading to protein truncation:  One nucleotide insertion to the above sequence gives ATG GGC AAC GAG TAG, which translated as Met-Gly-Asn-Glu-STOP.  This shortened protein may be misfolded, degraded, exhibit reduced activity, or not be affected at all, depending on protein structure.

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DNA replication and repair
DNA Replication
DNA must be replicated each time the cell divides, prior to division. It must be fast (because it has 6 billion base pairs to replicate!) and accurate (in our cells it makes about 1 mistake per billion base pairs). This is important because cells can divide frequently, and DNA carries the instructions for making the proteins, and hence the cell. DNA is a polymer of deoxynucleotide subunits linked to each other by a single phosphate group between deoxyriboses. DNA in one strand has the "complementary" sequence of the second strand. The double-stranded nature of DNA enables it to be faithfully replicated. The two strands are pulled apart and each strand acts as a template to synthesize a complementary strand. The enzyme that makes the second (complementary) strand is DNA polymerase. The free deoxynucleotide triphosphate (dNTP) precursor base-pairs in position and is the added to the 3' end of the DNA strand. Both strands are resynthesised before cell division, each new double stranded DNA going to a daughter cell. The precursors are dNTPs - dATP, dTTP, dCTP and dGTP (i.e. one for each nucleotide).

The rules of DNA replication

DNA polymerase has a number of requirements: 1. DNA is produced by copying a pre-existing strand 2. DNA polymerase cannot initiate a DNA strand - it requires a "primer" with a 3'-OH end. 3. DNA strand growth is in one direction only = 5' to 3'.

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The physical structure of DNA also provides a series of problems for replication, partly as a result of these 'rules' of replication: 3. Where to start? DNA synthesis begins at the replication fork, the site where the 2 DNA strands are separated. Eukaryotic DNA is very long, and therefore in humans, replication begins at multiple sites simultaneously (remember that it has to happen quickly). 4. DNA polymerase needs a 'primer' DNA strand with a free 2'-OH group to attach incoming nucleotides to. It cannot begin with a bare single stranded DNA alone. An enzyme called primase lays down a 'primer' strand for new dNTPs to be attached to by DNA pollymerase. This is made of RNA. 5. DNA polymerase can only synthesize in one direction (5' to 3'). Therefore, the two strands are made by slightly different mechanisms. For one strand, the leading strand, this occurs easily, while the other strand runs in the opposite direction, i.e. 3' to 5', the lagging strand and therfore has to be made in small sections. This is much slower, hence its name.

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Synthesis of the leading strand
The 2 DNA strands are pulled apart, a single RNA primer is laid down and nucleotides are added to the 3'OH by DNA polymerase.

Synthesis of the lagging strand

• • • •

The two strands are pulled apart, an RNA primer is laid down and DNA polymerase adds dNTPs onto the end of the primer for about 1000 bases. These are known as Okazaki fragments. A new RNA primer is then laid down closer to the replication fork as the DNA continues to open up, and a new Okazaki fragment is synthesized up to the previous fragment. The RNA primer is removed ad the 2 Okazaki fragments are joined or ligated together by an enzyme
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called DNA ligase. The lagging strand synthesis is thus discontinuous, while the other, 'leading' strand is continuous.

Mechanisms of errors during replication
We have (and need) exceedingly accurate copying of DNA - less than 1 mistake per billion bases is made in human cells. Why is this so accurate? We have about a trillion cells in the adult body, each derived from a cell division event and DNA replication, so there are plenty of opportunities for mistake to become established. There are two types of cell division, in which errors in DNA replication can cause problems: • Meiosis - formation of gametes. Errors here can lead to inviability of fetus or establishment of a genetic disease. • Mitosis - majority of cell divisions, e.g., to make body cells. Mutations to DNA (mistakes) are
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potentially less damaging since loss of some cells can be tolerated, unless the mutation establishes the neoplastic phenotype, which can lead to cancer.

DNA proof-reading
The chemistry of the DNA nucleotides can potentially interfere. A number of the bases can exist in alternate 'tautomeric' forms. There are isomers, a C=O bond becoming a C-OH, in cytosine for example. The majority of the time the bases exist in a single form, but rarely, about 1 in every 10,000 to 100,000 cytosine bases will be in alternate form and can switch transiently. This sort of base will hydrogen bond with adenine, not guanine, and potentially introduce a mutation into the daughter strand. If left in, a further round of replication will result in introduction of a T->C mutation. This is just a single example of mis-incorporation. This sort of mistake is avoided by the 'proof-reading' ability of DNA polymerases carried out as the DNA is being synthesized. • The incorrect dNTP is incorporated onto the 3' end of the DNA strand. • If base-pairing is disrupted, it doesn't proceed with synthesis, but removes the previous base with a 3'5' exonuclease activity. • With correct base-pairing established, synthesis continues.

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From DNA to protein - transcription and translation (Lecture 6.10)
DNA contains genes - the information coding for proteins. Each amino acid is coded for by a specific 3-base DNA sequence (a codon). The process by which the DNA code is converted into an amino acid code (or protein) involves 2 steps: 12. The DNA sequence is copied into an RNA sequence by a process called transcription. 13. The RNA sequence is converted to amino acid sequence by a process called translation.

RNA, or more specifically messenger RNA, is therefore an intermediate which links the DNA and protein codes. RNA is structurally similar to DNA except that: • it contains ribose instead of deoxyribose • it contains uracil instead of thyamine • it is single-stranded Why do we need an intermediate (or messenger) to convert the DNA code to protein? Several key reasons: 1. Proteins are constantly being degraded and replaced, at different rates depending on which protein it is. 2. Not all proteins are expressed in a given cell, i.e. cells express different sets of proteins (e.g. heart muscle cell contractile proteins versus RBC hemoglobin), even though all cells contain all the genes. Some selectivity is required. 3. Different proteins are expressed in different amounts, so proteins levels have to be selectively altered. This can be done most easily by regulating the amount of RNA expressed. Consequently, DNA is constantly being transcribed, and translated into protein. So these processes don't simply happen when the cell first develops, and then stops.

Transcription
Transcription is the first step in converting the DNA code to protein, and involves the formation of an mRNA copy of the DNA. mRNA is synthesised by RNA polymerase. The mRNA is complimentary to the antisense, or non-coding DNA strand (by convention drawn as the bottom strand) and is therefore identical to the sense strand. The DNA is pulled apart by RNA polymerase, and travels along the DNA using the antisense DNA strand as a template for the RNA. RNA polymerase:

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Most of our DNA does not contain genes (or protein coding regions) so the RNA polymerase must be able to work out which bits need to be transcribed, i.e., it needs to know where to start and stop transcription. The DNA contains 'signal' sequences indicating where the polymerase should start or stop. • The start signal is called the promotor - TATAAA (aka TATA box) • The stop signal is called the terminator - AAUAAA on the RNA, AATAAA on the DNA. Without these sequences, transcription of a strand of mRNA cannot occur and therefore a protein cannot be produced. The promotor or TATA box is recognised by a protein called TATA binding protein. It binds to the promoter, recruits other factors including the RNA polymerase, so that transcription can occur.

The rate of transcription of genes (or gene expression) is controlled by a large group of proteins called transcription factors:
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• •

• • •

increase or decrease the rate of transcription ("activation"/"repression"). Different transcription factors effect different genes, e.g., GATA is a transcription factor that alters expression of alpha-globin and beta-globin found in hemoglobin, but not contractile proteins, such as actin & myosin. Their expression may be cell-type specific, e.g., the transcription factor MyoD is expressed only in muscle cells. There are hundreds known Make up ~1% of the human genome

Transcription factors bind to specific DNA sequences These regulatory elements (sequences) are often referred to as 'enhancer elements' because they enhance the rate of transcription. They occur most frequently upstream, close to the TATA box, but can occur distantly, up to 50,000bp upstream or downstream from the coding sequence. Individual DNA regulatory elements are usually <20bp long.

Translation
Translation is the second step in converting the DNA code to protein, and involves the formation of an amino acid sequence from the mRNA. Translation requires three components: 3. mRNA - acts as the template for protein synthesis (generated during transcription) 4. transfer RNA (tRNA) - acts as an adaptor between the mRNA and the amino acid 5. ribosomes - the 'protein-making machines' tRNA:

• • •

A specialised RNA molecule. One specific amino acid is covalently bound to the 3' end. Folded into a 'clover leaf' shape exposing a 3-base "loop" (the anticodon loop) that base-pairs with the complementary base sequence in mRNA. The 'adaptor' molecule base-pairs with the mRNA, thus placing a specific aminoacid in position for addition to the peptide chain (protein) being synthesised on the ribosome. There are a variety of different tRNAs, each with a different aminoacid to one end, and a specific 3-base anticodon loop. Each 3 bases in the mRNA can therefore be translated into an aminoacid.

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Ribosomes

These are large, multiprotein particles. • They bring together the mRNA, C-terminal end of peptide, and the tRNA molecules. • They catalyse the addition of aminoacids from base-paired tRNA to C-terminal end of peptide chain. • They move along mRNA 5' to 3'. Proteins are synthesised on ribosomes where the RNA sequence is 'read' or translated into protein sequence.

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The entire mRNA molecule does not encode for the aminoacid sequence, so there must be a signal for where protein translation should start and stop. Start codon ATG = Methionine. Translation starts at the first ATG in the sequence. This is recognised by a tRNA which is linked to the Met aminoacid. Each subsequent group of 3 bases is read by a tRNA and an aminoacid is added to the polypeptide chain. Contrast this to the promotor in transcription. Stop codon There are 3 stop codons (TGA, TAA, TAG). Translation stops at the first 'in-frame' (i.e. proper, in-line group of 3) stop codon. This is not recognised by a tRNA but by a release factor which releases the peptide chain from the ribosome.

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General Pathology
Cellular responses to stress and noxious stimuli
The normal cell is confined to a fairly narrow range of function and structure, set by a number of factors, including: • its genetic programs of metabolism, differentiation and specialisation • constraints of neighboring cells • the availability of metabolic substrates. Stimuli may provoke the cell to change its responses, but still maintain a relatively normal level of homeostasis. More severe physiologic stresses, however, and some pathologic stimuli, may bring about a number of physiologic and morphologic cellular adaptations, which entail now but altered steady states. These states preserve the viability of the cell and modulate its function as it responds to such stimuli. Hyperplasia is one adaptive response; it entails an increase in the number of cells. Contrastingly, hypertrophy represents an increase in the size of individual cells. Atrophy is the opposite, it is an adaptive response in which there is a decrease in the size and function of cells. The following table outlines some cellular adaptations to injury and some causes. Cellular responses to injury
Nature and severity of injurious stimulus Altered physiological stimuli: • Increased demand, increased trophic stimulation (growth factors, hormones) • Decreased nutrients, decreased stimulation • Chronic irritation Reduced oxygen supply; chemical injury; microbial infection: • Acute and self-limited • Progressive and sever (including DNA damage) • Mild chronic injury Metabolic alterations, genetic or acquired Prolonged life spa with cumulative sublethal injury Cellular response Cellular adaptations: • Hyperplasia, hypertrophy • Atrophy • Metaplasia Cell injury: Acute reversible injury Irreversible injury  cell death o Necrosis o Apoptosis • Subcellular alterations in various organelles Intracellular accumulations; calcifications Cellular ageing • •

As detailed in the above table, some changes are more permanent than others. Cells have limits. If the limits of an adaptive response to a stimulus are exceeded, or if the cell is exposed to an injurious agent or stress, a sequence of events takes place that is loosely termed cell injury. Cell injury is reversible up to a certain point, but if the injurious stimulus is persistent, or is severe enough from the beginning, the cell reaches a point of no return and ultimately this ends in the death of the cell. Cells that are exposed to sublethal or chronic stimuli may not be damaged but may show a variety of subcellular alterations. Metabolic derangements in cells may be associated with intracellular accumulations of a number of substances, including proteins, lipids and carbohydrates. Calcium is often deposited at the site of cell death, resulting in pathologic calcification. Also, cell aging is also accompanied by characteristic morphologic and functional changes. The cellular response to an injury will always depend on the type, severity and duration of the injury, in addition to the type of cell (for example, neurons have different metabolic requirements than muscle cells). Some causative agents include: • trauma • thermal injury
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• • • • • • •

genetic abnormalities immunologic reactions poisons drugs infections organisms ionising radiation oxygen deprivation

There are a range of causative agents, but really only four actual cellular targets of injury: 1. External and internal cellular membranes 2. Aerobic respiration 3. Protein synthesis 4. Genetic apparatus

Common harmful cellular alterations
And as a consequence of the effect of the causative agents on the above four cellular targets of injury, some common harmful cellular adaptations can include: • decreased aerobic respiration • increased free radical species (oxygen-radical species) • increased intracellular calcium (too much will overactivate certain cellular pathways) • mitochondrial damage (mitochondria are very sensitive to cellular injury)

Decreased aerobic respiration
ATP is an essential energy source, derived from one of two pathways: 1. Oxidative phosphorylation in mitochondria (most important) 2. Glycolytic pathway (also known as anaerobic respiration) Ischemia (or lack of blood flow, and therefore oxygen and metabolic nutrients, to particular organs or cells), hypoxia, or toxic agents may cause impaired aerobic respiration. Cells with a higher ability to use the glycolytic pathway can withstand the causes of decreased aerobic respiration much easier (for example, brain cells versus muscle cells).

