Vibrio Cholera

Vibrio Cholera:

Rod/comma shaped bacterium, which can cause dehydration through diarrhoea, 

It can be ingested through 

• Contaminated water
• Food, or preparation

It affects the body through a toxin that it produces, the toxin only affects the upper region of the small intestine as this is the only region with membrane receipts that complement the toxin. 

How:

Many of the organisms are destroyed by the stomach acid, but in the case of a less acidic stomach some bacterium may survive into the small intestine, using their flagella in a corkscrew motion they will pass the mucus layer and anchor itself into the duodenum.

The bacteria will then produce an exotoxin, that binds to specific receipts of the cell surface membrane, and activates the chloride ion channel in the cell membrane. 

This causes the chloride ions to diffuse out of the cells into the lumen. 

Therefore the water potential in the lumen is lowered, so the water moves by osmosis, down its water potential, out of the cells and into the lumen. Producing diarrhoea and dehydration.

Treatment: 

Oral rehydration therapy (ORT), replaces lost water and salts, 

Drink a solution of sugar and salt and water, 

Which will be taken into the cell by the co transported protein, lowering the water potential in the cell, increasing the gradient between the cell and the gut. Therefore water is then taken up by osmosis, back into the cell. Rehydratic cells and reducing diarrhoea. 

Prokaryotes and Eukaryotes

Differences between Prokaryotic and Eukaryotic cells: 

Endotoxins are produced from the break down of bacteria (cell walls) and are lipopolysaccharides, 

Exotoxins are proteins, which are secreted from living cells. 

Prokaryote cells

• Cell membrane: regulates entry/exit, as it is selectively permeable, 
• Mesosome: respiration and cell division 
• Cell wall: Protection, prevents osmotic lysysis, (water moving into the cell), made of peptidoglycan. 
• Slime layer/capsule: Protection e.g. against antibiotics,
• Flagellum: Movement of cell, 
• DNA: circular, free in cell, containing genetic material. 

Eukaryotic Cells

Magnification

  

• Resolution: The ability to distinguish between two separate points, Electron microscopes have a higher resolution than light microscopes as they use electrons, as they use electrons (which has a shorter resolution than light). Shorter resolutions allow for better resolution than longer wavelength. Some times microscopes have blue filters to allow for this as blue has the shortest wavelength. 
• Magnification: Indicates how much bigger the image is than the original object. Simply given as a magnification factor e.g. x100, By using more lenses microscopes can magnify by a larger amount, but the image may get more blurred, so doesn't mean that more detail can be seen.
• Light Microscopes: Oldest, simplest, and most widely used microscopes. Specimens illuminated with light, which is focused using glass lenses and viewed using the eye or photographic film. Specimens can be alive or dead, but often need to be stained with colour to make them visible. 
• Electron Microscopes: This uses a beam of electrons, rather than electromagnetic radiation, to “illuminate” the specimen. Electrons behave like waves and can be easily produced (using a hot wire), focused (using electromagnets) and detected (Phosphor screen or photographic film). A beam of electrons has a very useful wavelength of less than 1nm, so can be used to view sub cellular specimens. 
• Transmission Electron microscopes (TEM): work like light microscopes, transmitting a beam of electrons through a thin specimen and then focussing the electrons to form an image on the screen or on a film. This is the most common form of electron microscopes and has the best resolution (<1nm)
• Scanning Electron microscopes (SEM): scan a fine beam of electrons onto a specimen and collect the electrons scattered by the surface, this gives poorer resolution, but means you can view the final image in 3D.

Cell Fractionation

The process of separating different parts and organelles of a cell, so that it can be studied in greater detail. The most common method is differential centrifugation. 

This is where the organelles are separated due to their different densities. 

There are three key stages: 

1. Homogenisation 
2. FIltration 
3. Centrifugation 

Homogenisation: 

Pestle and mortar or a blender to break open the cells. You will need and ice cold, isotonic buffer solution:  

• COLD: This reduced enzyme activity and will reduce autolysis (self destruction) of the organelle. 
• ISOTONIC: To prevent osmotic movement where swelling of organelle may cause lysis or shrinkage of the organelle, both processes will result in the destruction of its natural function. 
• BUFFER SOLUTION: Preventing any change of pH, which may damage the organelles, either by denaturing the enzymes or the proteins affecting its function. 

Filtration: 

This is to remove any debris (unbroken cells, cartilage etc). This will prevent contamination of the pellets resulting from the centrifugation. 

Centrifugation: 

Homogenate is centrifuge at different speeds, The speed and length of centrifugation will increase each time. The densest organelle sinks to the bottom, and the remaining solution, the supernatant, is separated for further centrifugation. 

