Molecular Biology Of The Cell Flashcards
Why is step 1 of glycolysis irreversible
Because glucose6phosphate produced is negatively charged therefore cannot leave the cell through glucose transporters. This commits the cell to subsequent reactions
Why is regulation of phosphofructose kinase important?
It is an important control step for the entry of sugars into the glycolysis pathway
What is reaction 4 of glycolysis
Fructose-1,6-bisphosphate is converted by Aldolase to glyceraldehyde 3 phosphate and dihydroxyacetone phosphate in a hydrolyitc reaction
Enzyme for reaction 5 of glycolysis
TPI —> triose phosphate isomerase
What type of reaction is reaction 6 of glycolysis and what does it produce
Redox and group transfer
Produces 1,3-bisphosphoglycerate and NADH
Enzyme for glycolysis reaction 7
Phosphoglycerate kinase
Net result of glycolysis
2 atp
2 nadh
2 pyruvate
What type of reactions do dehydrogenases catalyse
Redox
What 3 amino acids can be substrates of kinases
Tyrosine threonine serine
What can elevated LDH levels mean
Cell death and tissue damage —> diagnosis of stroke and MI
Which High energy bond joins the acetyl group onto CoA
Thioester bond - readily hydrolysed enabling acetyl coA to donate acetate (2C) to other molecules
Net products of TCA cycle?
2 Co2
3 NADH
1 FADH2
1 GTP
How can amino acids enter TCA cycle
Have their amine group removed by transamination reaction - resulting new ketone acid can join TCA cycle or production of glucose
(Amino group is removed as urea)
Degradation of all 20 amino acids only gives rise to 7 molecules:
Pyruvate, succinyl CoA, acetyl CoA, acetoacetyl CoA, oxaloacetate, fumerate, alpha ketoglutarate
Which TCA cycle defects lead to cancer
Defects in genes of Fumerase, succinate dehydrogenase, isocitrate dehydrogenase
What are important positively and negatively charged amino acids
Histidine: pKa of 6, can donate or accept proton depending on environment
Lysine and arginine: physiological pH of 7, are always protonated, are basic, are positively charged
Aspartate and glutamate: have acidic side chains so release H+ and are negative
Glycerol phosphate shuttle
In skeletal muscle and brain
Cytosolic glycerol 3 phosphate dehydrogenase transfers electrons from NADH to dihydroxyacetone phosphate to generate glycerol 3 phosphate (and NAD+)
(Membrane bound) Mitochondrial glycerol 3 phosphate dehydrogenase transfers the electrons from glycerol 3 phosphate to FAD to form FADH2 which passes the electrons to co enzyme Q which is part of electron transport chain
Also this produces dihydroxyacetone phosphate again
Malate aspartate shunt
Oxaloacetate in cytoplasm is reduced to form malate by MDH (malate dehydrogenase) and NAD+ is formed from NADH - redox reaction
The malate enters mitochondria through malate alpha ketoglutarate antiporter
Inside mitochondria the malate is converted back to oxaloacetate by reverse reaction also catalysed by MDH and this time producing NADH from NAD+
The oxaloacetate the undergoes transamination reaction with glutamate to form aspartate and the keto acid alpha ketoglutarate catalysed by AT (aspartate transaminase)
The alpha ketoglutarate exits the mitochondria into cytoplasm through malate alpha ketoglutarate antiporter
The aspartate exits mitochondria to cytoplasm through glutamate aspartate antiporter
In cytoplasm the aspartate undergoes reverse transmaination reaction with alpha ketoglutarate to reform glutamate and oxaloacetate
Glutamate enters mitochondria through glutamate aspartate antiporter
5 main classes of lipids
Free fatty acids Triacylglycerols (triglycerides) Phospholipids Glycolipids Steroids
What are the 3 primary sources of fats
Diet
De novo biosynthesis in liver
Storage depots in adipose tissue
What are triacylglycerols and why are they ideal for storage
Fatty acids are often stored as triacylglycerols
3 fatty acids joined to glycerol via ester linkages that help neutralise the carboxylic acid group and keep cell in normal ph range
Fatty acids are reduced and anhydrous making them ideal for storage
Why is fatty acid metabolism important
Caloric yield from fatty acids is about double than from carbs
More than half of body’s energy including liver but not brain comes from fatty acid oxidation - enhanced over long duration fasting
First step of beta oxidation of fatty acids
Fatty acids are converted to acyl coA spices by combination with Co enzyme A through acyl coA synthetase action
2 Hugh energy phosphoanhydride bonds of ATP are broken
Cartinine shuttle
Transports acyl CoA from where it’s made in outer mitochondrial membrane into matrix
Acyl group is transferred from acyl CoA to cartinine to form acyl cartinine by cartinine acyltransferase I
translocase imports acyl cartinine molecule into matrix
Cartinine acyltransferase II adds acyl group to coA by removing acyl group from acyl cartinine to form cartinine and acyl coA
