IMMS Flashcards
DNA structure
DNA double helix coils around nucleosomes, coils again into supercoils and then condenses further into chromosomes
Stain for chromosomes
Giemsa: G banding for complementary chromosome pair identification
Quinacrine: Q banding for individual light and dark bands on chromosome
Cell cycle
Interphase:
G1 growth phase
Synthesis phase
G2 growth phase
Mitosis
G0
Cells not in cell cycle (e.g. liver cells)
Cells that do not undergo mitosis
Myocyte cells in heart and neurones in the brain
Cells constantly in cell cycle
bone marrow cells
gut cells
What happens during S-phase?
DNA replication and centrosome replication
What occurs during prophase?
chromatin condenses into chromosomes and centrosomes nucleate microtubules which move to opposite poles of the nucleus
What occurs during prometaphase?
Nuclear membrane breaks down, microtubules invade nuclear space and chromatids attach to microtubules.
What occurs during metaphase?
Chromosomes align along equatorial place (metaphase plate)
What occurs during anaphase?
The sister chromatids separate, and chromatids are pulled to opposite poles of the cell
What occurs during telophase?
Nuclear membranes reform, chromosomes unfold into chromatin, and cytokinesis begins
What occurs during cyotkinesis?
2 identical daughter cells are produced
Clinical relevance of mitosis (epithelium)
One of the indicators of precancerous lesions is mitotic figures at multiple levels in a microscope sample. No mitotic figures in areas where it shouldn’t be means that the tumour is benign. The number of mitotic figures helps grade the severity of the malignant tumours.
Mitosis + drugs
Chemotherapeutic agents such as taxol and vinca alkaloids (vinblastine, vincristine) work by preventing the formation of the mitotic spindle.
What is ispinesib?
Monoclonal antibody therapy targeting spindle poles
Colchicine-like drugs:
chemotherapeutic agents which arrest mitosis in anaphase and give rise to abnormal mitotic figures where the chromosomes form a circle.
Process of meiosis summary
- interphase
- prophase 1 (crossing over)
- metaphase 1
- anaphase 1
- telophase 1
- prophase 2
- metaphase 2
- anaphase 2
- telophase 2
- cytokinesis into 4 zygote cells
Crossing over
Independent sorting of genes during prophase 1 of meiosis
Production of sperm
Primordial germ cells undergo a lot of mitosis to produce spermatogonia (precursor stem cells).
Meiotic divisions commence at puberty.
Production of eggs
Primordial germ cells undergo 30 mitoses to produce oogonia, the primitive gametes of women. Oogonia enter prophase of meoisis 1 by 8th month of intrauterine life, at which point the process is suspended. Cells enter ovulation 10-50 years later.
Meiosis 2 is only completed if fertilisation occurs.
Non-disjunction
Failure of chromosome pairs to separate in Meiosis I, or sister chromatids to separate properly in Meiosis II.
Monosomy
only one copy of chromosome
Trisomy
extra copy of chromosome
3 causes of disease
genetic
multifactorial
environmental
Classification of genetic disease
Chromosomal Mendellian (autosomal dominant or recessive, X-Linked) Non traditional (mitochondrial, imprinting, mosiacism)
Autosomal dominant inheritance definition, onset and example
Disease which manifests in the heterozygous state.
Adult onset.
Example: Huntingdon’s disease.
Autosomal recessive inheritance definition and example
Disease which manifests in homozygous recessive state i.e. no functioning copy of gene for the disease.
Example: sickle cell disease
X-linked recessive inheritance
caused by pathogenic variants in genes on the X chromosome.
Early onset in childhood.
Example: cystic fibrosis.
Mitochondrial DNA
Mitochondria have their own ring shaped mitochondrial chromosome and mitochondrial genome which code for m. enzymes.
Mitochondrial chromosome mutations
severe conditions such as epilepsy and brain conditions which can manifest from birth/early childhood
Imprinting
For some genes, only 1 out of the two alleles is active, the other is inactive. For particular genes, it is always the paternal or maternal allele which is inactivated. For example, growth genes inactivated by maternal allele keep fetus small to increase chance of survival.
Spinal bifidia
Multifactorial.
MTHFR gene function reduced; does not activate enough folic acid (needed for DNA synthesis and repair).
If the peri conceptual folic acid is reduced there is more likelihood child will get the congenital birth defect. An environmental reason for lack of folic acid is excessive alcohol consumption.
Familial Hypercholesterolaemia and Coronary Heart Disease
High levels of Low Density Lipoproteins are associated with increased risk of heart disease. Some people have a pathogenic variant in the gene for LDL receptors and have an increased genetic predisposition for coronary heart disease.
What doesn’t water interact with?
Non-polar substances
Lipids
Aromatic groups
Hydrophobic compounds
What is a monosaccharide?
Chain of carbons, hydroxyl groups and one carbonyl (C=O) group.
An aldose monosaccharide has an aldehyde.
A ketose has a ketone.
Sugar derivatives
Aminosugars (containing an amino group; glucosamine)
Alcohol-sugars (sorbitol)
Phosphorylated (containing phosphate groups; glucose-6-phosphate)
Sulphated (containing sulphate groups; e.g. Heparin)
Glycosidic bond
The hydroxyl group of a monosaccharide can react with an OH or NH group to form glycosides.
O-glycosidic bonds form disaccharides, oligosaccharides and polysaccharides.
N-glycosidic bonds are found in nucleotides and DNA.
Disaccharides
Disaccharides contain 2 monosaccharides joined by a glycosidic bond.
Oligosaccharides
Oligosaccharides contain 3-12 monosaccharides. They are the products of digestion of polysaccharies, or part of complex proteins/lipids.
Polysaccharides
Formed by thousands of monosaccharides joined by glycosidic bonds.
Starch
Glycogen
Lipids: Fatty Acids
Straight C chains with a methyl group and a carboxyl group at the end.
Melting point decreases with the degree of unsaturation. (number of C=C).
Phosphoacylglycerols
Derive from phosphatidic acid.
Formed from fatty acids esterified to glycerol and phosphorylated at C3.
Sphingolipids
Derive from ceramide (serine, plamitic aci and another fatty acid)
Eicosanoids
Synthesised from 20 C atoms
Acids with 3,4,5 double bonds
Nucleotides
Nitrogenous base
Sugar
Phosphate
Amino acids
Amino group
Carboxyl group
R side chain
Charge of amino acid
Determined by all three components and changes with the pH of the environment
Polarity of amino acid
Often determined by side chain (R).
Hydrophillicity (polarity) or hydrophobicity (non polarity).
Non polar amino acids
Glycine Alanine Proline Valine Leucine Isoleucine
Polar amino acids
Methionine Cysteine Glutamine Serine Threonine
Negatively charged AA
Aspartate
Glutamate
Positively charged AA
Arginine
Lysine
Histidine
Peptide bond
Proteins are formed by amino acids linked by peptide bonds
Examples of protein structure-function relationship
Immunoglobins Fibrous protein: collagen Enzymes Channel and carrier proteins Receptor proteins Neurotransmitters
Properties of peptide bonds
Very stable
Cleaved by proteolytic enzymes (proteases or peptidases)
Partial double bond
Flexibility around C atoms not involved in bond allowing multiple conformations. However there is usually one preferred native confirmation, determined mainly by the type of side chains and their sequence in the polypeptide.
Van der Waals forces
Weak attractive interactions between atoms due to fluctuating electrical charges. Only important when two macromolecular surfaces fit closely in shape.
Hydrogen bonds
Interactions between dipoles, involving a hydrogen and an oxygen/nitrogen. The partial negative charges on electronegative atoms such as O and N are bound to H, which then has a partial positive charge. These partial charges allow weak attractive interactions between amino acid side chains, main-chain oxygen and nitrogen and water.
Hydrophobic forces
Uncharged and non-polar side chains are poorly soluble in water and are repelled by water.
These hydrophobic side chains tend to form tightly packed cores in the interior of proteins, excluding water molecules. This attraction is the hydrophobic force.
Ionic bonds
Occur between fully or partially charged groups. Weakened in aqueous systems by shielding by water molecules and other ions in solution.
Disulphide bonds
Covalent bonding between side chains of cysteine residues.
Primary structure of proteins
Linear sequence of amino acids linked by peptide bonds. The primary structure determines its 3D conformation.
Secondary structure - the alpha helix
Hydrogen bonds between each carbonyl group and the hydrogen attached to the nitrogen which is 4 amino acids along the chain.
Secondary structure - the beta-sheet
Formed by H bonds between linear regions of polypeptide chains.
Chains from two proteins, or the same protein.
Paralell or antiparallel chains, pleated or not.
Tertiary structure
Overall 3D structure of protein.
Forces involved include electrostatic, hydrophobicity, H-bonds, and covalent bonds.
Can be affected by pH and temperature.
Quaternary stucture
3D structure of a protein composed of multiple subunits.
Same non-covalent interactions as tertiary structures.
Porphyrin Ring
(Haemoglobin)
At the core of a haemoglobin molecule is a porphyrin ring which holds an iron atom. An iron containing porphyrin is termed a ‘heme’. The iron atom is the site of oxygen binding.
Factors influencing haemoglobin saturation
Temperature
[H+]
PCO2
An increase in these factors will modify the structure of haemoglobin and thus alter its affinity for oxygen.
Factors influencing haemoglobin saturation
Temperature
[H+]
PCO2
An increase in these factors will modify the structure of haemoglobin and thus alter its affinity for oxygen. When these factors are increased Hb affinity for oxygen decreases and oxygen is unloaded more readily in the blood.
Decrease is vice-versa.
Immunoglobulins (Antibodies)
Antibodies are produced to bind antigens, typically toxins or proteins on the surface of microbial agents. These targets are consequently labelled for destruction by cells of the immune system or by lysis through the complement system.
Immunoglobulin structure
The immunoglobulin fold structure of antibodies comprises a supporting scaffold (framework regions) that serves to display highly variable loops of complementarity determining regions (CDRs).
The diverse nature of CDR regions enable a range of reversible bonding effects to act between antibody and antigen.
Antigen recognition
The close proximity of the antibody CDR regions and the antigen surface allows the combination of relatively weak interactions to produce a strong binding surface. The CDR loops have a sequence of amino acids that “complement” the surace of the antigen.
What is the name for the portion of the the antigen which is bound to an antibody?
epitope
Which direction is RNA read in?
5’ => 3’
3 characteristics of genetic code
- Degenerate (many amino acids specified by more than one codon)
- Universal (all organisms use same code)
- Non-overlapping (each nucleotide read only once)
Factors turning off gene expression
- activation of repressors
- each step of RNA transcription or processing finds no longer actively produced transcription and processing proteins
- complexes do not form anymore for lack of phosphorylation
- enzymes no longer activated
- RNA stability
Prokaryotic DNA
No nuclear membrane
DNA arranged in a single chromosome
In E.Coli PDNA is circular
Eukaryotic DNA
DNA in the nucleus bound to proteins (chromatin complex)
Eukaryotic DNA
DNA in the nucleus bound to proteins (chromatin complex)
Antiparallel DNA strands
One strand goes 5’ to 3’ and the other strand is read 3’ to 5’.
What is heterochromatin?
DNA which has condensed and is out of action. The histones have changed and methylation renders DNA inactive.
Enzymes involved with DNA and their function
polymerase: reads 3’ to 5’ and prints 5’ to 3’
helicase: opens helix
ligase: joins DNA together
nuclease: digests elements
primase: synthesises primers
topoisomerase: unwinds helix and relieves supercoiling
Editing function of DNA polymerase
It detecs the incorrect insertion of a base and will excise it and repeat
How is DNA damaged??
chemical damage
radiation damage
spontaneous insertion of incorrect bases during replication
What is benzopyrene?
Product of incomplete combustionof hydrocarbons. It is a DNA adduct, meaning it reacts with bases to form a bulky group that disrupts replication.
Effect of ionising radiation
damage to bases
causes breaks in the phosphate backbone
Effect of UV
Damages bases
In particular, causes the formation of thymine dimers
What is the most popular type of genetic testing in the NHS?
Multi-gene panels
What is the most effective genetic test?