Damage from free radicals
Free radicals are reactive oxygen species that can often come other cells in the body themselves. Free radicals damage lipids, proteins and nucleic acids and are known as ‘highly reactice’ with those substances. Cells possess their own defence mechanisms against free radicals, using free radical scavenging pathways and involve the enzymes, 1. catalase
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2. superoxide dismutase 3. glutathionine peroxidase Again, there are cells that vary in their ability to protect themselves from the effects of free radicals.

Damage from increased intracellular calcium
Calcium is an important component of many enzymatic pathways (for example, muscular contraction). Increased intracellular calcium may result in cell injury. This can arise from: • release from internal sources (mitochondria, endoplasmic reticulum) • exogenous source through channels, membrane toxins

Mitochondrial damage
Mitochondria are typically a common direct or indirect target of cellular injury Damage of mitochondria leads to the formation of an abnormal channel – a Mitochondrial Permeability Transition Pore (MPTP). Damage of mitochondria can also lead to the release of cytochrome C, a major initiator of the apoptotic pathways.

Organelles involved in the cellular response to injury

Lysosomes
Primary lysosomes in normal digestion fuse with vesicles containing material for digestion, and become secondary lysosomes, or phagoysosomes. Exposure to harmful stimulus could lead to repeated lysosomal fusing and digestion and swelling of the cell. Heterophagy is when material is taken up from the external environment. Autophagy is where cellular elements are bound up in an autophagic vacuole which fused with a primary lysosome and are digested. Lysosomes may contain indigestible material (such as lipofuscin pigment granules).

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Increased volume of smooth endoplasmic reticulum
The cell may increase its volume of smooth endoplasmic reticulum in order to metabolise various harmful compounds.

Mitochondrial alterations
Mitochondria, in response a stress or injurious stimulus, may: • Swell • Increase in number (this makes sense because a cell under stress requires more energy) (associated with cell swelling) • Reduce in number (associated with cell shrinkage) • Become abnormal (e.g. megamitochondria in hepatocytes)

Cytoskeleton
The proteasome and heat schock proteins have a role in binding to and degrading proteins. Heat shock proteins are ‘chaperone proteins’ and have a normal role in protein ‘folding’. The ubiquitin-proteasome system is involved in degrading proteins.

Cellular adaptations – intracellular accumulations
Intracellular accumulations are localised within either the cytoplasm, vacuoles or in the nucleus. They mark reversible or degenerative changes. Fatty changes (steatosis) as an example: • alcohol abuse is the most common cause of fatty change in the liver (resulting in ‘fatty liver’) • it is the abnormal accumulation of triglycerides (e.g. heart and liver) • the triglycerides accumulate in vacuoles Pigments, lipofuscin as an example: • lipofuscin is a complex of lipid and protein components • common in cells of the heart, liver and brain • associated with call ageing and atrophy

Tissue changes
Physiological adaptation is a process involving normal signalling mechanisms. Pathological adaptation, however, is a response to various forms of injury. ATROPHY = decrease in cell size HYPERTROPHY = increase in cell size HYPERPLASIA = increase in cell number METAPLASIA = change of cell type (e.g. from a squamous epithelial cell to a cuboidal epithelial cell)

Atrophy
This involves the loss of cell ‘substance’ leading to cell shrinkage. A cell that has undergone atrophy may have decreased function, but it is not dead.
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The causes of atrophy are: 1. decreased workload 2. loss of innervation 3. loss of endocrine situation 4. ageing Atrophy is a cell survival strategy as a response to diminished use, supply or signalling. It is a reduction in structural components of the cell, achieved through decreased synthesis and increased degradation.

Hypertrophy
Hypertrophy is the increase of the size of cells, and results in the increase in the size of an organ. Structurally, it is the opposite of atrophy. Hypertrophy can be physiological (for example, the cells of the uterus during pregnancy undergo hypertrophy), or it can be pathological (for example, the cells of the myocardium after hypertension). It results from an increased functional demand, or from extracellular signalling.

Hyperplasia
Hyperplasia is the increase in number of cells in a tissue or organ. Hyperplasia is often linked with hypertrophy. A hyperplastic response is usually controlled, but may be pre-cancerous.

Metaplasia
Metaplasia is the process whereby an adult cell type is replaced by another adult cell type. The change of cell type is intrinsically designed to resist stress. A key example is the effect of smoking on ciliated columnar cells of the upper respiratory tract; they become stratified squamous epithelial cells and have decreased mucous secretion. There is the suspicion that metaplasia and neoplasia may be linked.

Apoptosis and Necrosis
Apoptosis and necrosis are two forms of the death of a cell, and they have many differences but one key difference in function: apoptosis is a programmed, intentional cell death; necrosis is a cell death as a result of pathologic injury.

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The following table is an essential, basic summary of the key difference between the two processes.
Feature Cell size Nucleus Plasma membrane Cellular contents Adjacent inflammation Physiologic or pathologic role Features of Necrosis and Apoptosis Necrosis Enlarged (swelling) Pyknosis  karyorrhexis  karyolysis Disrupted Enzymatic digestion; may leak out of cell Frequent Always pathologic (culmination of irreversible cell injury) Apoptosis Reduced (shrinkage) Fragmentation into nucleosome size fragments Intact, but with altered structure (orientation of lipids) Intact; may be released in apoptotic bodies No Often physiologic (as a means of eliminating unwanted cells); may be pathologic after some forms of cell injury, particularly DNA damage

Apoptosis
Apoptosis is a pathway of cell death regulated by and induced by intracellular programming, in which, essentially, cells that intend to die activate enzymes that degrade the cell’s own DNA and the proteins inside its nucleus and cytoplasm. The cell’s own membrane remains intact, but changes in such a way that the apoptotic cell becomes a good target for the body’s phagocytes. The dead cell is rapidly cleared away before its contents leaks, so that the (cytokine-activated) inflammatory process does not begin.

Causes of apoptosis – physiologic conditions
Death by apoptosis is a normal even in the body that serves to eliminate cells that are no longer needed (like during development) and to maintain steady (not overwhelming) populations of the various types of cells in tissues. Apoptosis is important in the following normal physiologic conditions (with some examples): The programmed destruction of cells during embryogenesis • includes implantation, organogenesis, developmental involution and metamorphosis Hormone-dependent involution in the adult • such as endometrial cell breakdown during the menstrual cycle • ovarian follicular atresia in menopausee • regression of the lactating breast after weaning • prostatic atrophy after castration Cell deletion in proliferating cell populations • intestinal crypt epithelia are constantly sloughed After an acute inflammatory or immune response • after certain cells have served their useful purpose, they undergo apoptosis because they are deprived of necessary survival signals, such as growth factors • examples are neutrophils in an acute inflammatory response, or lymphocytes at the end of an immune response Elimination of potentially harmful self-reactive lymphocytes • either before or after they have completed their maturation Cell death induced by cytotoxic T cells
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a defence mechanism against viruses and tumours that serves to eliminate virus infected and neoplastic cells

Causes of apoptosis – pathologic conditions
Death by apoptosis is also responsible for loss of cells in a variety of pathologic states. Cell death produced by a variety of injurious stimuli • for example, radiation and cytotoxic anticancer drugs damage DNA, and if repair mechanisms cannot cope with the injury, the cell kills itself by apoptosis Cell injury in certain viral diseases • for example, viral hepatitis, in which loss of infected cells is largely because of apoptotic cell death Pathologic atrophy in parenchymal organs after duct atrophy • this occurs in the pancreas, parotid gland, and kidney Cell death in tumours • this occurs most frequently during regression but also in actively-growing tumours Also, even in situations where the cell death is mainly necrotic, the pathway of apoptosis can be a contributor.

Morphology of apoptosis
The following morphological features (seen mainly, or at least better, under an EM) characterise cells that are undergoing apoptosis. 1. Cell shrinkage • cell is smaller in size • cytoplasm is dense, • organelles, although relatively normal, are more tightly packed 2. Chromatin condensation • the most characteristic feature of apoptosis • the chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes • the nucleus itself may break up, producing two or more fragments 3. Formation of cytoplasmic blebs and apoptotic bodies • the apoptotic cell first shows extensive surface blebbing, then undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly-packed organelles, with or without nuclear fragments 4. Phagocytosis of apoptotic cells or cell bodies, usually by macrophages • the apoptotic bodies are rapidly degraded by lysosomes in the macrophages • adjacent healthy cells migrate or proliferate to replace the space occupied by the now deleted apoptotic cell

Mechanism of apoptosis
Apoptosis as a mechanism can be divided into four linked steps.
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1. Induction/Signalling phase a. Intrinsic b. Extrinsic (both discussed shortly) 2. Effector phase a. Cell becomes committed to die b. Activation of cell executioner pathway c. Point of no return d. May be linked to mitochondrial permeability and release of factors 3. Degradation phase a. Main set of enzymes involved in apoptosis – caspases b. Caspases are a family of aspartate-specific cysteinyl proteases c. Some caspases have initiator role (caspases 2, 8, 9 and 10) d. Other caspases have an effector or ‘downstream’ role (casoases 3, 6 and 7) 4. Phagocytic phase a. Cell fragments produced by apoptosis are recognised by macrophages and other phagocytic cells b. Expression of specific signals on the membrane may facilitate phagocytic recognition Initiation of apoptosis occurs principally by signals from two distinct but converging pathways: the extrinsic (or receptor-initiated) pathway, and the intrinsic (or mitochondrial) pathway. Both pathways converge to activate caspases.

The Extrinsic (Death-Receptor Initiated) Pathway This pathway is initiated by the engagement of cell surface death receptors on a variety of cells. The reception of certain ligands activates an enzyme that triggers a cascade of caspase activation by cleaving and thereby activating other pro-caspases, and the active enzymes mediate the execution phase of apoptosis. The Intrinsic (Mitochondrial) Pathway This pathway is the result of increased mitochondrial permeability and release of pro-apoptotic molecules into the cytoplasm, without a role for death receptors. Key principle – increased mitochondrial permeability  release of pro-apoptotic molecules that are normally sequestered within the mitochondria. Mitochondrial changes are critical to apoptosis for both pathways. Changes in mitochondrial membrane permeability result in the formation of a Mitochondrial Permeability Transition Pore Complex (MPTPC), also known as a mitochondrial megachannel. This results in:
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• releasing of cytochrome C and apoptosis initiating factor (AIF) • formation of apoptosome and activation of caspases Causes of the specific mitochondrial permeability include free radical species, high intracellular calcium and the aforementioned extracellular death signals. Additionally, the lecture notes stipulate two additional pathways leading to apoptosis: 1. Cell membrane damage 2. Irreparable DNA damage DNA Damage Pathway Damage to DNA results in the mobilisation of cellular pathways leading to repair. But, if repair fails, there is an increase in pro-apoptotic factors and the cell kills itself.

Necrosis
Necrosis refers to a spectrum of morphologic changes that follow cell death in living tissue, largely resulting from the progressive degredative action of enzymes on the lethally injured cell. That is, necrosis is not actually the death of the cell, it is the degradation that follows it. Necrotic cells are unable to maintain membrane integrity and their contents often leak out, which may elicit inflammation in the surrounding tissue. The enzymes that denature the cell can be derived either from the lysosomes of the cell themselves, in which the process is called autolysis, or from the lysosomes of immigrant leukocytes, during inflammatory reactions. Because of this, these processes require hours to develop, and so there would be no detectable changes in cells if, for example, a myocardial infarct caused sudden death (we would have to look for other clues, like the occlusion of an artery). Usually, necrosis is detectable at its earliest at 4 to 12 hours after the necrotic activity.

Morphology of necrosis
General changes: • Necrotic cells show increased eosinophilia, due in part to the loss of the dark-staining RNA in the cytoplasm, and due in part to the increased binding of eosin to the denatured proteins inside the cytoplasm • Usually, the cells have a more glassy, homogenous appearance, mainly as a result of the loss of glycogen particles • Calcification of the dead cells may occur • Under EM, necrotic cells are characterised by overt discontinuities in plamsa an organelle membranes, marked dilation of mitochondria with the appearance of large amorphous densities, intracytoplasmic myelin figures, amorphous osmiophilic debris, and aggregates of fluffy material probably representing denatured protein Nuclear changes: • These changes appear in the form of one of three patterns, all due to the non-specific (general) breakdown of DNA. The following may occur: • Pyknosis – nuclear shrinkage and increased basophilia, where the DNA apparently condenses into a solid, shrunken basophilic mass. • Karyolysis – the basophilia of chromatin may fade • Karyorrhexis – where the pyknotic or partially pyknotic nucleus undergoes fragmentation • With the passage of time (a day or two), the nucleus in the necrotic cell totally disappears Tissue changes: • Coagulative necrosis
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o This is characterised by when denaturation is the primary pattern o It implies preservation of the basic outline of the coagulated cell for a span of at least some days o Cellular detail and tissue architecture relatively preserved o Firm, pale, ‘cooked’ appearance o Characteristic of hypoxic cells in all tissues except the brain Liquefactive necrosis o A characteristic of focal bacterial (or occasionally fungal) infection because microbes stimulate the accumulation of inflammatory cells o Hypoxic death of cells in the CNS result in liquefactive necrosis, for reasons too complicated to go into now o Whatever the pathogen, liquefaction completely digests the dead cells o The end result is transformation of the tissue into a liquid, viscous mass o If the process is initiated by acute inflammation, the material is frequently yellow and creamy because of the presence of white cells and is termed pus Gangrenous necrosis o Combination of the features of ischemic coagulative necrosis plus a liquefactive component o Usually applied to a limb that has lost its blood supply and has undergone coagulative necrosis, and a bacterial infection has been superimposed Caseous necrosis o A distinctive form of coagulative necrosis o The term caseous is derived from the cheesy, white gross appearance of the necrotic area o On microscopic examination, the necrotic focus appears as amorphous granular debris, seemingly composed of fragmented, coagulated cells and amorphous granular debris enclosed within a distinctive inflammatory border known as a granulomatous reaction o Unlike coagulative necrosis, the tissue architecture is completely obliterated Fatty necrosis o Does not denote a specific pattern of necrosis, but rather is a descriptive term describing areas of fat destruction, typically occurring as a result of the release of activated pancreatic lipases into the substance of the pancreas and the peritoneal cavity o Occurs during acute pancreatitis, where lipids from cells are broken down and combine with calcium to produce grossly visible chalky white areas, called fat saponification o Histologically, the necrosis takes the form of shadowy outlines of necrotic fat cells with basophilic calcium deposits, surrounded by an inflammatory reaction Haemorrhagic necrosis o Features dead tissue suffused with extravasated red cells o Common when cell death is due to blockage of venous drainage, resulting in a massive congestion of blood and poor perfusion

Ischemic necrosis of the myocardium:

Left: coagulative necrosis; right: liquefactive necrosis
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Left: Tuberculous lung featuring caseous necrosis; right: foci of fat necrosis with saponification in the mesentery

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Inflammation
Inflammation is a complex reaction to injurious agents such as microbes and damaged, usually necrotic, cells that consists of vascular responses, migration and activation of leukocytes, and system reactions. The reaction of blood vessels is a unique feature of inflammation that leads to the accumulation of fluid and leukocytes in extravascular tissue. The process of inflammation is closely intertwines with the process of tissue repair, and inflammation is fundamentally a protective response, consisting of four main components: 1. a vascular reaction (dilatation of blood vessels – endothelial leakiness 2. movement of protein-rich exudate 3. recruitment of cells (e.g. leukocytes) 4. proliferative activity (tissue regeneration, granulation and healing) There are two types of inflammation: 1. Acute inflammation: the initial, transient response 2. Chronic inflammation: the subsequent, prolonged response Before describing in detail the processes of inflammation, we should make some definitions. EXUDATION = the escape of fluid, proteins and blood cells from the vascular system into the interstitial tissue or body cavities. EXUDATE = an inflammatory extravascular fluid that has a high protein concentration, cellular debris, and a specific gravity above 1.020. It may also contain: • immunoglobulins (e.g. to target microorganisms for neutrophil phagocytosis) • coagulation factors and precursor protein (fibrinogen leads to the formation of filamentous fibrin threads); helps immobilise microorganisms and assist the migration of neutrophils TRANSUDATE = a fluid with a low protein content (in contrast with an exudate). EDEMA = denotes an excess of fluid in the interstitial or serous cavities. The fluid can be exudate or transudate. PUS = a purulent exudate, is an inflammatory exudate rich in leukocytes (mostly neutrophils), the debris of dead cells and in many cases, microbes.

Acute inflammation
This section aims to equip the student with knowledge of: • vascular and fluid changes in inflammation • the mobilisation and role of leukocytes • the forms of acute inflammation • the beneficial and harmful consequences of inflammation • the outcomes following acute inflammation

Stimuli for acute inflammation
Many stimuli can trigger an acute inflammatory process, including: • infections (bacterial, viral, parasitic) and microbial toxins • trauma (blunt or penetrating) • physical and chemical agents (thermal injury, e.g., burns or frostbite, irradiation, some environmental chemicals) • tissue necrosis (from any cause) • foreign bodies (splinters, dirt, sutures) • immune reactions (also called hypersensitivity reactions.
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Vascular changes during acute inflammation
Vascular changes are essential during inflammation because the two most important components of the inflammatory response, antibodies and leukocytes, are normally carried in the bloodstream and need to leave somehow. In normal situations, these components are sequestered inside the blood vessels and move in the direction of normal blood flow. In inflammation, blood vessels undergo the following changes that allow plasma proteins and circulating leukocytes out of the bloodstream and into the affected tissue. Changes in vascular flow and calibre: • Vasodilatation o This is one of the earliest manifestations of acute inflammation. o Vasodilatation first involves the arterioles, and the results in opening of new capillary beds in the area. o This causes increased blood flow – the cause of heat and redness (two of the cardinal signs of inflammation). o Vasodilatation is induced by the action of several mediators, notably histamine and nitric oxide, on vascular smooth muscle • Increased permeability of the microvasculature o Accompanied by the outpouring of protein-rich fluid into the extravascular tissues (addressed further below) • Concentration of red cells in small tissues o This is caused by the outpouring of exudate o It causes increased viscosity of blood (and a condition called stasis) • Leukocyte accumulation along vascular epithelium o Leukocytes (principally neutrophils) stick to the endothelium o Soon after, they migrate through the vascular wall into the interstitial tissue The increased vascular permeability (vascular leakage) can be further expanded upon. It is a hallmark of acute inflammation; it allows exudate and cells to migrate to the target area. The loss of protein from the plasma reduces the intravascular osmotic pressure, and increases the osmotic pressure of the interstitial fluid. Together with increased hydrostatic pressure owing to increased blood flow through the dilated vessels, this leads to a marked outflow of fluid and its accumulation in the interstitial fluid. This net increase of extravascular fluid results in oedema. Blood vessels become leaky for the following proposed reasons: 1. Formation of endothelial gaps in venules • the most common method • venules only • elicited by histamine, bradykinin, leukotrienes and other chemical mediators • the mediators tell the cell’s cytoskeletal proteins to contract, and the cell contracts, leaving holes in the blood vessel’s epithelium due to holes in the intercellular junctions 2. Direct endothelial injury, resulting in endothelial cell necrosis and detachment • because of the injury, the blood vessels do not contain the blood well and exudate leaks outward • this effect is usually encountered in necrotising injuries, and is due to direct damage to the endothelium by the injurious stimulus • in this method, all levels of the microcirculation are affected, including venules, capillaries, and arterioles 3. Leukocyte-mediated endothelial injury • leukocytes bind to the endothelium early in inflammation • as a result, they may become activated and release toxic oxygen species and proteolytic enzymes which cause endothelial injury or detachment
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4. Increased transcytosis • across the endothelial cytoplasm • vascular endothelial growth factor (VEGF) appears to increase this 5. Leakage from new blood vessels • when endothelial cells proliferate at the healing stages of inflammation, the vessels remain leaky • this contributes to the oedema that is characteristic of the early phases of healing that follow inflammation

Cellular changes during acute inflammation
The classic feature of acute inflammation is the extravascular accumulation of neutrophils. Neutrophils • Neutrophils are produced by the maturation of their precursor cells in bone marrow. • They are also known as polymorphonuclear leukocytes (because of their many-lobed nucleus). • They have a short lifespan once activated in tissues, and usually last about 1-2 days. • Their movement is guided by chemical signals (chemokines) • They contain granules rich in proteases • They can generate free radicals to kill phagocytosed bacteria, known as the respiratory burst • Circulating cytokines can stimulate neutrophil production and guide their recruitment to the site of injury; this is known as chemotaxis (think ‘chemical taxi’) There are four stages to the extravascular accumulation of neutrophils: 1. • • 2. • Margination increased viscosity and decreased flow movement of cells from axial to plasmatic (peripheral) zone of vessels Adhesion (also known as pavementing) Achieved by cell adhesion proteins – selectins and integrins, that create a ‘lock and key’ configuration between membrane call adhesion proteins on neutrophils and endothelial cell membranes 3. Aggregation • Adjacent neutrophils adhere to each other and undergo a shape change 4. Neutrophil invasion • Active amoeboid movement through endothelial walls

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Role of neutrophils in inflammation
Neutrophils perform the following functions when in the affected tissue: • Phagocytose microorganisms and dead cellular material • Kill microorganisms • Degradation and secretion of lysosomal enzymes and activation of the oxidative burst • Secretion of cytokines, which amplify and regulate inflammatory reactions • Opsonisation. This is the process of coating a particle, such as a microbe, to target it for phagocytosis. They are coated with opsins, which can be antibodies, complement proteins, and lectins. • After opsonisation, the leukocyte can engulf the microorganism, fuse the endosome with a lysosome, and the microbes are killed and degraded.

Chemical mediators of acute inflammation
A range of chemical mediators are involved in the process of acute inflammation, and here are some examples: Histamine • Stimulates potent vasodilatation • Promotes the movement of cell from the axial zone to the plasmatic zone of the blood vessel Serotonin • In inflammation, it acts as a vasoconstrictor Leukotrienes • Affect vascular permeability Cytokines (e.g. interleukins) • Have a variety of stimulatory and inhibitory roles Prostaglandins • Increase vascular permeability

Plasma factors involved in the inflammatory reaction
There are four plasma enzymatic cascade systems 1. Coagulation • Fibrinogen flows from the vasculature in the initial stages of inflammation
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• Convert soluble fibrinogen into fibrin • Fibrin is a significant component of exudate 2. Kinin system • Structurally-related polypeptides • Activation of the kinin system results in the release of the vasoactive bradykinin, which increases vascular permeability and causes contraction of smooth muscle, dilation of blood vessels, and pain when injected into the skin (effects similar to those of histamine) 3. Thrombolytic (fibrinolytic) system • Generates plasmin to degrade fibrin 4. Complement system • A large family of proteins that are synthesized in the liver, and circulate in the vascular system • Dead cells, bacterial endotoxins, antigen-antibody complexes, products of the kinin and fibrinolytic pathways can all activate complement • Example roles in inflammatory reaction a. C5a: chemotactant for neutrophils, increase vascular permeability, increase histamine release from mast cells b. C4b, 2a, 3b: opsonisation of bacteria (facilitates phagocytosis)

Alterations in the lymphatic system during inflammation
The lymphatic system accommodates to the inflammatory process through dilatation (which has a role in the drainage of oedema).

Role of mast cells during inflammation
Mast cells are stimulated by complement factors (which are stimulated by cell fragments, bacterial endotoxins, antigen-antibody complexes etc.) to secrete inflammatory mediators (histamine, leukotrienes, prostaglandins) that initiate the vascular response.

The five cardinal signs of inflammation
Redness – erythema – rubor • Vascular dilatation, initial increased blood flow Heat – hyperaemia – calor • In peripheral tissues • Systemic fever may contribute Swelling – oedema – tumor • Accumulation of exudate in extravascular tissue Pain – dolor • Pressure, related to swelling • Kinins can stimulate pain receptors • Affects hypothalamus and thermoregulation Additionally, systemic effects may include malaise and nausea.

Tissue repair, fibrosis and regeneration
The aim of this section is to: • Gain knowledge of the outcomes of the inflammatory response • Understand healing by organization and repair, including o Roles of different cell types
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o Staging of cellular changes o Mediators of successful and unsuccessful healing Knowledge of the cellular features of healing by primary or secondary intention

The following diagram is a general schematic representation of tissue response to healing:

Outcomes of acute inflammation
There are four possible outcomes to acute inflammation. 1. Resolution and regeneration • the term where the tissue or organ returns completely to normal • restoration of normal tissue occurs, as does the removal of the harmful agent/microorganism 2. Suppuration • The formation of pus • Pus may become bounded by a pyogenic membrane, involving vascular (capillaries) and cellular (neutrophils and fibroblasts) • Abscess 3. Healing by organization and repair • Replacement by granulation tissue • Vascular (capillaries) and cellular (fibroblasts and macrophages) • Fibrosis and scar formation 4. Progression to chronic inflammation

Resolution and regeneration
In the process, the exudate is removed, by a combination of: • liquefaction by neutrophil enzymes • re-absorption of fluid by lymphatics • phagocytosis of material by macrophages The damaged cells regenerate, and normal structure and function is returned.

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However, this process is not common. It is limited to certain cell types (lung, liver kidney, and not the heart and brain). It results in the restoration of normal tissue, and the removal of the harmful agent/microorganism. Vascular permeability returns to normal.

Healing by organization and repair
If the injurious stimulus that evoked the acute inflammation creates substantial damage to the tissue, or if the tissue is unable to regenerate, it goes through the process of organization and repair. This usually results in scar formation. Most tissues will do this rather than resolution and regeneration. 1. Initial stages • Angiogenesis: pre-existing capillaries in the undamaged areas bud into damaged tissue • Infiltration by macrophages, fibroblasts and myofibroblasts – very important! • Macrophages have a dual role at this stage: i. to phagocytose exudate and dead tissue, and ii. to produce angiogenic and fibrogenic factors 2. Formulation of granulation tissue (the redness in damaged tissue) • This is a popular exam question • Angiogenesis (neovascularisation) • Fragile complex of interconnecting capillaries, macrophages and support cells • As fibroblasts proliferate and form collagen, some capillaries are ‘pruned’ back • Fibroblasts become the main cell type • Persisting vessels acquire smooth muscle, forming arterioles and venules 3. Fibroblasts’ role • Fibroblasts align and deposit collagen in a uniform pattern (reticulin) • This provides maximum strength relative to the forces on the wound • Myofibroblasts contract, and thereby contract the wound’s edges to decrease exposure and to minimise the area of damaged tissue • This provides the formation of a collagenous scar • The granulation tissue is replaced by a fibrous scar = fibrous repair Once sufficient collagen is produced, fibroblasts assume a resting state as a fibrocyte Scarring signifies the imperfect restoration of structure, but it may allow restitution of function. Scarring is the deposition of collagen. It may be substantial and cause impairment (e.g. myocardial infarct). Persistent injury leads to tissue destruction and substantial scarring, for example, liver cirrhosis, chronic pancreatitis. Summary of organization and repair (organization of the exudate): 1. Local vessels are stimulated to form outgrowths (angiogenesis) 2. Local support cells divide to form fibroblasts and myofibroblasts (mitogenesis) 3. Fibroblasts and myofibroblasts migrate towards the area of tissue damage (chemotaxis and motility) 4. Secrete collagen (fibrogenesis) 5. Produce collagen-degrading enzymes (remodelling)

Healing of skin
Healing by primary or first intention This occurs in closely apposed surfaces with a narrow space, minimal dead tissue (for example, when a wound’s edges are held together by sutures). Healing by second intention
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This occurs in the healing of open wounds; it takes longer, and results in a larger scar. It is aided by wound contraction by myofibroblasts.

Skin sutures and wound strength 1 week – sutures removed – 10% tensile strength of normal skin 1 month – 50% strength 3 months – 80% strength

Factors impairing the ability of tissue to repair
• • • • • • • • Inadequate nutrition (protein, vitamin C and zinc are required) Ischemia Infection – promotes inflammatory reaction Foreign material (nidus for infection and inflammation) Steroids interfere with granulation tissue formation and has immunosuppressive effects which predispose tissue to infection Denervation Radiation exposure Diabetes (susceptibility to vascular disease and ischemia)

Factors that promote repair
• • The removal of dead tissue allows apposition of healthy tissues Antibiotic therapy

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Summary diagram

Chronic inflammation
Chronic inflammation is difficult to define, but essentially it is inflammation of prolonged duration (weeks or months) in which: • active inflammation, • tissue destruction, and
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• attempts at repair are all occurring simultaneously. Although it may follow acute inflammation, it frequently begins as an insidious (often) asymptomatic response. This type of chronic inflammation is the cause of tissue disease in some of the most common and disabling human diseases such as rheumatoid arthritis, atherosclerosis, tuberculosis, and chronic lung diseases.

Chronic inflammation arises in the following circumstances:
• Persistent infections o by certain microorganisms and certain viruses, fungi, and parasites o these organisms have low toxicity and evoke an immune reaction called delayed type hypersensitivity o the inflammatory response sometimes take a specific pattern called a granulomatous reaction Prolonged exposure to potentially toxic agents, either exogenous or endogenous o an example of a toxic exogenous agent is silica, a nondegradable inanimate materia that can result in the inflammatory lung disease silicosis o endogenous toxic plasma lipid components cause, in part, atherosclerosis Autoimmunity o sometimes, under certain conditions, immune reactions occur against the individual’s own tissue, leading to autoimmune diseases o the autoantigens evoke a self-perpetuating immune reaction that results in chronic tissue damage and inflammation

Morphologic features of chronic inflammation
While acute inflammation is manifested by vascular changes, edema, and mainly neutrophilic infiltration, chronic inflammation is contrastingly characterised by: 1. Infiltration with mononuclear cells that include macrophages, lymphocytes and plasma cells 2. Tissue destruction induced by the persistent offending agent or by the inflammatory cells 3. Healing by connective tissue replacement of damaged tissue

Mononuclear (macrophage) cell infiltration
The macrophage is the dominant cellular player in chronic inflammation. They originate as monocytes in the blood (whose half life is about 1 day, as opposed to when they become macrophages, where their life in tissues is several months or years). Monocytes begin to emigrate into extravascular tissue quite early in acute inflammation, and within 48 hours they may constitute the predominant cell type. Extravasation of the monocytes is governed by the same factors that are involved in neutrophil emigration, that is, adhesion molecules and chemical mediators with chemotactic and activating properties. Macrophages may be activated by cytokines secreted by sensitised T lymphocytes or natural killer cells, bacterial endotoxins and other chemical mediators. Activation results in increased cell size, increased levels of lysosomal enzymes, more active metabolism, and greater ability to phagocytose and kill ingested microbes. Activated macrophages secrete a wide variety of biologically active products that, if left unchecked, result in the tissue injury and fibrosis characteristic of chronic inflammation. In short-lived inflammation, if the irritant is eliminated, the macrophages eventually disappear. In chronic inflammation, macrophage accumulation continues, and is mediated by different mechanisms:
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1. Recruitment of monocytes from the circulation with the expression of chemotactic factors (like the attraction of neutrophils in acute inflammation) 2. Local proliferation of macrophages after their immigration from the bloodstream 3. Immobilisation of macrophages within the sit of inflammation. Certain cytokines and oxidised lipids can cause such immobilisation The products of activated macrophages serve to eliminate injurious agents such as microbes and to initiate the process of repair, and are responsible for much of the tissue injury in chronic inflammation. These products can: • be toxic to microbes and host cells • be toxic to extracellular matrix • be chemotactic to other cell types • cause fibroblast proliferation, collagen deposition and angiogenesis. Therefore, tissue destruction is one of the hallmarks of chronic inflammation. Other factors, in addition to the products of macrophages themselves, may contribute to tissue damage in chronic inflammation. • Necrotic tissue can perpetuate the inflammatory cascade (through the activation of kinin, coagulation, complement and fibrinolytic systems) • The release of mediators from leukocytes responding to the necrotic tissue • Liberation of substances like uric acid from dying cells • In cellular immune reactions, T lymphocytes may directly kill cells • Thus, ongoing tissue destruction can activate the inflammatory cascade by diverse mechanisms, so that features of both acute and chronic inflammation may coexist in certain circumstances.

Other cells involved in chronic inflammation

Lymphocytes
Lymphocytes are mobilised in both antibody-mediated and cell-mediated immune reactions and even in nonimmune inflammation. Lymphocytes and macrophages communicate bidirectionally. Macrophages present antigens to T cells and produce factors that activate them. T cells produce cytokines, and one of them is a major activator of macrophages. Plasma cells develop from activated B lymphocytes and produce antibodies directed against either the persistent antigen in the inflammatory site or against altered tissue components. This can look like secondary lymphoid tissue in severe chronic inflammation.

Eosinophils
These cells are particularly abundant in immune reactions against parasitic infections. Recruitment of eosinophils is similar to recruitment of neutrophils. They contain granules of major basic protein that is toxic to parasites but may also cause lysis of epithelial cells.
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Mast cells
Mast cells are widely distributed in connective tissue and participate in both acute and chronic inflammatory reactions. They usually secrete histamine when activated, which helps the bogy during anaphylactic reactions to foods, insect venom or drugs, but can backfire (with catastrophic results). When properly regulated, the response can be beneficial to the host. In chronic inflammatory reactions, they may produce cytokines that contribute to fibrosis.

Granulomatous inflammation
Granulomatous inflammation is a distinctive pattern of chronic inflammatory reaction characterised by focal accumulations of activated macrophages, which often develop an epithelioid appearance, also known as, epithelioid histiocytes. It is encountered in a limited number of immunologically mediated, infectious and some non-infectious conditions. Tuberculosis is the key demonstrative example of granulomatous diseases, but others include sarcoidosis, leprosy and syphilis. Histological recognition of the granulomatous pattern in a biopsy is essential because it significantly narrows the scope of the diagnosis.

Granuloma
GRANULOMA = a focus of chronic inflammation consisting of a microscopic aggregation of macrophages that are transformed into epithelium-like cells surrounded by a collar of mononuclear leukocytes, predominantly lymphocytes and occasionally plasma cells. In usual staining, the epithelioid histiocytes have a pale pink granular cytoplasm with indistinct cell boundaries, often appearing to blend or merge into one another. They can be distinguished from lymphocytes because their nuclei is less dense, is oval or elongate, and may show folding of the nuclear membrane. Older granulomas develop an enclosing rim of fibroblasts and connective tissue.

Cells unique to granulomas
Frequently, the epithelioid histiocytes fuse to form giant cells in the periphery or sometimes in the centre of granulomas. These giant cells contain a large mass of cytoplasm, containing 20 or more small nuclei arranged: 1. peripherally, making it a Langerhans-type giant cell, or 2. haphazardly, making it a foreign body-type giant cell No functional difference is known between the two, but you should remember the difference anyway. They are distinguishable from tumour giant cells because the tumour giant cells have abnormal nuclei, whereas the granulomatous giant cells have normal nuclei. The lecture notes adds a third type, touton giant cells, that have a central ring of nuclei, and usually exist at sites of adipose tissue degeneration.

Two types of granulomas
Granulomas (not the giant cells, but the whole granuloma) come in two types: Foreign-body granulomas are incited by relative inert foreign bodies, like sutures. They form when the foreign body is large enough to make phagocytosis by a single macrophage impossible, and do not incite any
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specific inflammatory or immune response. Epithelioid histiocytes and giant cells surround and encompass the foreign body Immune granulomas are caused by insoluble particles, typically microbes, that are capable of inducing a cell-mediated immune response. The prototype example is the bacillus of tuberculosis, which is classically categorised by the presence of central caseous necrosis (which is rare in other granulomatous diseases).

The picture above is a slide typical of tuberculosis. Note the area of central caseous necrosis, the giant cells (Langerhans type), the epithelioid histiocytes, and the many lymphocytes.

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Neoplasia
General facts about neoplasia
Neoplasia literally means new growth and is synonymous with the term tumour. Cancer is the common term for all malignant tumours. A neoplasm is an abnormal mass of tissue: 1. the growth of which exceeds normal tissue 2. that is uncoordinated compared with normal tissue 3. that persists after the cessation of the original stimulus. The persistence of tumourous growth after the cessation of stimulus is due to the hereditable genetic factors that are passed down to the affected cell progeny. These genetic changes allow excessive, unregulated growth/proliferation that becomes autonomous. (Although tumours generally remain reliant on the host for nutrition and blood supply. The entire population of a tumour arises from a single cell that has incurred genetic change; hence tumours are said to be clonal. All tumours, benign and malignant, have 1. Parenchyma (proliferative neoplastic cells), and 2. Stroma (connective tissue and blood vessels) We have recognised cross-talk between stromal and parenchymal cells which directly influences the growth and morphology of the tumour. Eg.  stromal support scant?  neoplasm is soft and fleshy  parenchymal cells can stimulate the formation of abundant collagenous stroma (known as desmoplasia).

Nomenclature and general principles
Benign tumours of mesenchymal (non-epithelial) origin:
 Designated by adding the suffix ‘oma’ to the name of the cell of origin.  E.g. fibroblast tumour = fibroma  cartilaginous tumour = chondroma  bone tumour = osteoma

Benign tumours of epithelial origin:
ADENOMA = benign epithelial neoplasm that  forms glandular patters, or  that results from glandular tissue but does not necessarily form patterns that resemble glandular tissue PAPILLOMA = benign neoplasms on epithelial surfaces producing microscopically or macroscopically visible finger-like or warty projections CYSTADENOMA = benign epithelial neoplasms that form large, cystic masses POLYP = a benign neoplasm that produces a macroscopic projection above a mucosal surface
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Malignant tumours of mesenchymal (non-epithelial) origin:
The nomenclature for malignant tumours follows the same schema as benign tumours, with the addition of “sarc”. SARCOMA = malignant tumour arising in mesenchymal tissue. (‘sar’ = fleshy; because they have little connective tissue stroma and so appear and palpate fleshy). Examples – fibrosarcoma, liposarcoma, leiomyosarcoma (smooth muscle cancer).

Malignant tumours of epithelial origin:
CARCINOMA = malignant neoplasms of epithelial origin (derived from any of the three germ layers ADENOCARCINOMA = a carcinoma with a glandular growth patter (microscopically) SQUAMOUS CELL CARCINOMA = a carcinoma producing recognisable squamous cells arising from any epithelial tissue in the body Normally we would also specify (where possible) the organ of the carcinoma’s origin. For example.  renal cell adenicarcinoma  bronchogenic squamous cell carcinoma

Difference between carcinomas and sarcomas:
Epithelial malignancies Common occurrence Mainly uses lymphatic route of metastasis In-situ phase exists Usually found in patients > 50 Non-epithelial malignancies Rare occurrence Mainly uses the hematogenic route of metastasis In-situ phase does not exist Usually found in patients < 50

Other types of tumours:
Most of the time, both benign and malignant tumours show cells with similar levels of differentiation; but sometimes, divergent differentiation of a single line of parenchymal cells into a different type of tissue creates mixed tumours. MIXED TUMOURS = tumours from a single germ layer that differentiate into different types of tissue For example, mixed tumour of salivary gland origin, which can show epithelioid cells scattered within cells that resemble bone or cartilage. Most neoplasms are made up of cells representative of a single germ layer, even mixed tumours. Teratomas are different. TERATOMA = malignant tumour made up of a variety of parenchymal cell types representative of more than one germ layer, usually all three.  With teratomas, because they arise from totipotent cells (either in the gonads or in stem cell nests throughout the body) which can differentiate along various germ lines, they often grow many identifiable tissues such as skin, muscle, gut epithelium or teeth and hair; indeed, any tissue of the body. HAMARTOMA = a benign tumour made up of cells lacking autonomy, in which the elements are fully differentiated and are normally found in the organ of origin, but in which the constituent parts are
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abnormally organised. Examples include, haemangioma, lymphangioma, rhabdomyoma of the heart, adenomas of the liver, pigmented naevi. HETEROTOPIA (also CHORISTOMA) = the presence of normal cells or tissues located in abnormal places. Examples include the residue of pancreatic tissue found in the wall of the stomach or small intestine, endometriosis.

Exceptions to the nomenclature rules of neoplasia
Melanoma; seminoma; hepatoma; blastoma; eponymous tumours; APUDomas. (While their names suggest they are benign, they are all malignant).