Translocase moves cartinine out of matrix
Beta oxidation cycle
Fatty acyl co A enters cycle and undergoes oxidation (catalysed by acyl co enzyme A dehydrogenase) , hydration, oxidation then thiolysis (split into a fatty acyl coA shortened by 2C and and acetyl coA)
Each Cycle produces one NADH and one FADH2
Cycle keeps going until left with a 4C molecules which splits to form 2 acetyl coA
Beta oxidation of palmitic acid (16C)
Cycle happens 7 times overall
Palmitoyl coA + 7 FAD + 7 NAD+ + 7H2O + 7CoA —> 8 acetyl coA + 7FADH2 + 7 NADH
What happens when fatty acid degradation/oxidation predominates and there is loss of balance between beta oxidation and carb metabolism
Acetyl coA cannot enter TCA cycle as needs oxaloacetate (which is not there due to no carbohydrate metabolism)
Instead acetyl coA forms ketone bodies: acetone, acetoacetate, D-3-hydroxybutyrate
Where does lipogenesis happen in adults
Mainly liver, adipose tissue and lactating breast
But can happen in certain cancer cells - use fatty acids to fuel their proliferation - could target FA synthetase in cancer cells during treatment
Differences between lipogenesis and beta oxidation
Beta oxidation Carrier = CoA Reducing power provided by: FAD/NAD+ Location = mitochondrial matrix Oxidation hydration oxidation cleavage
Lipogenesis Carrier = ACP - acyl carrier protein Reducing power provided by: NADPH Location = cytoplasm Condensation (of acetyl coA and malonyl CoA) reduction (by ketoreductase) dehydration (by dehydratase) reduction (by enol reductase)
2 enzymes needed for lipogenesis
Acetyl coA carboxylase
Fatty acid synthase
How are vigorous contraction requirements met in skeletal muscle?
O2 becomes a limiting factor
Glycogen stores are broken down to produce ATP
Locate is formed under the anaerobic conditions and leaves the muscle to travel to liver via blood
What is the function of adipose tissue
Long term storage for fatty acids in the form of triglycerides
Give 3 main roles of the liver
Maintaining blood glucose at 4 - 5.5 mM
Body’s main carb store (in form of glucose) and a source of blood glucose
Lipoprotein metabolism - transport of triglycerides and cholesterol
What can excess glucose 6 phosphate and excess acetyl coA generate
Exc. G6P : glycogen in muscle and liver
Exc. AcoA : fatty acids that are stored as triglycerides in adipose tissue
What can pyruvate and TCA intermediates be used as a source for
Their backbones can be used to make nucleotides which can be used to make amino acids
What does acetyl coA do during fasting
Produce ketone bodies instead of entering TCA cycle
How is bulk of NADPH needed for and Oli can pathways eg cholesterol cynthesis produced
When glucose 6 phosphate enters pentode phosphate pathway to produce nucleotides
How can hypoglycaemic coma be avoided in short term by body
Breakdown of glycogen stores in liver to maintain plasma glucose levels
Production of ketones from acetyl CoA via liver
Release of fatty acids frontman adipose
(Last 2 help as the fatty acids and ketone bodies can be used by muscle leaving more plasma glucose available for the brain)
What non carbon precursors can enter gluconeogenesis pathway and how
Lactate : produced in skeletal muscle from pyruvate when rate of glycolysis > rate of TCA and ETC
Lactate is taken up by liver and converted back to pyruvate in Cori cycle by LDH (lactate dehydrogenase)
Amino acids: derived from diet or breakdown of skeletal muscle
Glycerol: triglyceride hydrolysis gives FFA and glycerol
Glycerol backbone can be used to make DHAP (dihydroxyacetone phosphate)
3 bypass reactions for gluconeogenesis
Pyruvate —> phosphoenolpyruvate
- pyruvate carboxylate for pyruvate to oxaloacetate
- Phosphoenolpyruvate carboxykinase for oxaloacetate to phosphoenolpyruvate (^ glycolysis way is pyruvate kinase)
Fructose 1,6 bisphosphate —> fructose 6 phosphate
- fructose 1,6 bisphosphatase
(Glycolysis way is phosphofructokinase)
Glucose 6 phosphate —> glucose
- glucose 6 phosphatase
(Glycolysis way is hexokinase)
What happens during aerobic respiration during moderate exercise when muscle contraction increases
Increased demand for glucose is met by increased number of glucose transporters on membranes of muscle cells
Adrenaline can have the effects:
- increases rate of glycolysis in muscle
- increases rate of gluconeogenesis in liver
- increases release of fatty acids from adipocytes
What are glucocorticoids
Steroid hormones which increase synthesis of metabolic enzymes concerned with glucose availability
What are the complications of diabetes
Hyperglycaemia - can cause progressive tissue damage
Hypoglycaemia- if insulin treatment dose is wrong
Acidosis (increased acidity if blood) - due to increased ketone bodies
Cardiovascular complications - due to build up of fatty acids in plasma and lipoproteins