Naughty Monkeys Like Eating Raspberries 

• Nuclei 
• Mitochondria 
• Lysosomes 
• ER 
• Ribosomes

Cells and Water

Since cells contain various Biological Molecules, such as Sugars and Salts, they have a Water Potential lower then 0 kPa. Water may move in or out of a cell depending of the Water Potential Gradient between the inside of the cell and its  environment.

 When water diffuses into a plant cell, when it is placed in a solution of higher Water Potential than inside it, the cell contents will expand. However, since plant cells are surrounded by a strong cell wall, they will not burst. The cell contents will push against the cell wall, and the cell will become Turgid.

If a plant cell is placed in a solution of lower Water Potential, water will diffuse out. This causes the Cytoplasm to shrink and become Flaccid. If enough water leaves, the Cytoplasm will pull away from the cell wall. The cell will become Plasmolysed.  

 Animal cells will also expand when they are placed in a solution of higher Water Potential. Since animal cells do not have cell walls, if this happens excessively the cell will burst open and become Haemolysed.

If water leaves an animal cell by Osmosis, it will shrink and appear 'wrinkled'. It will become Crenated.

Proteins

• Transport proteins: Most transport of small molecules across the membrane take place through integral proteins. This transport includes facilitated diffusion and active transport.
• Receptor proteins: Receptor proteins must be on the outside surface of cell membranes and have a specific binding site where hormones or other chemicals can bind to form a hormone-receptor complex (like an enzyme substrate complex) This binding then triggers other events in the cell membrane or inside the cell.
• Enzymes: Enzyme proteins catalyse reactions in the cytoplasm or outside the cell, such as maltase in the small intestine. 
• Recognition proteins: some proteins are involved in cell recognition. These are often glycoproteins such as the A and B antigens on red blood cell membranes. 
• Structural proteins: Structural proteins on the inside surface of the cell membranes are attached to the cytoskeleton. They are involved in maintaining the cells shape, or in changing the cells shape for cell motility. Structural proteins on the outside surface can be used in cell adhesion - sticking cells together temporarily or permanently. 

Movement of substances

Factors affecting the rate of diffusion:

• Surface area: increasing the surface area increases the rate of diffusion, 
• Concentration gradient: Increasing the concentration gradient increases the rate of diffusion, 
• Temperature: Increasing the temperature increases the rate of diffusion. 
• Diffusion distance: Increasing the diffusion distance decreases the rate of diffusion. 

Rate of diffusion = Concentration gradient x surface area / diffusion distance 

• Osmosis: Water molecules are small enough to pass between phospholipid molecules, down a water potential gradient. The molecules move from an area of high water potential to an area of low water potential across the membrane. 
• Active Transport: Water-soluble substances that must be moved against a concentration gradient require carrier protein and ATP. 
• Facilitated diffusion: Polar, water soluble substances that cannot pass through the membrane can be transported bus specific channel proteins down a concentration gradient.
• Diffusion: Small lipid soluble substances can pass throughout the lipid bilayer - between the phospholipid molecules - down a concentration gradient. 
• Facilitated diffusion: Large water soluble substances cannot pass between the lipid molecules so are carried through the membranes by carrier proteins down a concentration gradient.

Water Potential

Osmosis is the movement of water molecules down a water potential gradient across a partially permeable membrane. Water molecules will move from an area of high water potential (e.g. pure water), to an area of low water potential (e.g. saline or sugar solutions) Water molecules move from a position of less negative to more negative water potential. 

Fluid mosaic model

The fluid mosaic model for membranes:

Membrane components and their functions: 

• Phospholipid: Hydrophilic head (phosphate), Glycerol, Hydrophobic fatty acid tail, forms a semipermeable phospholipid bilayer. 
• Carrier protein: protein with a specific shaw that complements the shape of the substance to be transported across the membrane, it is used in active transport and facilitated diffusion. 
• Cholesterol: Regulates membrane fluidity.
• Glycoprotein: Cell signalling and recognition and binding cells together.
• Glycolipid: Carbohydrate attached to lipid/phospholipid, cell signalling and cell recognition 
• Channel protein: Protein with a specific shape that complements the shape of the substance to be transported across the membrane, used in facilitated diffusion. 
• Cell signalling: Receptors on cells bind to hormones, drugs and other cells leading to a series of reactions within the cell

Temperature and permeability:

• A high temperature boosts the kinetic energy of the component molecules of the membrane and the transported substance, therefore the membrane becomes more permeable. 
• Very high temperatures will denature the protein molecules, changing their shape and making the membrane more permeable, eventually the membrane will be destroyed. 