Tumour morphology, behaviour and growth
General features
Benign tumours: 1. Behavior  slow growth  no metastases  non-invasive 2. Histological features  well-differentiated  no pleomorphism/polymorphism  few mitosis, those that exist are normal 3. Macroscopic appearance  well demarcated from surrounding tissue  capsule Malignant tumours: 1. Behaviour  rapid growth  invasive and destructive  metastases 2. Histological features  variable differentiation  pleomorphism and polymorphism  hyperchromatism  mitoses  grade: low, intermediate, high 3. Macroscopic appearance  sometimes a pseudocapsule  stage classification: TNM (tumour, nodes, metastases)

Tumour history
The natural history of most tumours can be tracked under the following phases: 1. Malignant change in the target cell (transformation)
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2. Growth of the transformed cells 3. Local invasion 4. Distant metastasis We often separate benign and malignant neoplasms based on these phases, and usually, we can do so on the basis of morphology, with considerable confidence; some neoplasms, however categorisation.

Differentiation and anaplasia
Differentiation refers to the extent to which neoplastic cells resemble comparable normal cells. ANAPLASIA = a complete lack of differentiation (i.e., the cells/tissue resemble no mature cells/tissue). Also evident in anaplastic tissue is a lack of cells’ orientation to one another and to their axial framework and blood vessels. Well-differentiated tumours are composed of cells resembling the normal mature cells of the tissue of origin of the neoplasm. Poorly-differentiated or undifferentiated tumours have primitive-appearing, unspecialised cells. Benign tumours are well-differentiated in general, and examining the individual cells of a benign tumour for evidence of neoplasia (before you know it is a tumour) may be difficult because they so closely resemble the normal mature cells of the tissue. So, you need to look at the big picture. Malignant tumours range from well-differentiated to undifferentiated. That is, malignant tumours do not have to be poorly differentiated, but often are. Remember, if a neoplasm is completely undifferentiated then it is anaplastic, and by definition, malignant. Anaplasia is marked by the following morphological changes: 1. PLEOMORPHISM/POLYMORPHISM  variation in the size and shape of both the cells and their nuclei  cells may be very large or very small in comparison to their neighbours 2.      ABNORMAL NUCLEAR MORPHOLOGY nuclei contains extra DNA and are extra dark-staining (together – hyperchromatic) nuclei are disproportionately large nucleus-to-cytoplasm volume ratio up to 1:1, rather than the normal 1:4 or 1:6 chromatin often clumped and situated around the plasma membrane often many nuclei

3. MITOSES  anaplastic tumours usually possess large amounts of mitotic processes  however, the presence of mitoses do not necessarily indicate that a tumour is malignant or that a tissue is neoplastic, because many normal tissues exhibit high levels of mitotic activity (like bone marrow or tissues expressing non-neoplastic hyperplasia.  so, the most important morphological feature of a malignant neoplasia is mitotic figures that are: • atypical/bizarre in morphology • tripolar or multipolar spindles 4. LOSS OF POLARITY  the orientation of anaplastic cells usually indicates they have lost normal polarity or it has been markedly disturbed  this can arise in sheets or large masses of tumour cells 5. OTHER CHANGES
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 

Tumour giant cells: often possessing a single huge polymorphic nucleus, others having many hyperchromatic nuclei. (Don’t confuse these with inflammation-related macrophages whose nuclei will be small and round and appear normal) Sometimes blood supply is scant in tumours, and central areas may show signs of ischemic necrosis

There is a wide spectrum of levels of differentiation in tumours ranging from undifferentiated to fully differentiated, and the space between the two poles is termed moderately well-differentiated.

Dysplasia
Also important are a couple more terms in relation to tumour morphology. DYSPLASIA = a loss in the uniformity of the individual cells as well as a loss of their architectural orientation, with the following features:  encountered principally in epithelium  pleomorphism/polymorphism  hyperchromatic, abnormally large nuclei  normal, but increased mitotic activity (that may be in abnormal locations in epithelium)  usually considered a preinvasive state Distinguishing dysplasia from anaplasia is through knowing that dysplastic tissues still somewhat resemble their mature tissue of origin. Think of the basal cell carcinoma, which is malignant but not anaplastic; it is dysplastic because the cells still resemble the basal cells of the stratum basale of the epidermis. Dysplasia displays the morphologic features of malignancy at a stage when invasion of surrounding tissue is not evident. When referring to epithelial tissue, it implies a reversible histologic deviation from normal, with deranged differentiation but controlled proliferation. A premalignant legion. CARCINOMA IN SITU = when dysplastic changed are marked and involve the entire thickness of the epithelium, but where the lesion remains confined to the normal tissue. The carcinoma-in-situ is considered a preinvasive neoplasm; when tumour cells move beyond the confines of the basement membrane, the tumour is said to be invasive. Dysplasia often predates the appearance of cancer. Clinically, benign neoplasms and well-differentiated carcinomas of endocrine glands frequently elaborate hormones of their origin, and can therefore be picked up in testing. If this occurs, we know the tumour is not anaplastic but more likely dysplastic.

Some other definitions relating to morphology
METAPLASIA = complete change in differentiation from one fully differentiated tissue form into another fully differentiated tissue form. This transformation is not neoplastic. HETEROPLASIA = the differentiation of a part of an organ/tissue in a way that is foreign to that part of the body. The anomalous differentiation takes place at the stem cell stage. It occurs without stimulus. HYPERTROPHY = the increased bulk due to increase in cell size, where cell numbers remain constant. HYPERPLASIA = the increased bulk due to an increase in cell number; an increase in cell size may also be an accompanying feature.
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Surface occurrence morphology:  Sessile  Papillary  Polypoid  Fungating  Ulcerated  Annular SCIRRHOUS = refers to hard tumours. MEDULLARY = refers to softer lesions.

Premalignant stages
Some tissues show dysplastic (abnormal tissue development) changes for a long period and then chance to carcinoma-in-situ, before becoming invasive cancers (e.g. uterine cervix carcinoma-in-situ). This stage is more easily recognised in epithelial tissues rather than non-epithelial tissues.

Lesions that risk malignancy
Long standing inflammatory lesions, metaplasias, or benign neoplasias may become malignant, for example:  adenomas in colon  ulcerative colitis  atypical ductal hyperplasia  chronic skin ulcers/burns  metaplasia in oesophagus

Neoplastic angiogenesis
Angiogenesis is the process in which tumours stimulate the growth of host blood vessels. Neoplastic angiogenesis is required for tumours beyond 1-2mm thickness in diameter, presumably because this is the maximum distance across which oxygen and nutrients can diffuse from blood vessels. Neovascularisation (new blood vessels) has a dual effect on the growth of the tumour: • perfusion supplies nutrients and oxygen to the neoplastic cells • new endothelial cells stimulate the growth of adjacent tumour cells by secreting polypeptide growth factors such as insulin-like growth factors and PDGF Tumour-associated angiogenic factors, produced by tumour cells (or may be derived from inflammatory cells that infiltrate tumours) promote • vascular endothelial growth factor (VEGF) o induced by hypoxia, cytokines, GFs • basic fibroblast growth factor (bFGF) o promote fibroblast migration and proliferation Evidence exists to suggest that early in the growth of a tumour, its cells do not express angiogenic factors but remain in-situ. When some cells within the tumour undergo a small tumour change to angiogenic phenotype, known as the angiogenic switch.

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Anti-angiogenesis factors (inhibitors) are also produced by tumour cells, and therefore the production of new tumour blood vessels rests on the balance between angiogenic factors and anti-angiogenic factors. Antiangiogenic factors include: • thrombospondin-1 • plaminogen  angiostatin • collagens  endostatin, tumstatin (used experimentally to stop tumour angiogenesis in mice) A structural comparison between normal blood vessel networks and tumour blood vessel networks: Normal: • Stable • Structure and function of wall and network appropriate to location • Clear distinction between arterioles, capillaries and venules Tumour: • Unstable, evolving network • Abnormal structure • Abnormal function • Inappropriate to location • Arterioles, capillaries and venules not apparent

Local Invasion

Local invasion – benign tumours
Nearly all benign tumours grow as cohesive, expansile masses of cells that remain localised to their site of origin and do not have the capacity to:  infiltrate  invade  metastasise Because their growth and expansion is slow, they usually develop a thin rim of connective tissue, sometimes called a fibrous capsule, which separates them from the host tissue. This capsule arises primarily from two things: 1. the stroma of the host tissue 2. the parenchymal cells of the host tissue, atrophied under the pressure of the expanding tissue

Local invasion – malignant tumours
The growth of (malignant) cancers is accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Usually, a well-defined cleavage plane between malignant tumours and normal tissue is lacking. However, a slowly-expanding cancer may develop a capsule. Important: next to the development of metastases, invasiveness is the most reliable feature that differentiates malignant from benign tumours. As a side note, carcinomas-in-situ display the cytological features and signs of malignancy without invading the basement membrane. (Given time, however, most will invade, if left untreated). Processes of invasion:
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1. Invasion of extracellular matrix a. detachment of tumour cells from each other (loss of cadherins) b. attachment to components of the extracellular matrix c. degradation of the extracellular matrix d. migration of tumour cells (now it becomes metastasis) e.

Metastasis
Metastasis continues the process of invasion, at step two: 2. Vascular dissemination and homing of malignant neoplastic cells a. platelet-tumour aggregates b. adhesion to endothelium c. crossing through basement membrane and extravasation d. metastatic deposit, angiogenesis, and growth

Metastasis unequivocally marks a tumour as malignant because benign neoplasms do not metastasise. With few exceptions, all cancers can spread. The notable exceptions are the gliomas (in the CNS) and the squamous cell carcinomas of the skin. Both these cancers are highly locally invasive, but rarely metastasise. In general, the more aggressive, rapidly growing, and larger the primary neoplasm, the more likely it is to metastasise. But, there are plenty of exceptions. This is a general principle. The following diagrams represent the process of invasion, left, and metastasis via the hematogenic pathway (discussed later), right.

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Possible methods of metastasis transport include:  lymphatics spread  hematogenous spread  seeding of body cavities These mechanisms provide pathways to other tissues. Seeding of body cavities and surfaces  can occur anywhere there exists a natural ‘open field’ and the cancer has invaded in a way that exposes itself to the field  most often involved the peritoneal cavity, but many others also
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Lymphatic spread  most common pathway for the initial dissemination of carcinomas  sarcomas may also use this route, but it is less common  important: the pattern of lymph node involvement follows the natural route of lymphatic drainage  sometimes performing a full lymph node dissection close to the presence of a tumour can be beneficial to prevent its spread, but this surgery can present significant morbidity, so more commonly, the sentinel nodes will be biopsied.  A sentinel node is the first node in a regional lymphatic basin that receives lymph flow from the primary tumour  Englargement of lymph nodes under the system presence of cancer can be due to one of the following:  the spread of, and growth of, cancer cells, or  hyperplasia of the immune tissue, reactive to the cancer. Hematogenous spread  the most common pathway for the dissemination of sarcomas  veins are more often penetrated than arteries (because they generally have thinner walls)  with venous invasion, the blood-borne cancer cells follow the path of venous flow  hence, the liver and lungs are the most commonly involved organs in secondary dissemination, because  all portal area draining flows to the liver, and  all caval blood flows to the lungs

Aetiology
Environmental carcinogenic/tumourgenic factors:  chemical agents  radiation  viruses  bacteria  fungi  parasites  acquired pre-neoplastic disorders:  cirrhosis  pernicious anaemia (associated with vitamin B12 deficiency)  ulcerative colitis (ulceration of the colon and rectum, cause unknown)  leukoplakia (white patch of oral mucous membrane associated with pipe smoking)  altered immune states Hereditary neoplastic syndromes:  familial adenomatous polyposis (polyposis of the colon that usually begins in childhood, and increases with age)  familial retinoblastoma  cancers without recognisable genes or transmission pattern  autosomal recessive syndromes  increased susceptibility to environmental carcinogens

Epidemiology of neoplasms
More common in adults:  colorectal carcinoma  bronchial carcinoma
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 mammary carcinoma  prostatic carcinoma More common in children:  leukaemia/lymphoma  brain tumours  neuroblastoma  nephroblastoma

Host defence against malignancy
Tumour antigens:  tumour-associated antigens  tumour-specific shared antigens  viral antigens  proteins encoded by viruses  over-expressed antigens Immune surveillance  AIDS  immunosuppression  x-linked immunodeficiency  overgrowth of antigen-negative cells  loss of histocompatibility antigens  tumour-induced immunosuppression  tumour cells do not express necessary cofactors  apoptosis of cytotoxic t-cells

Factors affecting prognosis
General:  patient’s age  overall fitness  other medical conditions  location Tumour:  histological type  histological grade  stage  response to therapies

The Molecular basis of Cancer
Five fundamental principles underlie the molecular basis of cancer
1. Nonlethal genetic damage lies at the heart of carcinogenesis 2. A tumour is formed by the clonal expansion of a single precursor cell that has incurred the genetic damage (i.e. cancers are monoclonal) 3. Four particular classes of genes are the principle targets of genetic damage that causes cancer
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4. These genes affect cell proliferation or survival indirectly 5. Carcinogenesis is a multistep process at both the phenotypic and the genetic level 1. Nonlethal genetic damage lies at the heart of carcinogenesis  such genetic damage may be acquired by the action of environmental agents such as chemicals  some may be inherited by the germ line  not all damage has exogenous causes, some causes are spontaneous and stochastic 2. Clonal expansion  monoclonal cancers  certain molecular markers exist that indicate that cancers have such clonality 3. Four classes of regulatory genes are the principle targets of genetic damage 1. the growth-promoting oncogenes 2. the growth-suppressing tumour-suppressor genes 3. genes that regulate apoptosis 4. genes involved in DNA repair 4. DNA repair genes affect cell proliferation or survival indirectly  they do so indirectly by influencing the ability of the organism to repair nonlethal damage in other genes, including protooncogenes, tumour suppressor genes, and genes that regulate apoptosis 5. Carcinogenesis is a multistep process at both the phenotypic and geneotypic levels  a malignant neoplasm has several phenotypic attributes discussed earlier, like excessive growth, local invasiveness and the ability to form distant metastases  these characteristics are stepwise and are called, together, tumour progression  at the molecular level, progression results from the accumulation of genetic lesions that in some instances are favoured by defects in DNA repair

The essential molecular alterations for malignant transformation
1. Self-sufficiency in growth signals  tumour cells have the ability to proliferate without external stimuli, usually as a consequence of oncogene activation 2. Insensitivity to growth-inhibitory signals  tumour cells may not respond to molecules that are inhibitory to the proliferation of normal cells 3. Evasion of apoptosis  tumour cells may be resistant to programmed cell death, as a result of the inactivation of p53 or other changes 4. Defects in DNA repair  tumour cells may fail to repair their own DNA’s damage caused by carcinogens or unregulated cellular proliferation 5. Limitless replicative potential  tumour cells have unrestricted proliferative capacity, associated with altered telomere length and function 6. Sustained angiogenesis  tumours require a blood supply, induced molecularly by various factors, most commonly vascular endothelial growth factor (VEGF)

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It is widely believed that the occurrence of mutations in cancer-causing genes is conditioned by the robustness of the DNA repair machinery of the cell. When genes that normally sense and repair genetic damage are impaired or lost, the resultant genomic instability favours mutations in genes that regulate the other acquired capabilities of cancer cells.

Skin tumours
Objectives for this section: 1. understand the cellular changes that characterise benign melanocytic neoplasms (naevi) 2. understand the cellular features of a malignant melanoma and know how these are used diagnostically 3. know the cellular changes underlying common non-melanoma malignant skin tumours (squamous and basal cell carcinomas) There are three major malignant skin tumours: 1. Squamous cell carcinoma 2. Basal cell carcinoma 3. Melanoma

Naevi
NAEVI = congenital lesion of the skin A naevi is a congenital or acquired neoplasm of melanocytes, with the following characteristics: • uniform pigmentation • light to dark brown in colour • features elevation on skin (papules) • has well defined borders • is derived from melanocytes in the basal layer • is made up of oval cells that grow in aggregates (‘nests’) Junctional naevi • early form of the melanocytic naevi • grows along dermal-epidermal junction, giving pigmentation to the skin • flat, uniformly pigmented • usually develop in childhood/early adolescence • features rounded nests of melanocytes • shown below

Compound naevi • has a raised or dome-shaped appearance
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• • •

symmetrical and uniformly pigmented histologically, features nests and cords of melanocytes extending into the dermis shown below

Intradermal naevi • raised and usually smoothly domed • typically has a uniform flesh colouration • rarely occurs in children and adolescents • confined entirely to the dermis • may decrease in size with further age • shown below

The vast majority of naevi are benign, and have well-differentiated individual cells. The melanocytes are organised in nests or in a linear (lentiginous) patters. But a junctional or compound naevus is at risk of developing into a dysplastic naevus.

Dysplastic naevus
Dysplastic naevus are characterised by the following features: • relatively large in shape • irregular edge, irregular surface, and irregular pigmentation • often looks similar to a compound naevus, but the distinction is that it often has an asymmetric ‘shoulder’ • melanocytes show cellular atypia and increased mitoses • the dermis shows linear or lamellar fibrosis • there exists the possibility of an early malignant change • these lesions should be excised

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In the right picure, you can see the presence of cytologic atypia (the irregularly shaped, dark-staining nuclei – hyperchromatism). You can also see linear or lamellar fibroses in the dermis underneath the epidermis. Evidence exists to suggest that dysplastic naevi are the precursors of malignant melanomas.

Lentigo maligna
These lesions are common on the face of the elderly. They feature flat shape, with a variegated, irregular border. They have a brown or black colouration. Histologically, basal cells are replaced by a continuous line of atypical melanocytes.

Malignant melanoma
• • • • • This is the most dangerous malignant skin cancer. It is a malignant melanocytic neoplasm that is more common in adults than in children. It may develop from previous lesions (from the dysplastic naevi or the lentigo maligna) or it may develop spontaneously They are more common in fair-skinned individuals UV exposure is the most significant predisposing factor

Morphological and histological features: • Relatively large in size, and usually is presented accompanied by a history of a recent increase in size • Irregular border and striking variations in colour (variable pigmentation) • Some appear flat, some appear nodular • Has the potential for ulceration of the lesion Very important concepts to remember about melanomas: 1. This is the skin cancer most likely to spread to other organs by metastasis
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2. Vertical and radial pattern of growth is vitally important There are three main subtypes of malignant melanomas.

Lentigo maligna melanoma
• • nodular lesion emerging in pre-existing facial lentigo maligna. nodular growth heralds vertical growth

Superficial spreading malignant melanoma
This melanoma usually evolved from a precursor lesion, the dysplastic naevus. • is about 75% of all melanomas • flat • variable colour • irregular edges It has two subtypes: In-situ superficial spreading malignant melanoma • features atypical melanocytes confined to the epidermis Invasive superficial spreading malignant melanoma • features clusters of malignant melanocytes invading the dermis

Nodular malignant melanoma
• • • • is about 5% of melanomas features a raised brown/black nodule in the center spreading pigmentation is the most aggressive form of melanoma

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Growth characteristics of melanomas
Melanomas grow in radial and vertical patterns. The vertical penetration patterns are linked with nodule formation and the capacity for metastasis via lymphatics and the bloodstream. EXCISIONAL BIOPSY = allows for the measurement of the depth of growth (Breslow thickness). A better prognosis exists when the depth of growth is less than 1.5mm. Secondary metastases of melanoma is common at the liver, lungs, bones and brain. If secondary metastases of melanomas occurs, the five-year survival rate is less than 10% and death usually occurs within 6-12 months.

Squamous cell carcinoma
The SCC is the second most common tumour of the skin, and is more common in men as it is in women. It is a malignant tumour. SCCs are rarely metastatic, and if they metastasise, it is mainly to lymph nodes. They are the most locally-invasive skin tumour, but can be cured if completely excised. However, they may recur following excision. There exists a range of predisposing factors, including UV exposure, ionising radiation and industrial carcinogens. There are two forms: in-situ and invasive.

SCC in-situ
This is also known as Bowen’s disease. These lesions: • feature red, sharply defined, scaling plaques • do not penetrate epidermal-dermal junction

Invasive SCC
This tumour features:
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• • •

highly atypical cells in all layers of the dermis neoplastic cells penetrated through the basement membrane into the dermis cells which demonstrate variable levels of anaplasia and variable morphology, including: o polygonal squamous cells organised in lobules with keratinisation o rounded, highly anaplastic cells with limited keratin formation

Approximately 5% of these tumours metastasise to regional lymph nodes. Strong link to UV damage to DNA and/or suppression of repair mechanisms. Involves impairment of local immune cells, causing reduced antigen presentation and decreased immunosurveillance.

Basal cell carcinoma
• • • • • • • The most common malignant skin tumour Slow growing Rarely metastatic Features a pearly or waxy elevated (papular) structure Has dilated subepidermal blood vessels (telgangiectasia) that are often visible May be pigmented, resembling naevi May become ulcerative (“rodent ulcers”)

Histologically: • The neoplastic cells resemble basal cells • Mostly nodular lesions (some also cystic) o grows deeply into dermis o features multiple islands of basaloid cells o palisaded cells at periphery of islands o dark-stained nuclei, scant cytoplasm • Scant ulcers are o unusually aggressive o may invade sinuses or bone

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Superficial BCC
• • • • • A less common variation of the BCC Affects elderly, predom trunk Flat in structure Red, scaly, patchy appearance may have raised ‘pearly’ papules, resembling nodular form

Benign skin neoplasms
Some benign skin neoplasms have the potential to resemble malignant forms, and some “benign” structures may lead to malignant forms in the future.

Seborrheic keratoses
These are common in middle to older aged people. They are typically localised to the trunk, but also can occur on limbs, head and neck. It is common to find inflammation associated with a seborrheic keratoses.
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They appear as round, flat, coinlike, wazy plaques that vary in diameter from millimetres to several centimetres. They can have a melanoma-like appearance, but the thing that distinguishes them from melanomas is the presence of prominent keratin-filled ‘horns’.

Keratoacanthoma
Acanthosis refers to the thickening of the stratum spinosum. These are rapidly growing neoplasms that may mimic well-differentiated squamous cell carcinomas both histologically and clinically. They often heal spontaneously, without treatment. They usually appear on areas exposed to the sun, and can grow to 1cm in diameter or more. They have a central, keratin-filled crater surrounded by proliferating epithelial cells that extend upwards in a liplike fashion over the sides of the crater and downward into the dermis as irregular tongues.

Solar or actinic keratosis
Benign/premalignant skin neoplasm. This tumour is related to chronic UV exposure. It is common exposed surfaces (back of hand, scalp, face, arms). They have a rough, sandpaper-like, scaly appearance. These lesions feature substantial cellular dysplasia, and therefore are categorised as being in a malignant state. They may or may not transform into malignant tumours (usually SCC).

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The stratum corneum in an actinic keratosis is thickened, and features nuclei cells, unlike normal skin. This is a pattern termed parakeratosis. Some of these lesions produce so much keratin that a cutaneous horn develops, as seen in the picture.

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Homeostasis
Body fluid compartments
Learning outcomes: • Describe the major fluid compartments in the body • Explain how water moves between fluid compartments and how water may enter and leave the body • Discuss the pressures involved in the movement of fluid between capillaries and interstitial spaces. • State the roles of the major solutes in each fluid compartment • Link the above to fluid shifts that occur after burns Fluid compartments are separated by selectively permeable membranes and comprise of: Total body water volume - 40 litres - 60% body weight, comprised of • Intracellular fluid volume - 25 litres - 40% body weight • Extrecellular fluid volume - 15 litres - 20% body weight, which is comprised of • Plasma volume - 3 litres - 20% of extracellular fluid volume • Interstitial fluid volume - 12 litres - 80% of extracellular fluid volume Clinically, plasma is the first access to body fluid and has significant clinical application (IV access, fluid replacement, etc.)

Characteristics of body fluids
Each compartment has a distinctive patters of electrolytes. Electrolytes are the most abundant solutes in body fluids. Proteins, phospholipids, cholesterol and neutral fats are much larger molecules and account for up to 90% of the mass of the dissolved solute.

Composition of body fluids

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Extracellular and intracellular differences
Extracellular: • Protein in plasma • Na+ is the main cation • Cl- is the main anion Intracellular: • K+ is the main cation • Phosphate is the main anion The difference here is primarily due to ATP-dependent sodium-potassium pumps.

Water intake and loss

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Fluid input comprises: 7. metabolic water 8. ingested foods and ingested liquids Fluid loss comprises: • losses from the gastrointestinal tract • lungs • skin (sweating etc.) • kidneys (excretion) Water is in a state of constant flux between fluid compartments

Water balance
Water balance is maintained by: 5. Regulating water input (the thirst mechanism) 6. Regulating water output (the renal system, antidiuretic hormone)

Thirst mechanism

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Regulation of water output

Regulation of body water
This mechanism is a classic example of homeostasis working in a negative feedback system. The intake of water is regulated by the thirst centre in the hypothalamus. Outflow of water is regulated by hormones affecting the kidneys. Balance is influenced by vomiting, diarrhoea, perspiration, burns, etc.

Starling's Law of the Capillaries

Capillary hydrostatic pressure filtration (24 litres per day) and osmotic pressure reabsoroption (20 litres per day) are in near equilibrium. The near equilibrium between filtration from capillaries to the interstitial space and reabsorption from the interstitial space into the capillaries is referred to as Starling's Law of the Capillaries. The net outward force results in a flow of fluid that contributes to a moist interstitial space, skin turgor and a positive interstitial fluid pressure. This net outflow of fluid (3-4 litres per day) is collected by lymphatic capillaries and returned to the cardiovascular system.
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Bulk flow vs diffusion
The most important mechanism for solute movement is diffusion. The balance of Starling Forces determines net fluid movement. Water movement can aid in the diffusion process by carrying with it entrained solute - this is termed "bulk flow".

Movement between interstitial and intracellular compartments

Plasma to interstitial (some causes and outcomes):  Oedema  Filtration from capillaries favoured or reabsorption impaired  Inflammation (increases permeability) altered filtration coefficient  Increased blood volume (renal failure, fluid overload and congestive heart failure) increased capillary hydrostatic pressure  Decreased serum albumin (malnutrition, burns and kidney disease) decreased colloid osmotic pressure  Pulmonary oedema (congestive heart failure, inflammation)  Pitting oedema of lower leg
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 Weeping oedema Interstitial to plasma: • Short term adaptive response to a sudden decrease in plasma volume (PV) or high interstitial fluid volume (IFV) • Haemorrhage • Later stages of burns • Stabilisation of hemodynamic variables in the case of hypovolemia • Burns (hypervolemia) Review of key points so far 6. Capillary network supplies almost all tissues in body with substrate necessary for homeostasis 7. The balance of Starling Forces determines net fluid movement across capillary endothelium 8. The process of diffusion is most important for solute exchange 9. Burns lead to an increase in capillary permeability, loss of plasma proteins and an imbalance in Staling Forcess that promotes the formation of oedema and a loss of plasma volume

Burns
• • • • •

Due to thermal or chemical injury to the skin Rule of nines Categorised according to depth and extent First, second and third degree burns vs partial and full thickness burns More than 20% third degree burns = significant risk of death

First degree: only epidermis involved painful and moist (think why) heals without scarring Second degree: total epidermis, some dermis generally painful and moist, often blisters Third degree complete epidermis and dermis painless, varied appearance problems - shock, electrolyte, imbalance, infection

Movement between compartments
Hydrostatic pressure is the force generated by the heart on the vessel (capillary) wall Osmotic pressure is the pressure caused by the presence of plasma proteins within the capillaries (note that there is virtually no protein in the interstitial space) Hydrostatic pressure forces fluid out of the capillaries into the interstitial space Osmotic pressure forces fluid into the capillaries from the interstitial space (fluid is drawn towards the plasma proteins) Fluid movement between the intracellular space and the interstitial space is largely by diffusion and osmosis.

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Be aware that intravascular fluid (aka plasma) and interstitial fluid is collectively termed extracellular fluid.

Chloride
Major extracellular anion Balances osmotic pressure Combines with H+ to form HCl in gastric acid

Calcium
Most abundant ion Important in bone growth and metabolism Participates in blood coagulation Essential for muscle tone

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Homeostatic function of the skin
Body heat
• •

• •

Metabolic process produce heat as a by product. The efficiency of most human metabolic reactions is about 50%. Mammals and birds are able to store a portion of this heat in their body and so maintain their body temperature at a level above that of the surrounding environment. This classifies them as endotherms (produced its own heat) . Mammals (humans included) and birds are also able to maintain their body temperature within a fairly narrow range. This classifies them as homoiotherms. The tissues of the body producing the majority of the body heat make up the body core. The layer of tissues outside of the core, forming a 'buffer zone' between the core and the environment, is termed the body shell. The skin forms the outermost layer of the body shell.

This table shows the skin temperatures of an adult male resting in a thermoneutral environment (~29C). (A thermoneutral environment is one where the termperature is such that we do not have to create or lose heat, i.e., we don't require cooling or thermogenesis). Region Head (highest) Trunk (highest) Upper arms Forearms Hands (lowest) Thighs Calves Feet (lowest) Area (m2) 0.20 0.70 0.10 0.08 0.07 0.33 0.20 0.12 Total = 1.80 m2 Percent of total body surface area 6% 36% 8% 6% 5% 19% 13% 7% Skin temperature (degrees C) 34.6 C 34.6 C 33.0 C 30.8 C 28.6 C 33.0 C 30.8 C 28.6 C Mean = 33.0 C

Effect of variation of ambient temperature on skin temperatures

Vertical: temperature of various body parts Horizontal: ambient temperature As seen on the graph, temperature does not vary linearly in various body parts with ambient temperature. With a rise in ambient temperature, the temperature of different body parts will change differently to others. For example, feet will jump quite a lot in temperature, while rectal temperature stays fairly constant.
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Homeostatic control system for body temperature

We can see from the above diagram that skin plays the role of both sensor and effector. A sensor role is provided by the presence of thermoreceptors in the skin that respond to local changes in temperature. An effector role is provided by blood vessels supplying the skin and by sweat glands located within the skin.

Skin thermoreceptors
We now know that all temperature sensors in the skin are in the form of free nerve endings without any specialised structure whatsoever. Cold sensors increase their firing rate as skin temperature falls, whereas warm sensors increase their firing rate as skin temperature rises. Over most of the skin surface, cold sensors considerably outnumber warm sensors. In a few areas, however (e.g. the scrotum) the situation is reversed and warm sensors predominate. There is quite a lot of overlap in the temperatures at which cold and warm sensors are active. Both are generally active around a mean skin temperature of ~33 C. This means that cold and warm receptors are active together a lot of the time.

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Shown above are the recordings from a cold sensor in response to a sudden lowering of the local skin temperature. Note that the initial change in temperature evokes the greatest response. This is termed the dynamic response. The steady-state response related to the fixed skin temperature. This is termed the static response. Note that this becomes 'bursting' in character at the lowest steady-state temperature shown.

Dynamic and static aspects of skin thermoreceptors

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This illustration further shows the concept of dynamic and static responses from skin thermoreceptors. You can see that the nerve fibres show a greater firing rate at the initial drop in temperature (dynamic effect) than with the sustained steady temperature (static effect). Note also that as the skin temperature is raised again, the firing rate falls to zero.

Operating ranges of cold and warm sensors

White dots: cold receptors Black dots: warm receptors The static or steady-state firing rate of each individual skin thermoreceptor tends to peak at a particular skin temperature. The diagram above shows a cold sensor that happens to peak at around 30 C, and a warm sensor that happens to peak at 39 C. Note how the cold receptor fires again at 45 C and peaks for a second time at 50 C. This is a pathological temperature and relates to actual 'thermal pain'. Another piece of evidince that allows us to differentiate skin thermoreceptors is that we, as humans can clearly recognise a difference between a feeling of cold or warmth on our skin. Distribution of skin thermoreceptors is highly individualistic.

Heat production in the body core

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There is a clear difference between heat production of the body at rest and during exercise. Also notice that fraction of body weight has little bearing on the production of body core heat and these variables are reliant on other metabolic factors. (There are a couple of tables here that I didn't include from the lecture slides. Look them up if you want, I don't think they're really necessary.)

Passive heat transfer from the body core
Note the word passive! When the temperature of the environment is lower than that of the body core (as is usually the case), heat flows passively down a gradient from the body core to the body shell. The rate of passive heat transfer depends upon th thermal conductivity of the tissues. • Fat has a low thermal conductivity so tends to act as an insulator. • Skin has a high conductivity so tends to act as a radiator.

Heat transfer by the circulatory system
This can be controlled.

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Heat passes most rapidly from the body core to the body shell by the controlled circulation of the blood. Heat conservation can be aided by shunting blood through deep-lying tissues where heat passing from the core via arteries may be transferred to deep veins and kept from escaping so rapidly. Make sure you are able to explain this because it will very likely be on the exam! On the other hand, heat loss is aided by allowing blood to pass through more superficial vessels closer to the skin surface. Note the location of the shunt in the next diagram:

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Functional relationships within the skin vasculature

Skin vasculature is composed of a network of large arterioles and venules in the deep dermis, which send branches to a superficial network of smaller arterioles and venules. Arterio-venous anastomoses allow direct flow from arterioles to venules and greatly increase blood flow when dilated. When closed, arterio-venous anastomoses re... CHECKING WITH PROF MASKREY ON THIS POINT. Capillary loops into the dermal papillae beneath the epidermis are supplied and drained by microvessels of the superficial dermal vasculature.

Effect of temperature on skin blood flow (Q sk )
Thermoregulation is a major determinant of skin blood flow - "Qsk". At rest in a thermoneutral environment the average Qsk = ~250mL per m squared of body surface area. This amounts to ~400mL per minute for an adult, or ~8% of cardiac output. In cold conditions, Qsk may fall to just 50mL per minute, which is just enough to meet nutritive requirements of the skin. In hot conditions, Qsk may rise to as much as 2800mL per minute. This obviously demands an increase in cardiac output. Exercising in the heat requires that there is a fine balance kept between Qsk and blood flow to working muscles.
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Heat loss mechanisms - radiation
When the temperature of the body surface is greater than that of the surroundings, heat is lost by radiation. The amount of radiative heat loss depends upon the skin temperature, th thermal gradient and the radiating area (think clothing) of the body surface. Radiation is also a mechanism for gaining heat. Heat can be gained by radiation when ambient temperature exceeds the temperature at the body surface.

Heat loss mechanisms - Conduction and convection

Body heat is lost to the air and to other molecules in contact with the skin. Conduction of heat in water is ~20 times that in air of the same temperature. Heat so lost can then be removed from the vacinity of the skin surface by convection. Free convection occurs when air heats and becomes less dense, whereas forced convection occurs where wind or a similar force disrupts the boundary layer of air next to the skin. Free convection is passive, it relies on the surrounding air warming up, becoming less dense and more buoyant and moving away.

Heat loss mechanisms - evaporation

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Passive, uncontrolled evaporative heat loss occurs through insensible perspiration. 14. This is the diffusion of small amounts of water from the skin and upper respiratory tract 15. 600-900 grams of water are lost daily 16. Half from the skin, half from the upper respiratory tract Sweating, on the other hand, is highly regulated, active evaporative heat loss mechanism. • It relies on the presence of eccrine sweat glands located within the dermis of the skin. • When ambient temperature exceeds core temperature, this is the only means of losing heat for the body.

Characteristics of the sweating response
Human adults possess 2-5 million eccrine sweat glands capable, between them, of producing up to 2L of sweat per hour. Sweat rate is regulated through changes in temperature within the body core. This is very important. Efferent control is by way of sympathetic nerves releasing Acetylcholine as their transmitter. Effectiveness of sweating is reduced where humidity of the surrounding air nears saturation. Continual exposure to high ambient temperature, or regularly increased body core temperature, leads to acclimation of the sweating response, wherein sweating occurs more rapidly, more 'dormant' sweat glands are recruited and salt loss through sweat is limited.

Control of sweating rate

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The figure above demonstrates that it is indeed body core temperatures, not skin temperature, that determines sweat rate. This is appropriate as the role of sweat evaporation is to cool the skin. A cool skin would would inhibit sweating if this were controlled by skin thermoreceptor input. So to make it clear, nerve endings that exist in the skin that are responsible for cold and warm sensations are for sensory awareness and integration at the hypothalamus. The thermoreceptors that are responsible for the sweat response exist inside the body core, not in the skin.

Relative contributions from heat loss mechanisms in a resting subject

Note that the relative contribution from evaporation increases as ambient temperature rises towards that of the body core. (It goes from 0.13 to 0.27 to 1.0 of total heat loss mechanisms). Do you REALLY understand the difference between radiation and convection? If you don't, read up on it.

Relative contributions from heat loss mechanisms in an exercising subject

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Note that in exercise, as opposed to the situation while at rest, heat loss via convection and conduction always exceed that due to radiation. This is because the action of the body, particularly the limbs, moving through the air substantially increases the amount of heat loss through forced convection. As with the resting subject, the proportion of heat lost through evaporation increases as the body core temperature increases.

Introduction to Pharmacology
Pharmacological Principles
Aims of this section: • To define a drug • To define the terms pharmacodynamics and pharmacokinetics • To provide some basic pharmacology terminology • To understand how drug side effects occur • To understand what drug interactions are and why they can be dangerous What is so different about the elderly? Pharmacology in the elderly is unique, because old age is associated with chronic diseases and disabilities, which in turn require multiple medications.

What is a drug?
Is insulin a drug? Yes and no. Yes, when it has been administered into the body. No, when it is occurring naturally in the body (where it is simply termed a hormone). Pharmacodynamics: What the drug does to the body: binds to a receptor, causes a cellular change and then a systemic response. Specific to the drug, or class of drugs. Pharmacokinetics: What the body does to the drug; drug absorption, transport, metabolism, and elimination. Not specific to drug or class, describes a general process.

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Some basic pharmacological terminology
INDICATION = an illness or disorder for which the drug has been documented as useful. For example, bronchodilators are indicated in asthma. CONTRAINDICATION = a health condition in which a particular drug may not be administered. For example, pregnancy. CELLULAR EFFECTS = describes what the drug does to a cell (second messenger cascade, or intracellular signalling) • Drug binds to receptor (e.g. on the cell surface), activated another protein inside the cell, which activates several more proteins and/or enzymes to cause the cell to do something, or stop it from doing something • For example, if the cell is a muscle cell it may be caused to contract or relax SYSTEMIC EFFECTS = the resultant effect of a drug on the body. • For example, if each cardiac muscle fibre contracts more forcefully and faster in response to receptor activation, the systemic effect is increased heart rate and force of contraction resulting in an increase in cardiac output

Potential drug targets: membrane bound enzymes
Enzymes catalyse reactions at the inner or outer surface of the membrane. Dugs can modify the activity of these enzymes. • For example: the gas nitric oxide (NO) causes an enzyme in the cell, guanylate cyclase, to be activated, which then sets off a second-messenger cascade. In smooth muscle, this causes muscle relaxation. Drugs that work via this pathway include GTN and Viagra.

Potential drug targets: G-protein coupled receptors
This is the largest receptor family and hence, one of the most common drug targets. • For example: the asthma drug Ventolin causes bronchodilation via a G-protein-coupled receptor known as the beta-2 receptor.

Potential drug targets: ion channel receptor proteins
This class of drugs includes many drugs that target ion channels, like many of the ones that modulate heart action. (Other receptor types not shown here include the steroid receptors and receptor-tyrosine kinases).

Potential drug targets: transport proteins
Drugs may hijack protein mediated transport to reach their site of action.

More terminology
AGONIST = will stimulate the receptor into action. ANTAGONIST = will block the receptor, usually without causing any cellular effect. PARTIAL AGONIST/ANTAGONIST = only partially blocks or stimulates the receptor. REVERSIBLE = after drug dissociation, the receptor is still functioning.
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IRREVERSIBLE = the drug does not dissociate. The bound drug and receptor are usually internalised into the cell and broke down. The receptor function will not be restored until a new receptor protein is made and replaced into the membrane (e.g. aspirin). POTENCY = the amount of chemical required to produce an effect. The affinity or strength of binding will determine the concentration required to produce a response. Or, the better the drug binds to the receptor, the greater the effect, usually means less drug is required to be administered. i.e. drug potency is how well a drug works! SELECCTIVITY/SPECIFICITY = the narrowness of a drug’s range of actions, or how well the drug ‘recognises’ its intended receptor. SIDE EFFECT/ADVERSE DRUG REACTION = a drug effect that was not the primary purpose for administering the drug. Side effects may be beneficial or harmful, and may differ between patients. That is, not all patients will always get all known side effects. This may be due to the patient’s health, other medications, or other factors. • Can be due to pharmacodynamics (e.g. low drug specificity) • Can be due to pharmacokinetics (e.g. active drug metabolites) DRUG-DRUG INTERACTIONS = a health outcome caused by the interaction of two or more drugs in the system. May be due to pharmacodynamics or pharmacokinetics or both. • The effect of one or both drugs can be either ‘potentiated’ (enhanced) or inhibited • Pharmacodynamics example: two drugs that operate at the same receptor, or set off the same cellular signalling pathway (e.g., GTN and Viagra) • Pharmacokinetics example: two drugs that are metabolised by the same enzymes in the liver. One drug may inhibit or potentiate the enzyme (e.g., the broad spectrum antibiotic clarithromycin potentiates the anti-coagulant, warfarin

Polypharmacy
Polypharmacy is a huge problem in the aged. It is the administration of two or more drugs at the same time. The key example is the ‘triple whammy’: aspirin, diuretic and ACEI (anti-platelet and anti-hypertensives) – one of the most common causes of hospital admission for medication-induced (iatrogenic) renal failure, and often fatal.

Drug delivery
Aims of this section: • To gain knowledge of the different types of drug delivery o to understand why there are different oral formations o to understand the meaning of parenteral administration • To understand that different types of drug delivery are based on pharmacokinetics and pharmacodynamics • To gain a basic understanding of the factors that influence drug absorption • To understand the factors that need to be considered in drug prescription

Why are there different routes of drug delivery?
The optimal pharmacological treatment is one that gives the greatest therapeutical effect with minimal side effects. This is aided by targeting drug delivery to the site of action (e.g. inhalation, eye drops, ear drops, injections directly into the heart). This is not always possible, so most drugs are administered orally, which are then absorbed into the bloodstream.
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The route of drug delivery can be influenced by both pharmacodynamics and pharmacokinetics. • Pharmacodynamics: o what the drug does to the body o binds to a receptor, causes a cellular and then systemic response • Pharmacokinetics: o what the body does to the drug o drug absorption, transport, metabolism and elimination (e.g. the liver inactivates many drugs, so they cannot be administered orally)

Different routes of drug administration
Oral Intrathecal Intravenous Intraarterial Intramuscular Subcutaneous Nasogastric Inhalation PO IT (into the spinal canal) IV IA IM SC NG

Drug delivery devices include inhalers, needles, and tablets and capsules.

Different forms of medication
• • • • • • • • • Pills Liquids taken orally Liquids for injection Aerosols Powders Patches Suppositories Creams Ear or eye drops

That is, drugs may be liquids, solids or gases. Pharmaceutics is the science of formulating drugs into different types of preparations.

How drugs get to their site of action
Drugs must cross certain membranes to get to their required site of action, and this means the drug has to be absorbed. How easily a drug is absorbed depends on: • drug structure (e.g. water or lipid soluble? proteins versus steroids?) • drug size (e.g. lithium has an atomic weight of ~7, compared to large protein drugs such as insulin or the influenza vaccine) o most drugs have a relative molecular mass between 100 and 1000  caffeine mw = 194  testosterone mw = 288  penicillin mw =373  insulin mw = 5700

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Oral administration
Used for about 75% of all drugs. Can be solid or liquid.

Solids (tablets)
• • • • About 60% of all oral drugs Must disintegrate before dissolving Once dissolved, the drug is available for absorption The more rapid the dissolution, the more readily available it is for absorption

Tablets are compressed mixture of the active ingredient, plus excepients. Excepients are inert chemicals, that include: • diluents • lubricants • absorbents • binders • adhesives • disintegrants • flavours • colours • sweeteners all covered in a film to mask the taste. Also used is sublingual (dissolution) administration, and effervescent tablets that dissolve in water. The rates of release of oral tablets can be controlled by their formation: • sustainable release or controlled release preparations o the active ingredient is mixed with a resin or compound which slows release • enteric coatings o tablet is coated with a substance that protects it from the digestive action of the stomach o this prevents decomposition of drugs sensitive to gastric acid (e.g. penicillin and erythromycin are unstable in an acidic pH) o prevents dilution of the drug before it reaches the small intestine o prevents nausea and vomiting induced by some drugs that effect the stomach o delays the action of the drug

Parenteral administration
These are where drugs are administered by injection. This is the most rapid form of systemic therapy, and it avoids the delay in drug absorption. Drug formation for parenteral administration must be: • sterile • particle free o (injections of solids can cause granulomas, inschemia or phlebitis) • isotonic with body solutions (e.g. diluted in normal saline) • buffered to body pH

Drug prescription quantities
Things to assess when deciding what quantity of a drug should be prescribed: What is the problem? • are there alternate therapies such as lifestyle modifications that may help? • is there are drug-based therapy?
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o o o o o o

what does the drug do and how does it act? how long will the patient need to be treated for? how will you monitor therapy? how much drug should be given? what is special about this patient? what other medications are they taking?

Some common abbreviations and symbols used in pharmacology
Abbreviation AC PC bd, bid prn ad lib IU Unabbreviated form Ante cibum Post cibum bis die, bis in die pro re nata ad libitum International unit Meaning Before meals After meals Twice daily As needed Freely Unit of pharmacological activity

Basic principles of receptors, ligands, and second messenger systems
Pharmacodynamics is what the drugs do to the body and how they do it. It involves the interaction of drug molecules with • target receptors or cells • second messengers o biochemical and physiological effects o adverse effects  cellular effects (receptors, cell behaviour)  systemic effects (what happens to the body after the cellular effects

Drug action interaction

at

the

receptor

can

vary

depending

on

the

drug-target

• • •

Agonist (stimulating) or antagonists (blocking) o Both are either competitive (reversible) or irreversible, where the drug binds, but does not let go, so the receptor needs to be replaced by the cell. Aspirin is an example of this. Partial agonists Allosteric modulators can modulate a protein (change how it acts) Enzyme inhibitors/activators o Enzymatic inhibitor – normal reaction inhibited o Fals substrate – abnormal metabolite o Pro-drug – active drug produced Non-specific

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Use-dependent drugs • Drugs that only exert their therapeutic effect at receptors that are or have been activated. Examples: o Beta-blockers, commonly used to treat hypertension and angina  Block the action of adrenaline and noradrenaline at beta-receptors  They have no antagonistic function in a person at rest because there is no adrenaline or noradrenaline present o Sodium channel blockers, commonly used to treat dysrhythmias in the heart  Block the sodium channels when they are in their open or refractory (unable to be activated) state, not when they are at rest

The four receptor superfamilies
There are four superfamilies of receptors: 1. Ligand gated ion channels (IONOTROPIC RECEPTORS) 2. G-protein-coupled receptors (METATROPIC) (~80% of drugs) 3. Kinase-linked receptors a. membrane-bound b. dimerisation
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4. Nuclear-linked receptors Each of the four receptor superfamilies have defined modes of signal transduction.

Ligand-gated ion channels (nicotinic and Ach)
These channels have an alpha, beta, delta, a second alpha and a gamma subunit, that all join together to create a pore in the centre. When acetylcholine binds to the receptor, the receptor opens and lets sodium in.

Ligand gates is the same as chemical-gated. Chemically-gated, voltage-gated and leakage ion channels govern changes in membrane potential. Chemically-gated (ligand-gated), voltage-gated and leakage ion channels govern changes in membrane potentials.

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Ion channel opening and closing mechanisms • Receptors may be activated by binding at specific sites: o for example, calcium channel blockers o important for hypertension, angina and some heart arrythmias o three classes, each act at a separate binding site on the same subunit

G-protein-coupled receptors

G-proteins have seven proteins wound through the plasma membrane • C-terminus – (tail) inside the cell (the –COOH end) • N-terminus – (head) outside the cell, ligand binds to this (the –NH2 end) Very important, likely to be on the exam: 1. GDP is exchanged for GTP and the alpha and beta-gamma subunits split, activating 1 or 2 targets 2. GTP hydrolysis deactivates the g-protein subunits 3. the GTPs can either activate targets 1 and 2 or inhibit them 4. Target 1 and 2 can be: a. ion channels b. guanylate cyclase c. adenylate cyclase, or d. phospholipase C

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The above diagrams will most likely be on the exam.

Kinase-linked receptors
Dimerisation and autophosphorylation activates kinase-linked receptors.

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Nuclear-linked receptors
Steroid receptor activation can stimulate the up- or down-regulation of protein synthesis.

Exam question examples (from all Dr Foa’s lectures)
• • • • Is it true that activation of G-protein coupled receptors only activates muscle contraction? Explain your answer. Explain how x receptor is activated and how activation of x can cause changes in muscle contraction or cellular response Define the terms pharmacodynamics and pharmacokinetics Explain the resting membrane potential:
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o Why is it important? o Which ion is most important? o What is an electrochemical gradient?

The dynamic and excitable cell membrane
Aims of this section: • To understand why membranes are excitable • To understand the ionic basis of the excitable membrane o the membrane potential o cell membrane proteins  role of ion pumps  role of channel proteins o molecules that help maintain the membrane potential  sodium  potassium  chloride  anions • To understand what the chemical gradient is

Why are membranes dynamic and excitable?
They are dynamic because they need to facilitate growth, change shape (as many cells are on the move and need to change as they grow and/or migrate), and because proteins have a finite lifetime and need to be replaced. They are excitable to facilitate communication, which allows movement, contraction, or generally a response to a stimulus. Cells are dynamically structured and their membranes are continually remodelled, hence the plasma membrane fluid mosaic model.

What is a membrane potential?
The regulation of ion movement by cells causes a difference in charge across the membrane. The outside of the membrane is positive with resect to the inside. This means that there are more positively charged ions immediately outside the membrane and more negatively charged ions immediately inside the membrane.

The proteins responsible for maintaining the membrane potential

Ion channels
Ion channels form the basis of cell communication. • They are integral membrane proteins (usually five joined together) • They have hydrophilic regions of the proteins lining the inside of the channel, and the hydrophobic region exposed to the lipid membrane • The charge of the hydrophilic region determines which ions may pass • Nongated ion channels are open all the time and are termed leakage channels • Gated ion channels have some sort of barrier Voltage-gated ion channels: open in response to a change in the membrane voltage, and are essential in action potential generation and conduction Ligand-gated ion channels: open in response to chemical stimuli, such as neurotransmitters, hormones or other ions that bind the channel, causing it to open; involved in synaptic transmission.
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Leakage channels: have gates that randomly open, and are involved in the maintenance of the membrane potential at rest. Mechanoreceptors: gated ion channels that open in response to mechanical stimulation, such as touch and vibration.

Cell communication: ion channel receptor proteins
These are integral proteins that have an exposed receptor site for binding specific ligand molecules. Ligand binding changes the shape of the protein so that the channel opens, allowing the passage of ions. For examples, a muscle contraction in your leg starts when acetylcholine binds to a sodium channel on the surface of a muscle fibre and opens the sodium inside the muscle fibre.

Cell communication: membrane-bound enzymes
Enzymes catalyse reactions at the inner or outer surface of the membrane. Example: the sodium-potassium ATPase pump.

The resting membrane potential
The charge difference across the membrane of a cell at rest, or unstimulated, is the resting membrane potential. • Muscle resting membrane potential is -85mV • Nerve cell resting membrane potential is -70mV The resting membrane potential is maintained by: • The Na+-K+ ATPase pump o This pump moves Na+ out of the cell and K+ into the cell o This results in a higher concentration of Na+ outside the membrane • The cell membrane is more permeable to K+ than Na+ ions or anions o Therefore, K+ diffuses out of the cell  As a result, the inside of the cell is more negative with respect to the outside  The membrane is poralised The Na+-K+ ATPase pump is crucial in maintaining the resting membrane potential. Against their concentration,

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Potassium is essential for the resting membrane potential. The resting membrane potential is established when the tendency for K+ to move out of the cell because of the concentration gradient, is equal to the tendency for K+ to move into the cell because of the electrical gradient (the attraction of the positively charged K+ to the negatively charged proteins and ions within the cell).

Electrical activity within neurons
The electrochemical gradient is the basis for all electrical activity within neurons. Passive oin diffusion across the membrane is down the chemical gradient. When ions move to a region of opposite charge, they are moving along their gradient. So, for each ion, there is an electrochemical gradient. • • • • The resting membrane potential is maintained by the difference in membrane permeability to sodium and potassium ions Neuronal membranes are about 75 times more permeable to K+ rather than Na+ The Na+/K+ ATPase pump actively pumps 3 Na+ molecules out of the neuron for every 2 K+ molecules it pumps in Chloride also slowly leaks into the cell
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At rest, the membrane is polarised

Why cells need a membrane potential
The charge difference across the membrane of a cell at rest, or unstimulated, is the resting membrane potential. Ion influx of efflux is the basis of changes to the membrane potential and membrane excitation (via voltage or ligand gated ion channels) that allows action potentials in nerve cells, and muscle contraction.

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