(Body acts as if it’s in starvation bc the glucose can’t be taken up by cells)
What is michaelis constant (Km)
The conc of substrate at which an enzyme functions at a half maximal rate (half of Vmax)
How is glucose metabolism in liver and muscles controlled
Hexokinase catalysed first irreversible step of glycolysis (glucose to G6P)
It has 2 isoforms which catalyse same reaction but are maximally active at different glucose conc (different Km values)
Hexokinase I : in muscle, Km is 0.1 mM so is active at low glucose conc and operates at max most of time
Is highly sensitive to inhibition by G6P so during anaerobic conditions where there is no TCA cycle and glycolysis is slow, the buildup of G6P can inhibit Hk I
Hexokinase IV : in liver, Km is 4mM so is less entice to blood glucose conc and less sensitive to inhibitory effects if G6P
G6P produced by hk IV is used to make glycogen
What happens after meal?
Blood glucose levels rise
Insulin secreted from islet cells of pancreas
Reduced glucagon secretion
Effects: increased glucose uptake and glycogen synthesis in muscle
Increased triglyceride synthesis in adipose tissue
Increased use if metabolic intermediates
What happens some time after meal
Glucose levels decrease
Glucagon secreted from islets
Reduced insulin secretion
Effects: glycogenlysis and gluconeogenesis in liver
Fatty acid breakdown - as alternative substrate for ATP production and preserving glucose for brain to use
What happens after prolonged fasting? Longer than can be covered by glycogen reserves
Adipose tissues hydrolyses triglycerides to provide fatty acids
TCA cycle intermediates are reduced in amount to provide substrate for gluconeogenesis
Protein breakdown provides amino acid substrates for gluconeogenesis
Ketone bodies are produced from fatty acids and amino acids in liver to partially substitute brains requirement for glucose
What is the site of production of ketone bodies
The liver
In what tissues is CK (creative kinase) present
In all cells at low levels But at high levels in: Brain - BB (homodimer) muscle - MM (homodimer) Heart MB (heterodimer)
How to establish a diagnosis of myocardial infarction
Do blood test
Do electrophoresis
Check MB (just measuring CK activity - eg by coupled enzyme assay - isn’t enough as could be any of the isoenzymes of CK. brain only produces B so has dimer BB. Muscle only produces M so has dimer MM. heart can produce B and M so produces all three isoenzymes - BB MM and MB. So if MB is detected it means that there has been death of heart cells)
Could also use immunological approach: artificial manufacture of antibodies against CK-MB
This would be done with other tests and is used Tod determine size and age of infarct
Other markers for myocardial damage (apart from CK)
SGOT: serum glutamate oxaloacetate transaminase
LDH: lactate dehydrogenase
Cardiac troponin: troponin is the calcium switch in muscles. Cardiac troponin I and troponin T is are only found in heart tissue so their presence in heart tissue is a specific marker for cardiac infarction - typically appears in serum 48hr after infarction and persists for ~5 days)
When and why is CK found in the blood
Atherosclerosis and blockage of blood vessel stops blood flow and oxygen delivery to tissue
Cells need oxygen for end part of respiration - TCA and ox phos
Cells don’t receive oxygen therefore can’t make as much ATP. Need ATP to pump things in and out of cell and maintain a balance between outside and inside environment eg with ions. Active expulsion of things like Na+ ions by protein pumps in membrane which are membrane ATPases - use energy in form of ATP to pump ions
Balance is lost so cells die
When they’re dying their membrane becomes leaky so they release their contents
So levels of proteins like CK or LDH in serum can be used as indirect indicators of cell death
How can the three isoenzymes of Ck be separated by electrophoresis
They have approx same molecular weight but different pI (Isoelectric point) : pH at which they have neutral charge
Therefore they have different charge at same pH
What is time course of serum CK after infarct
Around 4 days
SGOT is about 5/6 days
LDH is about 10/11 days
Does an increase in serum Ck relate to size of myocardial damage
Yes the levels of CK-BM in serum is directly proportional to amount of cell death in heart
Bc each monocyte has approx same volume so each cell releases a “quantum” of Ck into extra cellular fluid and serum when it dies
3 main steps of cholesterols synthesis and their locations
Synthesis of isopentenyl phyrophosphate, an activated isoprene which is a key building block - CYTOPLASM
Condensation if six molecules of isopentenyl pyrophosphate to form squareness - CYTOPLASM
Cyclisation and demethylationa of squareness by monooxygenases to give cholesterol - ER
What does isoprene do
Confers lipophilicity to biomolecules to allow them to sit inside the lipid layers of membranes
Eg dolichol phosphate and Oxidised coenzyme Q (ubiquinone)
The same lipophilic properties of isoprene unit confine ubiqonine to the inner membrane of mitochondria
How else can proteins gain affinity for lipid bilayers
They can undergo lipid modifications like prenylation
Describe cholesterol synthesis
Condensation of 2 acetyl co A to for acetoacatyl co A
Condensation of another acetyl co A with this to form HMG coA
HMG coA is reduced by HMG coA reductase to form mevalonate (this intermediate of mevalonate and end product cholesterol, as well as bile salts, inhibit HMG coA reductase - negative feedback control)
Mevalonate undergoes series of sequential phosphorylations to at hydroxyl groups at positions 3 and 5 then decarboxylation to produce 3-isopentenyl pyrophosphate (an activated isoprene unit which is useful building block for further synthesis)
Isomerization of 3-isopentenyl pyrophosphate to produce dimethylallyl pyrophosphate
Condensation of that with an isopentenyl pyrophosphate to produce C10 geranyl pyrophosphate
Condensation of that with an isopentenyl pyrophosphate to produce C15 farnesyl pyrophosphate
2 molecules of Farnesyl pyrophosphate condense to form C30 squalene and 2 molecules of pyrophosphate
Squalene is reduced in the presence of o2 and NADPH to produce squalene epoxide
Squalene epoxide lanosterol cyclase enzyme catalysed the formation of Lanosterol from this
Lanosterol is reduced and demethylated to form cholesterol
Describe 3 things that cholesterol can be made into
Bile salts: cholesterol can be broken down to form bile salts eg glycocholate and taurocholate
Vitamin D: in presence of UV light- 7 dehydrocholesterol is activated by UV light to form previtamin D3 which forms Vitamin D3 which forms Calcitriol
Steroid hormones: desmolase converts cholesterol to pregnenolone which is the precursor for all 5 classes of steroid hormones ( progestagens, glucocorticoids, mineralocorticoids, androgens, oestrogens)
What is calcitriol
The most active vitamin D metabolite
Plays a key role in calcium metabolism
What does deficiency of vitamin D3 in childhood lead to
Rickets - defect of bone development in children
Describe genetics of FH (familial hypercholesterolaemia)
It is a mono genie dominant trait
Describe difference in having 1 or 2 copies of mutant gene for FH
1 copy: serum cholesterol levels are 2/3 times higher than normal, increased risk of developing atherosclerosis in middle life
2 copies: serum cholesterol levels are 5 times higher than normal, severe atherosclerosis and coronary infarction seen in adolescence
Visual symptoms of FH
Orange yellow xanthomas seen on skin due to skin macrophages engulfing plasma LDL derived cholesterol
Disease mechanisms underlying FH
Patients with severe FH lack function LDL receptors (LDLR) due to mutations so therefore LDL is not taken up by the cell surface receptors and remains in serum
The mutations can be one of many that have the same effects but can be classes into 5 classes of mutation
- mutation that stops LDLR synthesis
- mutation that stops movement of LDLR from ER to Golgi causing low cell surface expression
- mutation that causes LDLR to not bind LDL effectively
- mutation that stops the LDLR:LDL complex from clustering in the pits on the cell surface, which needs to happen for endocytosis
- mutation that stops LDL from being released from receptor in endoscope inside cell so LDLR is not recycled and returned back to cell surface
What are the 2 main strategies for controlling FH
1) inhibition of de novo synthesis of cholesterol by liver by using HMG coA reductase inhibitors aka STATINS
eg Lovastatin - has a similar structure to HMG coA so is a competitive inhibitor for the enzyme
2) reduction of dietary cholesterol absorption by intestines by using RESINS or SEQUESTRANTS like CHOLESTYRAMINE
they bind/sequester bile acid-cholesterol complexes so prevent their reabsorption by intestine
They can lower LDL and raise HDL levels
Where in the mitochondria does TCA cycle occur
On the inner membrane - which has inward folds called Cristae
In terms of energy, how does oxidation of NADH and FADH2 lead to several ATP being produced
NADH + H+ + 1/2 O2 —> NAD+ + H2O
Delta G = -223 kJmol-1
FADH2 + 1/2O2 —> FAD + H2O
Delta G = -170kJmol-1
Delta G for ATP hydrolysis is -31 so the energy released from replication of NADH and FADH2 is enough to generate several phosphoanhydride bonds
What does the electron transport chain consist of
4 membrane proteins
Complex I: NADH dehydrogenase / NADH Q oxidoreductase
Complex II: succinate dehydrogenase / succinate Q reductase
Complex III: Q cytochrome c oxidoreductase
Complex IV: cytochrome c oxidase
(Complexes 1 3 and 4 accept electrons and a origin from aqueous solution)