Whole exome/genome sequencing
Why does targeted mutation anaylsis not sequence the whole genome?
It is done with a known gene/mutation in mind
What is a suitable genetic test for cystic fibrosis and why?
Single gene sequencing because genetic diseases such as cystic fibrosis are caused by a single gene.
What is a suitable genetic test for a child with a epilepsy?
Chromosomal microarray analysis, which picks up microdeletions.
Sanger sequencing
Uses PCR to amplify regions of interest followed by sequencing of products. There is a single start point (primer).
Benefits of Sanger sequencing
Useful for single gene testing
Very accurate
Downsides of Sanger sequencing
slow
expensive
Next-Generation sequencing
Rapid sequencing of targeted gene panels. Multi gene panels so whole genome can be sequenced.
Method = massively parallel sequencing.
Benefits of NG sequencing
Fast
Can sequence a whole human genome in one day
Downsides of NG sequencing
Huge amounts of raw data to interpret
expensive
Only moderately accurate
Variants can either be …
Pathogenic variants, varients of unknown significance, normal variants
Types of mutation:
nonsense
frame shift
spice site
missense
What cell types are used in genetic testing?
Blood (T-lymphocytes) Skin/umbilical cord Bone marrow Solid tumour Amniotic fluid/Chorionic villus
Trisomy
Extra chromosome
Down’s Syndrome
Edward’s Syndrome
Patau Syndrome
Monosomy
Single chromosome
Turner syndrome
Polyploidy
Three copies of each chromosome
Sex Chromosome abnormalities
Klinefelter Syndrome
47, XXY in males
(slower development, reproductive issues, feminine body shape)
Deletion of 15q
Prader Willi
Angelman Syndrome
F.I.S.H
Fluorescence in situ hybridisation using DNA probes labelled with fluorophores. They are hybridized directly to the chromosome preparation or interphase nuclei.
Nonsense mutation
If a mutation puts a STOP codon earlier than it needs to be, disrupting gene function. A nonsense mutation takes place when an out of frame deletion produces a stop codon either at the deletion site or further along.
Splice-site variant
A SSV affects the accurate removal of an intron. Intron could be translated into protein (retained) which causes problems with protein structure and function. It can cause the presence or removal of an exon where it should not be.
Mis-sense variant
single base substitution which changes the type of amino acid in the protein. This may or may not be pathogenic i.e. it may be a polymorphism of no function.
Allelic heterogeneity
lots of different variants in one gene
Dominant-negative variants
where the protein from the variant allele interferes with the protein from the normal allele.
Multifactorial inheritance definition
Disease due to a combination of genetic and environmental factors
How do you identify that a condition has a genetic component?
By clinical observation: family studies, twin studies, adoption studies
Family Studies
compare the incidence of a disease amongst the relatives of an affected individual with the general population.
In a multifactorial condition, the risk of the condition in relatives of an affected individual is —– than in the general population.
higher
Twin studies
compare genetically identical (monozygotic) with genetically non-identical (dizygotic) twins
How to calculate the concordance rate in twin studies?
The percentage of twin pairs in the study that both have the condition.
How to calculate the concordance rate in twin studies?
The percentage of twin pairs in the study that both have the condition.
What effect would a genetic component have on concordance rate?
If the condition had a genetic component you would expect the concordance rate to be higher in monozygotic twins than dizygotic.
Adoption studies
Adopted children of a biological parent with a multifactorial condition have a high risk of developing the disease.
What is the definition of hereditability?
The proportion of the etiology (cause) that can be ascribed to genetic factors as opposed to environmental factors.
How is hereditability expressed?
As a proportion of 1/as a percentage.
Characteristics of multifactorial inheritance
The incidence of the condition is greatest amongst relatives of the most severely affected patients. The risk is greatest for the first degree relatives and decreases rapidly in more distant relatives. If there is more than one affected close relative then the risks for other relatives are increased.
Sex and multifactorial inheritance
If the condition is more common in one particular sex, then relatives of an affected individual of the less frequently affected sex will be at higher risk than relatives of an affected individual of the more frequently affected sex.
What is a neural tube defect?
Defective closure of the developing neural tube during the first month of embryonic life
What is the genetic component of neural tube defects?
About 10% of cases can be attributed to mutations in the MTHFR gene which leads to decreased plasma folate levels
What is the environmental component of neural tube defects?
Environmental factors include poor socioeconomic status, multiparity and valproate. Periconceptional folate supplementation reduces recurrence risk to about 1%.
Environmental agents acting on embryogenesis
Drugs and chemicals (thalidomide, alcohol) Maternal infections (rubella) Physical agents (radiation) Maternal illness (diabetes)
Definition of metabolism
Metabolism refers to the sum of the chemical reactions that take place within each cell of a living organism
Dietary components are metabolised in cells through 4 main pathways
Biosynthetic
Fuel storage
Oxidative process
Waste disposal
Definition of anabolic metabolic process
Synthesis of larger molecules from smaller components
biosynthetic, fuel storage
Definition of catabolic metabolic process
Break down of larger into smaller
oxidative
What is a cofactor?
A cofactor is a non-protein chemical compound/metallic ion that is required for an enzyme’s activity as a catalyst (“helper molecules”)
Adipose tissue
85% fat (mostly triglycerides)
Main function = storage of energy rich molecules
Function of the liver
Gluconeogenesis (production of glucose for catabolic reactions)
Removal of toxins
Metabolism of xenobiotics to get them into a state ready for the disposal of toxins from the body, usually urine.
The Cori Cycle brief description
In the Cori Cycle, lactate produced by the muscles is converted to glucose by the liver, and is fed back to the muscles.
Why does the Cori cycle take place?
It is a metabolic route to get rid of lactate produced in the anaerobic breakdown of glucose.
What happens to glucose generated in the Cori Cycle if the muscle activity stops
Glucose undergoes glycogenesis to replenish the glycogen stored in the muscles.
Limitations of the Cori Cycle
Liver uses up 6 molecules of ATP to carry out gluconeogenesis but glycolysis only makes 2 mols of ATP.
What is the name for the accumulation of excess lactic acid in the system?
Lactic acidosis
This brings down the pH of the blood, which can lead to tissue damage.
3 main dietary energy sources
carbohydrates
lipids
proteins
Storage of fat, carbohydrates and proteins?
Fat = adipose tissue Carb = glycogen in liver and muscles Protein = muscle
Which energy source has the most energy per gram
lipid
BMR stands for:
basal metabolic rate
What is the BMR?
A measure of the energy required to maintain non-exercise bodily functions (e.g. respiration, contraction of cardiac muscles).
What is the rate limiter of glycolysis
phosphofructokinase
What is the rate limiter of glycolysis
phosphofructokinase
Hydrolysis reaction of ATP
ATP -> ADP + Pi + H+
Glycolysis step 1: glucose is converted to ..
glucose-6-phosphate
Which enzyme converts glucose into glucose-6-phosphate?
hexokinase (phosphorylation of glucose by ATP)
glucokinase (liver)
Glycolysis step 2: glucose-6-phosphate is converted to …
Fructose-6-phosphate
Which enzyme converts glucose-6-phosphate into fructose-6-phosphate
phosphoglucose isomerase
Glycolisis step 3: fructose-6-phosphate is converted to..
fructose-1,6-biphosphate
Which enzyme converts fructose-6-phosphate into fructose-1,6-biphosphate
phosphofructokinase-1
Glycolysis step 4: fructose-1,6-biphosphate is converted into …
Glyceraldehyde-3-phosphate
Dihydroxyacetone phosphate
Which enzyme converts fructose-1,6-biphosphate into the two triose phosphates?
aldolase
Glycolysis step 5: dihydroxyacetone phosphate is converted into …
Glyceraldehyde-3-phosphate
Which enzyme converts dihydroxyacetone phosphate into glyceraldehyde-3-phosphate?
triose phosphate isomerase
Glycolysis step 6: two glyceraldehyde-3-phosphate molecules are converted into …
two 1,3-biphosphoglycerate molecules
Glycolysis step 6: two glyceraldehyde-3-phosphate molecules are converted into …
two 1,3-biphosphoglycerate molecules
Which enzyme converts two glyceraldehyde-3-phosphate molecules into two 1,3-biphosphoglycerate molecules
glyceraldehyde-3-phopshate dehydrogenase
Glycolysis step 7: two 1,3-biphosphoglycerate molecules are converted into …
two 3-phosphoglycerate molecules
Which enzyme converts two 1,3-biphosphoglycerate molecules into two 3-phosphoglycerate molecules
phosphoglycerate kinase
Glycolysis step 8: two 3-phosphoglycerate molecules are converted into …
two 2-phosphoglycerate molecules
Which enzyme converts two 3-phosphoglycerate molecules into two 2-phosphoglycerate molecules?
phosphoglyceromutase
Glycolysis step 9: two 2-phosphoglycerate molecules are converted into …
two phosphoenolpyruvate molecules
Which enzyme converts two 2-phosphoglycerate molecules into two phosphoenolpyruvate molecules?
enolase
Glycolysis step 10: two phosphoenolpyruvate molecules are converted into …
2 pyruvate molecules
Which enzyme converts phosphoenolpyruvate into pyruvate?
pyruvate kinase
Which glycolysis reactions require the conversion of ATP into ADP?
glucose –> glucose-6-p
fructose-6-p –> fructose-6-bip
Which glycolysis reactions require the conversion of ADP into ATP?
1,3-biphosphoglycerate –> 3-phosphoglycerate
phosphoenolpyruvate –> pyruvate
Which glycolysis reaction requires an oxidation reaction?
Glyceraldehyde-3-phosphate is oxidised by NAD+.
Glycolysis symmary
Glucose + 2NAD+ + 2Pi + 2ADP –> 2 Pyruvate + 2NADH + 2H+ + 2ATP + 2H2O
What is pyruvate converted to in anaerobic conditions?
Lactate
Pyruvate -> Lactate reaction
glucose + 2ADP + 2Pi –> 2 lactate + 2ATP + 2H2O + 2H+
Which tissues are reliant on anaerobic glycolysis?
Mature erythrocytes, lymphocutes, white blood cells, renal medulla, tissues of the eye, skeletal muscle, skin
What are the various functions of glycolysis?
Glycolysis provides ATP, generates precursors for biosynthesis because the intermediates can be converted to ribose-5-phosphate (i.e. nucleotides) or amino acids like serine, glycine, cytesine.
Pyruvate is transaminated to alanine.
Pyruvate is a substrate for fatty acid synthesis.
Glycerol-3-P is backbone of triglycerides.
Glycolysis when there is low [ATP]
When there is a low concentration of ATP and then the concentration is increased, the reaction will increase because ATP acts a substrate.
Glycolysis when there is high [ATP]
When there is a high concentration of ATP, increasing ATP concentration will result in a decreased in reaction speed because the catalytic site of PFK-1 is saturated and ATP causes allosteric inhibition which is opposed by AMP.
Citrate in regulating rate of glycolysis
Citrate is an allosteric inhibitor of PFK-1.
What is the reaction for the conversion of pyruvate to acetyl-CoA
pyruvate + CoA + NAD+ —> acetyl-CoA + CO2 + NADH + H+
What is malnutrition?
A state of nutrition with a deficiency, excess or imbalance of energy, protein or other nutrients, causing measurable adverse effects.
Adverse side effects are on tissue, body size, body function and clinical outcome.
Re-feeding syndrome risks
Re-distribution of electrolytes (phosphate, potassium, magnesium etc) due to insulin causing irregular concentrations in blood stream.
Body switch back to carbohydrate use as the main fuel which requires phosphate and thiamine, that can be used pretty quickly.
Essential fatty acids
(acids that the body cannot synthesize)
omega-3
omega-6
Essential vitamins:
Vitamin C (ascorbic acid) which is useful for heat labile, collagen synthesis, iron absorption, antioxidant
Vitamin B12 useful for protein synthesis, DNA synthesis, folate regenration, folic acid synthesis, energy
Vitamin B1 / thiamine useful for energy production
Vitamin D increases calcium and phosphorous absorption, making teeth and bones stronger and healthier
Overall Krebs reaction
acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O –> 2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+
Reaction for conversion of pyruvate to acetyl-CoA
pyruvate + CoA + NAH+ –> Acetyl-CoA + CO2 + NADH + H+
Enzyme action in conversion of pyruvate to acetyl-CoA
pyruvate dehydrogenase
pyruvate dehydrogenase multienzyme complex (3 enzymes + 5 co-factors) within the mitochondrial matrix
Inhibited by high concentrations of acetyl-Coa and NADH.
Inactivated by phosphorylation
Activated by phosphate removal
1: acetyl-CoA –> …
citrate
1: acetyl-CoA –> citrate enzyme
citrate synthase
1: acetyl-CoA –> citrate cofactors and products
cofactors: +h2O
products = CoA
2:citrate –> …
isocitrate
2:citrate –> isocitrate enzyme
aconitase
3: isocitrase -> …
alpha-ketoglutarate
3: isocitrase –> alpha-ketoglutarate enzyme
isocitrate dehydrogenase
3: isocitrase –> alpha-ketoglutarate products
products = NADH + H+
and CO2
4: alpha-ketoglutarate –> ..
succinyl-CoA
4: alpha-ketoglutarate –> succinyl-CoA enzyme
alpha-k… dehydrogenase
4: alpha-ketoglutarate –> succinyl-CoA c+p
cofactors: CoA
products = NADH + H+
and CO2
5: succinyl-CoA –> …
succinate
5: succinyl-CoA –> succinate enzyme
succinyl-CoA thiokinase
5: succinyl-CoA –> succinate CF+P
CF: Pi (GDP –> GTP)
P = CoA
6: succinate –> …
fumarate
6: succinate –> fumarate enzyme
succinate dehydrogenase
6: succinate –> fumarate CF + P
P = FADH2
7: fumarate –> …
malate
7; fumarate –> malate enzyme
fumarase
7: fumarate –> malate CF
H2O
7: malate –> …
oxaloacetate
7: malate –> oxaloacetate enzyme
malate dehydrogenase
7: malate –> oxaloacetate P
P = NADH + H+
Citrate synthase regulation
ATP and NADH allosterically inhibit citrate synthase by reducing the affinity of citrate synthase for its substrates. This reduces the rate of reaction of Krebs.
Succinyl CoA competetively inhibits citrate synthase (competetive feedback of Krebs’ Cycle).
Oxidative phosphorylation causes a decrease in the NADH: NAD+ ratio. This increases conversion of malate to oxaloacetate which is a substrate for citrate synthase.
Increased [citrate] inhibits citrate synthase, reduces speed of cycle
Isocitrate Dehydrogenase Regulation
Isocitrate DH activation decreases [citrate] which increases the citrate synthase reaction rate.
ICDH is made up of subunits. Isocitrate binds IDCH subunit leading to a conformational change in other IDCH subunits to active form.
ICDH is inactivated by phosphorylation when concentrations of pyruvate are low.
ADP is an ICDH allosteric activator and binds to it when all ICDH subunits are in active form.
ICDH inhibited by high [ATP] and [NADH].
Α-ketoglutarate DH Regulation:
Inhibited by its products NADH and succinyl-CoA as well as GTP, ATP, and reactive oxygen species.
Activated by Ca2+ which may be useful in generating ATP during intense muscle exercise due to increased calcium flux.
Fatty acid components/structure
Hydrophillic carboxylate acid head group with hydrophobic aliphatic tail.
Saturated C-C or unsaturated C=C
Lipid absorption and transport
Fatty acids are not soluble.
The gall blader secretes bile salts which emulsify fatsinto micelles (phospholipids encapsulating the oils) within the small intestine. Intestinal lipases hydrolyse triglycerols. Fatty acids and other products are taken up by the intestinal mucosa and converted into triglycerols. The triglycerols are in corporated, with cholesterol and apoproteins, into chylomicrons. Chylomicrons move through the lympathic system and blood stream to tissues. Lipoprotein lipase, activated by apoC-II in the capillaries, releases fatty acids and glycerol. FA enter cells and are oxidised as fuels or re-esterified for storage.
Fatty Acid activation
Fatty acid => Acyl Adenylate => Acyl-Coa (using acyl-CoA synthase)
Movement of fatty acids into mitochondria
If Acyl-Coa has less than 12 carbons can diffuse through mitochondrial membrane.
More dietary fatty acids have more than 14 carbons so are taken through the mitochondrial membrane using the carnitine shuttle.
Fatty acids must be activated in the cytoplasms before they can be oxidised in the mitochondria.
Utilisation of Acetyl-CoA
- Krebs Cycle (normal conditions)
- converted into ketones (ketogenesis)
Ketogenesis overview
During high rates of FA oxidation, primarily in the liver, large amounts of acetyl-CoA are generated. These exceed the capacity of Krebs and one result is ketogenesis.
Ketone bodies (acetone, acetoacetate and beta-hydroxybutyrate) are synthesised in the mitochondrial matrix from acetyl-CoA generated from beta-oxidation of fatty acids.
Ketogenesis process
two Acetyl CoA ’s are converted by the enzyme thiolase (used in b-oxidation), back to Acetoacetyl CoA. Under the action of two further enzymes the Acetoacetyl CoA can be converted to Acetoacetate, acetoacetate can then enter the blood or it can be converted to b-hydroxybutyrate under the action of another enzyme, which can then enter the blood. When the level of glycogen in the liver is high the production of b-hydroxybutyrate increases. Another fate of the acetoacetate is that it can spontaneously be converted to acetone. Since acetone is volatile it is rapidly expired by the lungs
Utilisation of acetoacetate and beta-hydroxybutyrate
Acetoacetate & b-hydroxybutyrate can both be oxidised as fuels in most tissues, including skeletal muscle (see diagram).
Cells transport the acetoacetate and b-hydroxybutyrate from the blood into the cytosol then into the mitochondrial matrix. Here b-hydroxybutyrate is oxidised back to acetoacetate.
Acetoacetate can then be activated to Acetoacetyl CoA which can then be cleaved into two molecules of Acetyl CoA by the thiolase enzyme (same enzyme involved in b-oxidation).
Then the Acetyl CoA can be used in the Kreb’s cycle to produce ATP
Why does the liver not have the enzyme succinyl CoA: acetoacetate CoA in sufficient concentrations?
This enzyme is used to convert acetoacetate to acetoacetyl CoA.
As a result of the liver not having this enzyme, it CANNOT utilise ketone bodies as fuel. This ensures that extrahepatic tissues have access to ketone bodies as a fuel source during starvation.
Regulation of Ketogenesis (glycerol-3-phosphate conc.)
A high [glycerol-3-phosphate] results in triglyceride production, whilst a low level results in increased ketone body production.
Regulation of Ketogenesis (ATP demand and ATP levels)
High demand for ATP means that most acetyl-CoA will be further oxidised in TCA cycle.
High ATP levels means that more ketones are produced.
Regulation of ketogenesis (glucagon and insulin levels)
Fat oxidation is dependent on theamount of glucagon (activation) and insulin (inhibition) present.
Insulin is produced when there is too much glucose in the blood, removes glucose and stores it in the liver.
Glucagon is produced when there is not enough glucose in the blood. It is converted to glucose.
Clinical significance of ketogenesis
Under normal feeding and normal physiological state, ketone body production occurs at a relatively slow rate.
Carbohydrate shortages cause the liver to increase ketone body production from acetyl CoA.
In the early stages of starvation, the last remnants of fat are oxidised, the heart and skeletal muscle will consume primarily ketone bodies in order to PRESERVE GLUCOSE FOR USE BY THE BRAIN - when glucose in the brain decreases then the brain CAN use ketone bodies for energy
Ketoacidosis (clinical)
Occurs in insulin-dependent diabetics when dose is inadequate or because of increased insulin requirement (infection, trauma, acute illness).
Is often the presenting feature in newly diagnosed type 1 diabetics.
Occurs in chronic alcohol abuse or starvation.
Patients present with hyperventilation and vomiting.
Diabetic Ketoacidosis overview of the three routes
Insulin deficiency leading to either hyperglycaemia or increased production of acetoacetate and beta-hydroxybutyrate.
ID»_space; inhibition of glycolysis and stimulation of gluconeogenesis»_space; hyperglycaemia.
ID»_space; glycogen breakdown and inhibition of glycogen synthesis»_space; hyperglycaemia
ID»_space; increasing lipolysis (increased free fatty acids)»_space; increased production of acetoacetate and beta-hydroxybutyrate
Alcoholic Ketoacidosis
High blood ethanol concentration and depleted protein and carbohydrate stores lead to impaired gluconeogenesis and decreased insulin and increased glucagon production. As a result there is increased lipolysis (increased free FAs). This leads to increased ketone production.
Consequences of Ketoacidosis
Ketones are relatively strong acids (pH/pKa ~ 3.5). Therefore excessive ketone levels lower the blood pH. This impairs the ability of haemoglobin to bind to oxygen.
Ketoacidosis treatment
Sliding scale of insulin
IV fluid Hydration (10%dextrose, 0.9% saline)
Monitor fluid balance closely
40 mmol potassium
Pabrinex (injection containing vitamin C, B1, B2, B3, B6)
What are some impermeable substances which require transport proteins and energy to pass through membrane?
Large uncharged polar molecules
Ions
Charged polar molecules such as glucose
Features of membrane channels
Narrow aqueous pores from intracellular to extraceullular Selective for size and charge Passive May be voltage or ligand gated Usually ions or water (aquaporins)
Features of carriers in membrane
Specific binding site
Carrier undergoes a conformational change
Active or passive
3 types of carrier protein
Uniport (single substance/simple diffusion)
Symport (two substances in the same direction)
Antiport (two substances in the opposite direction)
Driving forces behind movement across membranes
Chemical
Electrical
Electrochemical
Based on gradient
Chemical driving force
Based on concentration gradient
Electrical/membrane potential driving force
Based on the distribution of charges across the membrane
Electrical/membrane potential driving force
Based on the distribution of charges across the membrane.
Only charged substances such as sodium and potassium ions are involved.
Force depends on:
-size of membrane potential
- charge of the ion
Electrochemical driving force
Combines the chemical and electrical forces. Net distribution is equal to the sum of chemical and electrical forces.
Only charged substances
When passive diffusion goes wrong:
GLUT1 is a carrier protein present in many cells, including in the brain, where it transports glucose across the blood-brain barrier via facilitated diffusion. GLUT1 Deficiency syndrome is a very rare disorder where there is a mutation in the gene that encodes GLUT1. Less functional GLUT1 reduces the amount of glucose available to brain cells. Symptoms include seizures, microephaly, developmental delay.
Sodium-potassium pump in resting state
Open on the internal side of the cell
Has an affinity for sodium ions.
When active transport goes wrong
ATP7B protein is a Cu2+-ATPase present in the liver that transports copper into bile. Wilson’s Disease is a rare disorder where mutations occur in ATP7B gene resulting in deposition of copper in the liver and other tissues such as the brain and eyes. Symptoms include liver disease, tremor, Kayser-Fleischer rings (dark ring pigmentation around iris of eye).
Co-transport/Secondary active transport
One ion goes against gradient coupled to a different ion (Na+ or H+) which moves down its gradient. This method uses energy from the generation of the ions electrochemical gradient by primary active transport. When the sodium ions and glucose bind to the co-transport protein, only then will a conformational change occur releasing the products.
Example of co-transport
Intestinal lumen/renal tubules co-transport of sodium and glucose.
Glucose moves from low to high concentration. Na+/K+-ATPase generates a sodium gradient to enable co-transport for glucose by allowing sodium to diffuse across membrane.
What is cellular signalling?
Communication between cells takes place via signalling molecules such as hormones, neurotransmitters and growth factors.
What do signalling molecules bind to? Give two examples.
Steroid hormones bind to intracellular receptors.
Peptide hormones bind to cell-surface receptors.
What are some secondary messengers?
cAMP
IP3
DAG
Calcium ions
What is a ligand-gated ion channel?
The binding of a ligand either opens or closes the channel
What is a G protein-coupled receptor
When an external signaling molecule binds to a GPCR, it causes a conformational change in the GPCR. This change then triggers the interaction between the GPCR and a nearby G protein.
Enzyme linked receptor
Ligand activates and releases enzyme
Intraceullar receptor
Ligand moves through bilayer and binds to inside receptor
Cholera
Vibrio cholerae bacteria produce the cholera toxin. This crosses the cell membrane and modifies a subunit of the G-protein with increase adenylate cyclase activity. This results in increased cAMP levels. This stimulates several transporters in the cell membrane of intestinal cells. Resulting in massive secretion of ions and water into the gut. This leads to severe diarrhoea and dehydration that can be fatal.
Endocytosis
Transports molecules into cells through:
- phagocytosis
- pinocytosis
- receptor-mediated endocytosis
Exocytosis
transports molecules out of cell through:
- constitutive secretion receptors on vesicle
- regulated secretion needing an external signal
Cystic fibrosis
Hereditory disorder
Mutation in CFTR protein meaning chloride channel cannot be released. Found in many tissues such as pancreas, lungs, skin. Abnormal function results in sticky, viscous mucus where bacteria can spread.
Drugs targeting membrane transporters
Cardiac glycosides
Proton pump inhibitors
Loop diuretics
Thiazide diuretics
Major communication systems and their signals
Endocrine (hormones) nervous (electrical) and immune (ions).
Autocrine definition
cells talking to themselves
Paracrine
cells talking to neighbouring cells a short distance away
signal diffuses across the gap between cells
Inactivated locally, so doesn’t enter the blood stream - to prevent too much signal build up e.g. acetylcholine at neuromuscular junctions
Endocrine
cells talking to other cells elsewhere in the body using hormones travelling through blood stream
Endocrine organs
Hypothalamus Pituitary Thyroid Parathyroid Thymus Adrenals Pancreas Ovaries Testes
General system of endocrine activity
- Hypothalamus releases 6 main hormones into blood stream.
- Anterior pituitary gland stimulates 6 main hormones (Some send directlyto target tissue)
- Sends to various endocrine glands.
- Sends hormone to target tissue
Hormone
Molecule that acts as a chemical messenger
Classified according to structure
- peptide hormonne - AA derivatives
- steroid
Peptide hormones
Peptide and AA hormones produce a quick reaction in the body e.g insulin, growth hormone. Peptide hormones are made up of amino acids and vary in size, depending on the number of amino acids. Some have carbohydrate side chains. They are hydrophillic.
TSH
hormone premade and stored in pituitary cell ready to be released when needed. It dissolves into blood since its a protein and can dissolve in water. It acts on receptors on cell membrane and chemical reaction produces a quick response in thyroid cell.
AA hormones
synthesised from tyrosine
Steroid hormone response speed
slow
What are steroid hormones made from?
cholesterol
Steroid hormones + enzymes
Different enzymes modify molecule to produce a variety of hormones
Solubility of steroid hormones
Can dissolve in lipids
Can’t dissolve in water
Examples of steroid hormones
Testosterone
estrogen
cortison
Extracellular fluid components
Sodium ions Chloride ions Bicarbonate ions Glucose Urea Protein colloid
ICF components which ion is dominant
Potassium
Plasma osmolality
total number of particules dissolves in a solution
What is plasma osmolality determined by
[Na+ and associated anions]
When can plasma osmolality increase?
Diabetes
Kidney failure
Alcohol
Normal plasma osmolality
275-295mmol/kg
Antidiuretic hormone effect
Opens aquaporins in collecting duct within nephron. Kidney creates a large gradient of osmolality. In the medulla the ICF and ECF are not equal, producing a gradient. Water channels are opened up in the collecting duct. This produces a small volume of concentrated urine.
Where is renin enzyme produced?
kidneys
1) Kidneys detect decrease in ECF volume and …
less blood is delivered to kidney causing a pressure decrease. Renin is released.
2) Once renin is released, what is its effect
Angiotensinogen => angiotensinogen I
3) What happens to angiotensin I?
Converted in lungs by ACE into angiotensinogen II
What is the function of angiotensinogen II?
Angiotensinogen II helps restore blood pressure. A2 causes vasoconstriction and an increased production of aldosterone produced in the adrenal gland allowing for more absorption of sodium/water and excretion of potassium ions. Stimulates ADH from pituitary gland to reabsorb water and restore ECF.
Water depletion causes
Reduced intake Sweating Vomiting Diarrhoea Diruesis/diuretics
Consequences of water excess
Hyponatraemia (low sodium ion concentration)
Cerebral overhydration (headache, confusion, convulsions)
ECF volume expansion (heart and kidney failure, cirrhosis w/ascites)
Loss of intravascular fluid
Low effective circulating volume stimulates RAAS and ADH
Renal sodium retention and water retention
Oedema
Oedema
Excess accumulation of fluid in interstitial space.
There is disruption of the filtration and osmotic forces of circulating forces. Obstruction of venous blood and lymphatic return.
Inflammation; increased capillary permability.
Loss of plasma protein.
Serous effusion
Excess water in body cavity
Formation of ISF - capillary bed
High hydrostatic pressure at arteriole end of capillary and low at venule end. The fluid gets pushes out into interstitial space. Oncotic pressure acts as a balancing force and proteins cause reabsorption of water at venule end. The small amount that isnt reabsorbed is taken by lymphatics.
Hydrostatic pressure
Pressure difference between plasma and ISF. Water moves from plasma => ISF.
Oncotic pressure
pressure caused by the difference in protein concentration between plasma and ISF. Water moves from ISF to plasma.
Pathogenesis
Increased fluid leakage into ISS/impaired reabsorption of fluid. Caused by oedema and serous effusion.
Inflammatory oedema
Causes gaps between endothelium. More water leaves capillary into ISS. Albumin leaves.
Venous oedema
blockage at venous end, water can’t leave to heart causing backwards pressure effect, not enough water reabsorption, overwhelming lymphatics.
Lymphatic oedema
blockage in lymphatic and water cant enter lymph
Hypalbuminaemia oedema
Less albumin => less water reabsorption
How much fluid is contained in normal pleural space
~10ml
What is a pleural effusion
A pleural effusion results from the disruption of the balance between hydrostatic+oncotic forces in the visceral + parietal pleural vessels and lymphatic drainage. In pleural effusion, different fluids can enter the pleural cavity.
What is transudate?
Fluid pushed through the capillary due to high pressure within the capillary. It has low protein content.
What is exudate?
Fluid that leaks around the cells of the capillaries caused by inflammation and increases the permeability of pleural capillaries to proteins. It has a high protein content.
Why is pleural fluid protein measured?
To differentiate between exudative and transudative effusions
Conditions associated with transudative/exudative effusions respectively?
Transudative: left ventricular failure, cirrhosis, hypalbuminaemia, peritoneal dialysis
Exudative: malignancy, pneumonia
Normal range of plasma sodium concentration
135-145mmol/L
Concentration is a ratio, not a measure of total body content
What is changes in plasma sodium concentration usually a result of
Gain/loss of water rather than gain/loss of sodium
Clinical effects of disorders of plasma sodium
On the brain due to constrained volume in the skull
Causes of hypernatremia (high Na+)
Water defecit
- poor intake
- osmotic diuretics
- diabetes inspidus
Na+ excess
- Minteralcorticoid (aldosterone) excess
- salt poisoning
hypernatremia effects
Cerebral intercellular dehydration (tremors, irritability and confusion)
Causes of hyponatremia (low Na+)
Artefactual
Na+ loss
- Diuretics
- Addison’s disease
Excess water
- IV fluids (iatrogenic)
- SIADH (excess ADH production)
Excess water and Na+
- oedema (congestive cardiac failure, liver disease)
hyponatremia effects
Cerebral intracellular overhydration (headache, confusion, convulsions)
Fertilisation and early cell division
Sperm + egg => morula => blastocyst (Day 7)
What does ectoderm end up becoming
Epidermis of skin, hair, nails Mammary sweat and sebaceous glands Central Nervous System Peripheral nervous system Pituitary gland Enamel of teeth Lens of the eye/parts of inner ear Sensory epithelium of nose, ear and eye
What does endoderm end up becoming
Epithelial lining of GI tract, respiratory tract and urinary bladder
Parenchyma of the thyroid gland, parathyroid gland, and pancreas
Epithelial lining of the tympanic cavity and auditory tube
Plays a part in the development of the notochord
What does mesoderm end up becoming
Musculoskeletal system Deep layers of the skin Abdominal and chest walls and lining Walls of the bowel Urogenital system
How is mesoderm formed?
As ectoderm grows a folding occurs along the caudal midline (primitive streak). Cells from the base of the primitive streak break off and migrate to lie between the layers of the ectoderm and endoderm, forming the mesoderm. On day 17 the mesoderm separates the whole of the ectoderm from the endoderm except for 2 small areas, one at each end of the trilaminar disc.
Formation of notochord
A tube develops from the end of the primitive stream and extends towards the cranial end. The tube fuses with the endoderm to become a groove in the endoderm layer. The plate folds to become a tube again - the notochord. This plays a central role in further developments in the midline.
Ectoderm - formation of CNS
Neuroectodermal tissues differentiate from the ectoderm and thicken into the neural plate. The neural plate border separates the ectoderm from the neural plate. The neural plate bends to form a u shape (dorsally) with the two ends eventually joining at the neural plate borders, which are nowreferredto as the neural cret. The closure of the neural tube disconnects the neural crest from the epidermis. Neural crest cells differentiate to form most of the peripheral nervous system. The notochord degenerates and only persists the nucleus pulposus of the inervertebral discs. Other mesoderm cells differentiate into the somites, the precursos of the axial skeleto nand skeletal muscle.
Day 22-23
Somites become the myotomes, the endoderm overlying them are the dermatomes.
Day 22-23 embryology
Failures of fusion
If fusion does not extend all the way to the CAUDAL end of the embryo, the child is born with spina bifida.
If fusion does not extend all the way to the CRANIAL end of the embryo, the child is born without the cerebral cortex - ANENCEPHALY.
Mesoderm splits into… (brief overview)
- Paraxial mesoderm
- Intermediate mesoderm
- Lateral mesoderm
Paraxial mesoderm
Along the neural tube. Somites form the: - myotome (muscle tissue) - sclerotome (cartilage and bone) - dermatome (Dermis of the skin N.B not superficial layer)
What is each somite supplied by?
A single spinal nerve
Intermediate mesoderm
More lateral
Forms the urogenital system
- the kidneys, the gonads, and their reproductive duct systems
Lateral mesoderm
extending to the edge of the embro
Somatic parietal layer mesoderm: the outer layer covers the inside of the chest and abdominal walls
Splanchic visceral layer mesoderm: the inside layer covers the organs in the thorax and abdomen
Where does lateral folding start?
The developing trilaminar disc. Cuts across embro in an axial slice.
What happens to yolk sac?
Portion of the yolk sac lined with endoderm is incorporated into the embryo to form the primitive gut; the remaining part of the yolk sac and allontois remain outside, connected through the umbilicus. Folding of the yolk sac lead to formation of body cavities.
Where does cephalo-caudal folding take place?
Starting with bilaminar disc.
Cuts across length of embro in saggital slice.
What does the cranial area of the embryo contain??
The buccopharyngeal (oropharyngeal) membrane, the cardiogenic area (heart) and the septum transversum.
Cranial flexion
Cranial flexion brings the buccopharyngeal membrane, cardiogenic area and septum transversum ventrally, forming the surface of the future face, neck and chest. It brings the heart into its thoracic position and septum transversum to the diaphragm.
Caudal flexion
Caudal flexion brings the cloacal membrane onto the ventral surface of the embryo.
Summary of first 8 weeks of embryology
1st week: fertilization, formation of the morula and blastocyst
2nd week: implantation of the blastocyst and formation of bilaminar embryonic disc and early placenta
3rd weel: differentiation of cell layers to form the trilaminar embryonic disc
4th week: folding of the embryo and continuing development of the 3 germ layers
5th - end of 8th week: development of all organs
By the end of 8th week: the embryo looks like an adult and is called the foetus; growth becomes the predominant feature