Specialised membranes:

• Different cells’ membranes have varying properties, functions and capabilities. These depend on the glycoproteins, glycolipids, channel proteins and carrier proteins that are present. 
• Some membranes are folded to increase surface area for transport or absorption, e.g. microvilli.
• Membranes are fluid, so they can be folded by an organisms cytoskeleton to for vesicles. The active process (meaning it requires ATP) is part of endocytosis.
• Vesicles can fuse with the membrane as part of exocytosis. 

The structure of a cell membrane and phospholipids: 

Fluid mosaic model: Fluid - molecules within the membrane are free to move in relation to each other.

Mosaic - mixture of phospholipids and proteins.

Double layer of phospholipid molecules, phospholipid consists glycerol, to which are joined two fatty acids, and a phosphate, formed by a condensation reaction. The phosphate head is hydrophilic and the fatty acid tail is hydrophobic, meaning in the membrane the phospholipids are arranged as a bilayer, heads on the outside and tails on the inside. 

• Intrinsic proteins pass through the entire bilayer, some of the proteins have channels/pores, and some have binding sites and are carrier proteins. These proteins allow the transport of water soluble molecules.
• Extrinsic proteins are only in one layer, those on the outer side ofter act as receptors for hormones. 

Many of the proteins and phospholipids have carbohydrates attached forming glycolipids and glycoproteins that make up the glycocalyx. 

Movement: 

• Most molecules move across the membrane by diffusion down a concentration gradient, 
• Small molecules (water/gases) and lipid soluble molecules diffuse between the phospholipid, 
• Polar molecules require channel or carrier proteins to move them.
• Channels are water filled pores that can be open at all times or they can be gated. 
• Carrier proteins have a specific binding site for the molecules/ions, This cal me facilitated diffusion, a passive (no ATP required) process,
• Some molecules are actively transported across the membrane (against the concentrate gradient). This requires ATP (released in respiration). The ATP changes the shape of the protein to move the molecule across the membrane. 

Lactose intolerance

Lactose is a sugar found in milk, it is digested by an enzyme called Lactase, found in the intestines. So if you don't have enough lactase, you wont be able to fully digest the lactose. 

Undigested lactose is fermented by bacterial, which releases gasses, meaning you have symptoms such as, stomach cramps, wind and bloating. 

Diarrhoea is caused by having a high concentration of lactose in the intestines, this causes the water to move out of the blood and into the intestines (by osmosis). The increase in water means you get runny faeces. 

Milk can be artificially treated with purified lactase to make it suitable for lactose intolerant people. Its fairly common to be lactose intolerant.

Digestion and the small intestines

Absorption of Glucose in the intestines: 

• When Carbohydrates are first broken down there is a higher concentration of glucose in the small intestine than there is in the blood. Therefor glucose moves across the epithelial cells of the small intestine, into the blood, by a process called diffusion. When the concentration in the lumen becomes lower than in the blood, there is no longer a concentration gradient, therefore diffusion stops. 
• The remaining glucose is absorbed by active transport with sodium ions:
• Sodium ions are actively transported out of the epithelial cells into the blood, by the sodium potassium pump, creating a concentration gradient, theres a higher concentration of sodium ions in the lumen than the cells.
• This concentration gradient causes sodium ions to diffuse into the cell from the lumen, this is done by sodium-glucose co-transporter proteins. The co-transporter carries glucose into the cell with the sodium, the increase of glucose in the cell increases the concentration.
• The glucose then diffuses down its concentration gradient into the blood, through a protein channel, via facilitated diffusion.

How is the small intestine adapted to its digestive and absorptive functions:

• Large surface area provided by villi and microvilli, 
• Thin epithelium gives a short diffusion pathway, 
• The dense capillary network for absorbing amino acids and sugars and the lacteal for the absorption of digested fats; which ensures a steep concentration is maintained,
• Many mitochondria in the epithelial cells provide ATP/ energy for active transport,
• Carrier proteins (in membranes) provide a path for polar molecules to pass through the membrane,
• Enzymes built into the epithelial membrane make it more likely for enzyme substrate complexes to form and ensure products for absorption are released close to the channel and carrier proteins, 

Digestion of Carbohydrates and Disaccharides:

Food moves through the digestive system by peristalsis. 

Mouth = digestion of carbohydrates,

Stomach = digestion of protein,

Duodenum = most digestion, receives pancreatic juice from the pancreatic duct, and bile from the gall bladder (produced in the liver.)

Ileum = most absorption, 

Colon = absorption of water and minerals,

Enzymes

Enzymes break down substances by: 

• Lowering the activation energy, a substance with a complementary shape to the enzyme entries the active site, forming an enzyme substrate complex, the active site changes shape to mould around the substrate (induced fit), this weakens the bonds in the substrate (lowering the activation energy) by stretching and distorting them, the bonds are broken, the products then leave the active site, leaving the enzyme unchanged. 