2 mobile electron carriers:
Coenzyme A aka ubiquinone
Cytochrome C
What is a redox couple
A substrate that can exist in both oxidised and reduced forms eg NADH / NAD+
Fe3+ / Fe2+
FADH2 / FAD
What is a redox/reduction potential and what do certain values mean
“Eo”
It is the ability of a redox couple to accept or donate electrons using hydrogen electrode as a reference
Negative value = substrate has a greater reducing power than hydrogen
Positive value = substrate has a greater oxidising power than hydrogen
What is respiratory control
The uptake of O2 by mitochondria can be controlled by the components of ATP production - Pi and ADP
This allows the body to adapt O2 consumption to actual energy requirements
What are metabolic poisons
Molecules that interfere with either the flow of electrons along the ETC or the f,ow of protons through ATP synthase so interrupt ATP synthesis
What do cyanide (CN - ) and azide (N 3-) do
They bind w high affinity to the ferrous (Fe3+) form of the haem group in the cytochrome complex (complex IV) blocking final step of ETC
What does malonate do
It resembles succinate so acts as competitive inhibitor of succinate dehydrogenase
It slows down from of electrons from succinate to ubiquinone by inhibiting oxidation of succinate to fumerate
What does rotenone do
It inhibits the transfer of electrons from complex I to ubiquinone
What does oligomycin do
It is an antibiotic produced by treptomyces that inhibits ox phos by binding to stalk of ATP synthase and therefore blocking flow of electrons through the enzyme
What does Dinitrophenol do
It is a proton ionosphere which can shuttle proteins across inner mitochondrial membrane which uncouples ox phos from atp production as prions go through DNP instead of ATP synthase
It increases metabolic rate and temp
Increase in metabolic rate induces weight loss but it is very dangerous and can kill
What does an oxygen electrode do
It measures the o2 conc in a solution that’s out in a small chamber. The base of the chamber is made of a Teflon membrane permeable to o2, underneath which is a compartment with a platinum cathode and a silver anode.
A small voltage of 0.6 volts is applied between the electrodes and oxygen diffuses through the Teflon membrane and is reduced to water at the platinum cathode
o2 + 4H+ + 4e- —> 2 H2O
The circuit is completed at the silver anode which is lowly oxidised to AgCk by the KCl electrolyte
4Ag+ + 4Cl- —> AgCl + 4e-
The resulting current is proportional to the o2 conc in the sample
How can you use oxygen electrodes to analyse mitochondrial respiration
The o2 consumption can be measured so we can see the effects of various substrates/inhibitors on the ETC
1) place suspension of mitochondria into the chamber of the o2 electrode and start recording
2) measure the baseline respiration of the suspension over a minute - the o2 in the electrode will steadily decrease over time
3) add adp to the suspension which will cause a lot of o2 to be used up
4) find the ratio of the amount of ado phosphorylated by the mitochondria to the amount of o2 consumed - the adp-oxygen index
5) after all the adp is consumed the mitochondria return to the basal respiration rate and the o2 continues to decrease until it’s all used up
4 reasons for cell communication / signalling
1) to process information - eg sensory stimuli - sight/sound
2) for self preservation - eg spinal reflexes
3) voluntary movement - eg getting from A to B
4) homeostasis - eg glucose homeostasis and thermoregulation
4 types of cell communication / signalling
Endocrine
Paracrine
Autocrine
Signalling between membrane attached proteins
Describe endocrine signalling and examples
Hormone travels within blood to act on sit at target cell
Examples
1) hypoglycaemia - glucagon is secreted by alpha cells of islet of langerhans in pancreas and travels in blood to act on liver to increase gluconeogenesis and glycogenolysis
2) adrenaline produced in adrenal glands travels to act on the trachea
3) insulin produced in pancreas travels to act on liver, muscle and adipose tissue
Describe paracrine signalling and examples
Hormone is secreted and acts on adjacent cells
Examples
1) hyperglycaemia- insulin is secreted by beta cells of islet of langerhans in pancreas and acts on adjacent alpha cells to inhibit their glucagon secretion which decreases gluconeogensis and glycogenolysis (insulin also has endocrine effects in liver)
2) osteoclast activating factors secreted by adjacent osteoblasts
3) nitric oxide made by endothelial cells in blood vessels causes vasodilation
Describe autocrine signalling and examples
Signalling molecules act on the same cell
Examples
1) T cell receptor (TCR) activation initiates a cascade of reactions within T cell and causes the cell to express interleukin 2 receptor on its surface
The activated cell also secrets IL-2 which binds to its receptor on the same cell it was secreted from as well as on adjacent cells
2)acetylcholine binds to presynaptic M2 muscarinic receptors
3) growth factors (eg TGF beta) from tumour cells can bind to and act in tumour cells to cause mitogenesis (initiate mitosis)
Describe signalling between membrane attached proteins
Interaction between plasma membrane proteins on adjacent cells
Examples
1) blood borne viruses eg hepatitis C detected in blood by APC- antigen presenting cell
APC digest the pathogen and expresses major histo-compatibility complex class II (MHC II) molecules on its surface
circulating T lymphocytes engage with MHC molecules through TCR interaction
2) HIV GP120 glycoprotein binds to CD4 reciter on T lymphocytes
3) bacterial cell wall components bind to toll like receptors on haematopoitic cells
What is the most abundant cation in plasma
Na+
Most abundant cation in cells
K+
High internal conc is neutralised by anions in the cell eg proteins, phosphorylated proteins, nucleic acids
Describe difference in Cl- conc in cell and in plasma
Cl- has higher conc in plasma than in cell
What is the main intracellular anion
Organic phosphate
Describe charge of proteins
Proteins are anions
Describe difference in pH and osmolarity between blood/plasma and cell
Inside cell is slightly more acidic than plasma - the intracellular H+ conc is double the conc in plasma
Osmolarity between blood and intracellular compartment is identical - no significant osmotic effect - expect for in regions of kidney where fluid is concentrated
What is an osmole
The number of moles of a solute that contribute to the osmotic pressure of a solution
What is osmolarity and how do you work it out
A measure of the concentration of all the solute particles in the solution
Eg NaCl - conc = 150 mol/l
^2 ions
So osmolarity = 2 x 150 = 300 mosmol/l
(Calculate from number of ions not atoms)
Eg glucose is one ion so would be 1 x conc
CaCl2 is 3 ions so would be 3 x conc
What is Tonicity, hypertonic, isotonic and hypotonic
The strength of a solution as it affects the final cell Volume - depends on both cell membrane permeability and solution composition
Hypertonic = osmolarity of impermeable solutes is greater outside the cell than inside so cell shrinks in solution
Isotonic = osmolarity of impermeable solutes outside cell is identical to inside cell so cell volume stays same
Hypertonic = osmolarity if impermeable solutes is less outside cell then inside so cell swells in solution
Why don’t cells burst if conc of impermeable solutes is much higher in them than in plasma
Na+k+ atpase maintains conc of Na-0+ lower inside cell than outside - makes membrane effectively impermeable to Na+ as actively transports Na+ back out of cell whenever it enters
Conc of impermeable solutes (can’t cross membrane) inside and outside cell balance each other to prevent too much water leaving or entering cell
Which substances can diffuse across lipid bilayer
Gases and hydrophobic molecules
Describe how tissues are preserved for transplantation
Need to be cooled to 4 degrees so that they are hypothermic in order to slow their degradation
Can be done by perfusion with cold solution through tissues arterial supply
But if tissue is cooled below 15 degrees the Na+K+ATPase will stop working. Also there is no circulation so little oxygen and little ATP produced
So Na+ ions can enter cell so water enters which causes cell to burst
Instead, use a UW solution to perfume the tissue
UW solution:
Has no Na+ or Cl- ions so they can’t enter cell
Has colloid (starch) and extra cellular impermeable molecules so water stays outside cell
Has Allopurinol and Glutathione antioxidants to protect tissue from reactive oxygen species
How do molecules traverse the endothelial layer of loom vessels
Lipid soluble molecules - pass through endothelial cells
Small water soluble molecules - pass through pores between endothelial cells
Exchange proteins - moved by vesicles
Plasma proteins - cannot enter
Balance of which 2 pressures determines solute and fluid movement into and out of blood vessel and what are normal conditions like for a capillary
Balance between COP (colloid osmotic pressure) and hydrostatic pressure
COP - due to higher conc of plasma proteins inside capillary than out - causes movement of water and other solutes into vessel
Hydrostatic pressure - due to flow of blood through vessel - pressure is greater in vessel than tissues around it kneading to tendency to push molecules through capillary pores
In normal capillary the hydrostatic pressure is slightly greater than COP so overall net leakage of molecules from capillary
What is oedema and how do lymphatics help to prevent it
Accumulation of tissue fluid due to increased permeability of blood vessel walls - eg enlarged pores
Allows plasma proteins to leave the capillary do reduces COP
Results in hydrostatic pressure»_space; COP so fluid is lost more easily from vessel
Lymphatics take up interstitial fluid and return to blood circulation - higher pressure in intertstitium than lymph vessel so fluid moves in
Also lymph nodes are bind ended (open at only one end) which also allows fluid to move in
Fluid is retuned to blood at lymph ducts in subclavian region or lymph nodes
Odeoma happens when the leakage of fluid exceeds the capacity of the ,y oh vessels to take up the fluid so fluid accumulates in interstitial space
What are 3 types of oedema
Inflammatory oedema - due to inflammatory or infectious stimuli
Hydrostatic oedema - due to high blood pressure which increases hydrostatic pressure in blood vessels and therefore increases fluid leakage
Compromised lymph system - eg in breast cancer axillary lymph node is removed - reduces fluid take up
In elephantiasis, parasitic worms block lymph vessels - prevents drainage of lymph
What is a biopsy
A small section of tissue taken from patient
1) placed in formalin solution to preserve it - as causes cross linking of the proteins
2) embedded in paraffin wax - allows it to be cut into thin slices
3) cut into v thin sections using a microtone and mounted onto glass microscope slide
4) can be stained to help identify certain cells etc
Eg heamotoxylin and eosin - detect nucleus and cytoplasmic fragments of leukocytes
Ziehl Neelsen - stains acid-fast bacteria red, used for diagnosis of TB
Biopsy can be used to see if tissue is inflamed or cancerous - USED FOR DIAGNOSIS
Usually takes 2-3 days to get results
What is a resection specimen
Piece of tissue removed during surgery - sample can be taken from it like a biopsy
USED TO LOOK AT STAGE OF DISEASE AND ITS PROGRESSION
can be donated to bio banks and used for research
What is a frozen section
Small sample taken during surgery can be examined and analysed in real time and results can be relayed back to surgeon and use to inform the surgery
Freshly taken tissue sample is frozen in a cryostat, cut, mounted onto glass slide and then can be stained like a biopsy
Results take 30 mins
Tissue must be free of preservatives like formalin and must be fresh
USED TO SEE IF TISSUE IS CANCEROUS, IS THERE CANCEROUS TISSUE LEFT, WHAT OTHER PATHOLOGICAL PROCESSES ARE GOING ON
What is a fine needle aspirate and it’s advantages and disadvantages
It is use of fine needles to suck(aspirate) cells from a lesion to be analysed as a smear
+ve : needles can penetrate inaccessible tissues and assess tissues without need for surgery
-ve : only gives view of cells and cytologies can’t comment on architecture if the whole tissue
What are conjugations
Things we at the to Fc region of antibodies to make them useful in diagnosis Enzymes Fluorescent probes Magnetic beads Drugs
Difference between direct and indirect election using antibodies
Direct: conjugate attached to primary antibody - the one that binds to the antigen
Indirect: conjugate attached to secondary antibody- the one that attaches to the primary antibody
Uses of manufactured antibodies
Blood group serology
Immunoassays - detection of hormones in blood
Immunodiagnosis - detection of IgE or antibodies from infectious diseases
What is ELISA and what is it used for
Can be used with standard curves to find levels of molecules in sample
Clinical sample adheres to plastic plate
Probe with specific antibody raised against molecule of interest
Enzyme conjugation generate coloured product
Refer to standard curve to determine precise conc of molecule in sample
What is flow cytometry
Allows detection of specific cells esp lymphocytes
There are fluorescent LDH conjugated antibodies specific for leukocyte antigens - normally surface antigens - but if different co,ours
Run a stream of single cells through laser beam which excites the flurophores
Colour of light admitted and the forward or side scatter of laser beam denotes the identity of the cell surface molecules expressed and the size/granularity if the cells
Difference between lethal and sub lethal cell injury
Lethal = causing cell death Sublethal = not amounting to cell death - may be reversible but may also progress on to cause cell death
8 causes of cell injury
Oxygen deprivation Chemical agents Physical agents Ageing Infectious agents Immunological reactions Nutritional imbalances Genetic defects
What about the injury does the cell response depend on and what do the consequences of the response depend on
Response depends on
Type of injury
Duration of injury
Severity of injury
Consequences depend on
Type of cell
Status of cell - whether it’s proliferating or not
What 4 intracellular systems are particularly vulnerable to cell injury
Cell membrane integrity
ATP synthesis
(These 2 can cause immediate damage if impaired - they also link to each other eg loss of cell membrane integrity causes loss of internal environment which impairs ability of cell to carry out metabolic reactions such as making ATP)
Protein synthesis (can begin to cause injury once the cell uses up its protein reservoir)
Integrity of genetic apparatus (effects aren’t visible until later on)
When do you get morphological changes to the cell
After cell death , after loss of function
1 = loss of function
2 = death
3 = morphological change seen
What is atrophy and examples
Shrinkage in size of cell (or organ) by loss of cell substance
Eg Dementia brain is atrophic
Eg muscle after denervation is atrophic
What is hypertrophy and examples
Increase in the size of cells and consequently an increase in the size of the organ
Can be physiological or pathological
Physiological is either by increased functional demand or specific hormone stimulation
eg = uterus during pregnancy must grow in size so the cells that make it up grow
Pathological is caused by disease
eg growth in size of heart and therefore heart cells due to hypertension or valve disorder
Hyperplasia and examples of it
Increase in the number of cells in an organ
Physiological = hormonal or compensatory
Eg proliferative endometrium - during menstrual cycle after shedding of lining in endometrium, more cells are formed to replace the lining
Pathological = excessive/ abnormal hormonal or growth factor stimulation
Eg cancer
Metaplasia and examples of it
A reversible change where one adult cell type is replaced by another
Physiological
Eg the endocervix is lined with columnar epithelial cells. The cervix opens up during pregnancy so some of the cells which were inside the endocervix get exposed to the outside. The vaginal pH makes them change to squamous epithelial cells. After pregnancy when cervix closes up and they go back into endocervix they change to columnar cells again
Pathological
Eg Barrett’s Oesophagus. Cells lining oesophagus are squamous epithelial cells and those lining stomach are columnar. Acid reflux can cause cells in oesophagus to change to columnar. After weight loss/taking antacids and consequent reduction in acid reflux, the cells can change back
Dysplasia and examples
Precancerous cells that show the genetic and cytological features or malignancy but don’t invade underlying tissue
Signs = big/misshaped nuclei, increased mitosis
Can happen in barretts oesophagus - first metaplasia then dysplasia
What are the 2 light microscope changes associated with reversible cell injury
Both are degenerative changes
1) Cellular swelling eg ballooning degeneration: cytoskeleton damage causes protein accumulation in cells causing them to swell
2) fatty change eg big white spots seen in hepatocytes after excessive alcohol consumption. But if you stop drinking they can go away
What is necrosis and what are the 4 types
Confluent cell death associated with inflammation
1) coagulative necrosis: cells become solidified and fixed in place after they die, not broken down by enzymes
2) liquefactive necrosis: cells are broken down by enzymes eg in brain damage this happens
3) caseous necrosis: “cheesy” - cells become structureless and oozy
4) fat necrosis eg in acute pancreatitis: enzymes like lipase become activated in pancreas when they should be activated in duodenum. Lipase starts to break down fat in pancreatic tissue
Which type of necrosis happens in myocardial infarct
Coagulative necrosis
What type of necrosis happens in brain damage
Liquefactive necrosis
What type of necrosis happens in pulmonary TB
Caseous necrosis
What is necroptosis
Programmed cell death associated with inflammation eg in viral infections
What is apoptosis
Programmed cell death if individual cells
Causes of apoptosis
Embryogenesis
Auto reactive T cells in thymus
Hormone dependant physiological involution
Cell deletion is proliferating populations
Injurious stimuli that cause irreversible DNA damage that in turn cause cell suicide
Differences between apoptosis and necrosis
- necrosis is associated with inflammation but apoptosis is not
- usually see sheets of cells dying in necrosis but only individual cells dying in apoptosis
- apoptosis is physiological (but may be pathological eg in cancers) but necrosis is only usually pathological
- apoptosis is active energy reindent process so cells maintain membranes and are packed into apoptotic bodies - necrosis isn’t active
- in necrosis, bits of cells membrane break off and contents of cell eg enzymes are released
but in apoptosis, bits of the cell bleb off to form apoptotic bodies with fragments of cytoplasm/nucleus etc in them - but cell membrane for each of them remains intact. These bodies are destroyed by macrophages - very controlled process
How many ATP do NADH amd FADH2 produce
rexodation of NADH produces 3 ATP
reoxidation of FADH2 produces 2ATP