The effect of pH on the rate of enzyme activity: 

• Changes in pH affect the charges on the R groups of the amino acids at the active site. Therefore the interactions between the enzyme and the substrate are disrupted, reducing the frequency of enzyme substrate complexes forming, 
• Mote extreme pH conditions can cause the bonds (hydrogen, ionic, disulphide) holding the tertiary structure, to break. 
• If the enzyme denatures then the active site is no longer complementary to the substrate, therefore no enzyme substrate complexes can form. 

How the shape of the enzyme/protein molecules is suited to its function:

• Enzyme has a specific primary structure (amino acid sequence), this sequence determines where the hydrogen bonds will form during development of the secondary structure.Proteins have a unique tertiary structure held together by ionic, hydrogen and disulphide bonds. Globular proteins have an active site with unique structure. 
• The shoe of the active site is complementary to the substrate, so it'll only fit that substrate, so that enzyme substrate complexes can form.

The effect of temperature on the rate of enzyme activity: 

• As temperature increases dodoes the rate of the reaction, as the substrates gan kinetic energy and therefore collide more frequently, therefore there are more enzyme substrate complexes forming.
• A further increase of temperature can cause bonds (ionic, disulphide and hydrogen) which are holding the tertiary structure of the enzyme inlace to begin to break, this means the enzyme denature. 
• The active site no longer complements the substrate, therefore no enzyme substrate complexes can form.

How does an enzyme catalyse a condensation reaction: 

• The enzyme has a complementary shape to the substrate, meaning an enzyme substrate complex can be formed, the reactive groups are brought close together, the change in the active site (induced fit) lowers activation energy, water is removed and a bond is formed (glycosidic, peptide or ester bond) The products leave the active site, the enzyme remains unchanged

How do inhibitors affect enzyme activity:

• There are two types of inhibitors, competitive and non-competitive:  

• Competitive inhibitors -  have a similar shape to the substrate they can enter and bind with the active site, preventing enzyme substrate complexes forming, this problem can be  overcome by increasing the substrate concentration, 

• Non-competitive inhibitors -  have a different shape and bind to the enzyme at any point other than the active site, causing a change in the shape of the active site meaning the enzyme is no longer complementary to the substrate, preventing the formation of enzyme substrate complexes.

Biochemical Tests

Biochemical Tests:

Proteins

 Proteins: 

• Primary structure - The amino acid sequence
• Secondary structure - Folding of the polypeptide chain, held by hydrogen bonds, (alpha helix and beta sheet)
• Tertiary structure - Further folding of the secondary structure, held by hydrogen bonds, ionic bonds, and possibly disulphide bonds.
• Quaternary structure - Two or more polypeptide chains join together, 

Chains of amino acids are formed by condemnation reactions, producing peptide bonds, and releasing a water molecule. 

Globular proteins: 

Most proteins are globular, meaning they have compact ball-shaped structures. These include enzymes, membrane proteins, receptors and storage proteins.

• They have complex tertiary and sometime quaternary structures,
• They are folded into spherical (globular) shapes,
• They are usually soluble as the hydrophobic chains are in the centre of the structure,
• They play roles in metabolic retains,
• For example, enzymes and haemoglobin in the blood, 

Fibrous (or filamentous) proteins:

Fibrous proteins are long and thin, they tend to have more structural roles, such as collagen (bones), Keratin (hair), tubulin (cytoskeleton) and actin (muscle). They are always composed of many peptide chains.

• Little or no tertiary structure,
• long parallel polypeptide chains, 
• cross linkages at intervals forming long fibres or sheets,
• usually insoluble
• many have structural roles,
• for example, hair and outer layer of skin, collagen (a connective tissue) 

Carbohydrates and Triglycerides

Isomers: same molecular formula but different structural formula 

Carbohydrates: 

• Monosaccharides - sweet, water soluble, reducing sugars (test-benedicts),
• Disaccharides - sweet, water soluble, all but sucrose are reducing sugars,
• Polysaccharides - long chains of repeating subunits (monosaccharides) joined by condensation reactions,

• Sucrose —> Glucose + Fructose
• Lactose —> Glucose + Galactose
• Maltose —> Glucose + Glucose

Triglycerides:

 Fatty acid chains can be SATURATED (they contain no carbon-carbon double bonds)  or UNSATURATED (they do contain carbon-carbon double bonds). The more unsaturated the fatty acid the lower the melting point. Fats are insoluble in water. 

Formation of a Triglyceride: