Cells Flashcards
Alcohol fermentation?
Pyruvate -> acetaldehyde (using pyruvate decarboxylase)
Acetaldehyde -> ethanol (using alcohol dehydrogenase, forming an NAD+)
Lactate production?
Pyruvate -> lactate (using lactate dehydrogenase, forming an NAD+)
Acetyl CoA production from pyruvate?
Pyruvate + HS-CoA -> acetyl Co + CO2 (using pyruvate dehydrogenase complex, forming an NADH).
Acetyl group joins CoA group by high energy thioester bond which is readily hydrolysed so acetate can be donated to other molecules.
Thiamine pyrophosphate a co-factor of PDC, deficiency in thiamine = Beri Beri
Useful products from TCA with steps?
Isocitrate -> a-ketoglutarate (NADH)
a-ketoglutarate -> succinyl Co-enzyme A (NADH)
succinyl Co-enzyme A -> succinate (GTP)
succinate -> fumarate (FADH2)
maltate -> oxaloacetate (NADH)
overall 3NADH, 2CO2, 1GTP, 1FADH2
Solubility of enzyme in TCA?
All soluble and in mitochondrial matrix apart from succinate dehydrogenase which is insoluble and in inner mitochondrial membrane.
Products from glycolysis and steps?
- Glucose -> glucose-6-phosphate (ATP -> ADP + Pi).
- Fructose-6-phosphate -> fructose-1,6-bisphosphate (ATP -> ADP + Pi, enzyme phosphofructose kinase is rate limiting enzyme).
- GLAP -> 1,3Bisphosphoglycerate (2NAD+ -> 2NADH, GLAP dehydrogenase [redox and group transfer]).
- 1,3Bisphosphoglycerate -> 3-phosphoglycerate (2ADP -> 2ATP, phosphoglycerate kinase).
- Phosphoenolpyruvate -> pyruvate (2ADP -> 2ATP, pyruvate kinase, group removal).
net gain of 2ATP, 2NADH and 2pyruvate
Degradation of all 20 amino acids gives rise to?
Pyruvate
Acetyl CoA
Acetoacetyl CoA
Alpha-ketoglutarate
Oxaloacetate
Succinyl CoA
Fumarate
Example Transamination?
Alanine + a-ketoglutarate -> pyruvate + glutamate
C3 alanine undergoes Transamination by action of enzyme alanine aminotransferase.
glutamate can return to a-ketoglutarate using glutamate dehydrogenase
Warburg effect?
Mutations in genes of fumarase, succinate dehydrogenase or isocitrate dehydrogenase, decreased TCA activity, increases anaerobic glycolysis and preference to lactate production.
Glycerol phosphate shuttle?
DHAP -> G3P (NADH -> NAH+, using cytosolic glycerol-3-phosphate dehydrogenase)
G3P -> DHAP (FAD -> FADH2, using mitochondrial glycerol-3-phosphate dehydrogenase)
Electrons from FADH2 passed onto Co-enzyme Q -> QH2, part of ETC.
skeletal muscle and brain
Malate-aspartate shuttle?
Aspartate -> oxaloacetate -> malate (using aspartate transaminase and malate dehydrogenase, NADH -> NAD+)
malate through antiporter
Malate -> oxaloacetate -> aspartate (NAD+ -> NADH in mitochondria)
aspartate back through antiporter into cytoplasm
Transamination reaction in MAS - oxaloacetate + glutamate -> a-ketoglutarate + aspartate
in liver, kidney and heart
Fatty acid -> fatty acyl CoA?
Fatty acid + Coenzyme A -> fatty acyl CoA + H2O (acyl-CoA synthetase, ATP -> AMP +PPi)
in outer mitochondrial membrane
Carnitine shuttle?
Carnitine -> acyl carnitine (acyl CoA -> CoA, carnitine acyltransferase I)
carnitine through translocase into mitochondrial matrix
Acyl carnitine -> carnitine (CoA -> acyl CoA, carnitine acyltransferase II)
carnitine through translocase into cytoplasm
gets acylCoA into mitochondrial matrix for beta oxidation
Acyl CoA -> Acetyl CoA?
Oxidation, hydration, oxidation, thyolysis.
Per cycle, 1 FAD, 1NAD+ and 1 H2O used up.
1 Acetyl CoA, 1FADH2, and 1 NADH made.
Enzymes used in beta oxidation?
Short chain acyl-Coenzyme A dehydrogenase - anything less than 6C
Medium chain acyl-Coenzyme A dehydrogenase - 6C-12C
Long chain - 13C-21C
Very long chain - >22C
Ketone bodies from acetyl CoA?
Acetoacetone, acetone, D-3-hydroxybutyrate
Lipogenesis?
Enzymes - acetyl CoA carboxylase, fatty acid synthase.
1. Condensation (decarboxylative condensation, reaction of acetyl CoA + malonyl CoA, elongation)
2. Reduction (by ketoreductase)
3. Dehydration (by dehydratase)
4. Reduction (by enol reductase)
growing fatty acyl group linked to acyl carrier proteins
in adults, only in liver, lactating breasts and adipose tissues
Beta oxidation vs fatty acid synthesis?
Beta oxidation carrier is CoA, fatty acid synthesis is ACP.
Reducing power in beta oxidation is FAD/NAD+, in fatty acid synthesis is NADPH.
Location of beta oxidation is mitochondrial matrix, fatty acid synthesis is cytoplasm.
elongation of acyl group into fatty acids longer than 16C occurs in mitochondria and endoplasmic reticulum instead of cytoplasm
desaturation of fatty acids needs fatty acyl-CoA desaturases
Metabolic poisons?
Cyanide and azide - bind with high affinity to ferric form of haem group in cytochrome oxidase complex (IV), blocking final step in ETC.
Rotenone - inhibits transfer of electrons from NADH dehydrogenase (I) to ubiquinone.
Oligomycin - binds to ATP synthase and blocks flow of electrons through enzyme.
Malonate - resembles succinate, competitive inhibitor of succinate dehydrogenase (II). Inhibits oxidation of succinate -> fumerate, so slows flow of electrons from succinate -> ubiquinone.
DNP?
Dinitrophenol - proton ionophore, shuttles protons across inner mitochondrial membrane, uncouples oxidative phosphorylation from ATP synthase. Increases metabolic rate and temperature so weight loss.
Cell cycle phases?
G1 - decision point (interphase)
S - DNA replication (interphase)
G2 - decision point (interphase)
M - mitosis and cytokinesis, most vulnerable period, gene transcription silenced.
G0 - quiescent phase, cell cycle machinery dismantled, stays until triggered externally to G1
cyclical due to degradation and reformation of cyclins
Signalling cascade?
Growth factors, signal amplification, MAP kinases.
Mitogen activated protein kinases - Ras, Raf, MEK, ERK. Increase protein synthesis + decrease protein degradation.
cMyc?
Oncogene
Induced by growth factor signalling
Promotes G0 -> G1 and increases concentration of Cyclin D.
Cdk?
Cyclin dependent kinases, regulated by interactions with cyclins and phosphorylation. Cdk 1, 2, 4 +6.
phosphorylate + dephosphorylate serine, threonine and tyrosine
sequentially activated + stimulate synthesis of genes for next phase, giving direction and timing
Cyclins?
Transiently expressed at specific points in cell cycle, regulated at level of expression, synthesised then degraded.
Order of cyclin-Cdk complexes?
- Cyclin D-Cdk 4/6 complex leads progression through G1 restriction point.
- Cyclin E-Cdk 2 complex drives initiation of DNA replication in S phase.
- Cyclin A-Cdk 2 complex initiates mitosis by driving from G2 -> M phase.
- Cyclin B-Cdk 1 complex controls initiation and progression of mitosis in M phase.
Cdk activation?
Activated by binding to cyclins, phosphorylation of Cdk-cyclin complexes at inhibitory and activating sites, then dephosphorylation by phosphatases which removes inhibitory Cdk. Positive feedback as activated Cdk activates more phosphatases to remove inhibitory phosphates.
Deactivated by ubiquitylation.
Rb?
Retinoblastoma - molecular brake, found in every nucleated cell.
1. Binds to E2F transcription factor, prevents from promoting expression of genes for cell cycle (like DNA polymerase and thymidine kinase).
2. Mitogen signalling - cyclin D-Cdk 4/6 complexes phosphorylated Rb in G1 phase which inactivates Rb and releases E2F transcription factor so genes for cell cycle can be made.
3. In S cycle, Rb dephosphorylated.
p53?
Tumour suppressor, activated by phosphorylation, binds to and activates transcription and translation of p21. Enzyme formed by expression of p21 inhibits cyclin-Cdk complexes.
arrests cells with damaged DNA in G1
Reasons for cell communication?
- Process information
- Self preservation
- Voluntary movement
- Homeostasis
Intercellular signalling examples?
Endocrine - glucagon -> liver to stimulate glycogenolysis + gluconeogenesis
Paracrine - insulin in islets of Langerhans going from beta -> alpha cells to inhibit glucagon secretion.
Autocrine - growth factors (TGF) stimulating mitogenesis.
- T lymphocyte activation (activated TCR initiates cascade of reactions within T cell + T cell expresses IL-2 receptor on surface. Also secretes IL-2 which binds to receptor on same cell and adjacent cells).
- Ach-> presynaptic M2 muscarinic cells.
Membrane attached proteins - HIV GP120 glycoprotein binding to CD4 receptor on T lymphocytes.
Types of receptors with examples?
- Ligand gated ion channels (ionotrophic) - Nicotinic ACh, GABA A, NMDA.
- G-protein coupled receptors - beta1-adrenergic receptor, muscarinic M2 receptor, dopaminergic receptor, angiotensin 1 receptor.
- Enzyme linked receptor - insulin receptor (CD220 antigen)
- Intracellular receptors - cytoplasmic (glucocorticoid and oestradiol, associated with chaperone molecules, usually heat shock protein [hsp]) and nuclear (thyroid hormone complex).
Mechanism for G-protein coupled receptor?
- 7-TM receptor + heterotrimeric G protein inactive until ligand binding, when conformational change causes GDP -> GTP phosphorylation, in which alpha subunit and beta-gamma subunit components of G protein dissociate and act on secondary messengers. Can activate further heterotrimeric G proteins whilst active.
- When ligand dissociates, GTP -> GDP, alpha subunit dissociates from target protein and binds to beta-gamma subunit.
Enzyme linked receptor mechanism?
- Ligand binding -> receptor clustering which activates enzyme within cytoplasm. Enzymes phosphorylate receptors so signalling proteins bind to cytoplasmic domain and recruit other signalling proteins.
- Signal terminated when phosphatase dephosphorylates receptor.
Types of adaptation with examples?
Atrophy - shrinkage in size of a cell. E.g dementia brain, muscle atrophy after denervation or lack of use.
Hypertrophy - increase in size of a cell. E.g pathological (hypertension or valve disorders) or physiological (muscles grow after exercise or uterus during pregnancy).
Hyperplasia - increase in number of cells. E.g. pathological (cancer) or physiological (liver growing after resection).
Metaplasia - reversible change in cell type. E.g. pathological (Barrett’s oesophagus- squamous -> columnar when in contact with stomach acid) or physiological (cervix in cervical expansion squamous -> columnar, vaginal pH a factor).
Dysplasia (not adaptation) - precancerous cells, slow genetic and cytological features of malignancy but not invading underlying tissues. Large nuclei, high nuclei:cytoplasm ratio, high number of mitoses.
neoplasia = malignancy
Types of necrosis with examples?
Necrosis - confluent cell death associated with inflammation.
Coagulative - structure becomes fixed after death. E.g. myocardial infarct.
Liquefactive - tissue liquified. E.g. liquefactive cerebral infarct (brain doesn’t have much connective tissue to keep cells in place)
Caseous - cheesy, cells become structureless and oozy. E.g. lungs in pulmonary TB.
Fat - fat becomes liquefied. E.g. acute pancreatitis - enzymes activated in pancreas rather than duodenum.
Causes of apoptosis?
Embryogenesis, deletion of auto-reactive T cells in thymus, withdrawal of hormone, cell deletion in proliferation, DNA damage.
Immune cell recruitment chemokines?
Chemokine CXCL8 (IL-8) works on receptor CXCR1 + CXCR2 (G couples 7-transmembrane proteins) on neutrophils.
Neutrophil extravasation?
- Chemo-attraction - cytokines (TNF-alpha) act on endothelial layer to promote upregulation of adhesion molecules (selectins).
- Rolling adhesion - carbohydrate ligand in low affinity state on neutrophils bind selectins and migrate along blood vessel (e.g. PSGL-1 [selectin P ligand] binds P + E selectins).
- Tight adhesion - chemokines promote low -> high affinity switch in integrins like LFA-1 and Mac-1 to enhance binding to ligands (ICAM-1).
- Transmigration - cytoskeletal rearrangement and extension of pseudopodia to move cell into interstitium. Mediated by PACAM interactions on both cells.
How do neutrophils recognise pathogens at site of inflammation?
Use of TLR-4 + CD14 to identify lipopolysaccharides present in gram-negative bacteria.
Phagocytosis?
- Large particles engulfed into membrane bound vesicles (phagosomes)
- Phagosomes fuses with lysosome (which releases vesicles containing enzymes like elastase and lysozyme) to form phagolysosome.
reactive oxygen species - phagocyte NADPH oxidase
antimicrobial peptides - defensins
T and B cells in chronic inflammation?
T cells - allow specificity, pro-inflammatory (TNF, IL-17, IFN-gamma), cytotoxic (granzymes and perforin), regulatory (TGFbeta).
B cells - secrete antibodies, inflammatory, local and remote.
Components of ECM?
Collagen - Type I, II, III, IV.
Multi-adhesive glycoproteins - laminins, fibronectins, fibrinogen.
Proteoglycans - versican, decorin, aggrecan, perlecan.
Elastic fibres - elastin, fibrillin.
ECM protein mutations?
ECM protein mutations:
- Osteogenesis imperfecta (Type I collagen)
- Marfan’s syndrome (Fibrillin I)
- Alport’s syndrome (Type IV collagen, alpha 5)
- Epidermolysis Bullosa (Laminin 5)
- Congenital muscular dystrophy (Laminin 2)
ECM catabolism disorders?
Inability to degrade glycoaminoglycans (MPSs)
Hurlers syndrome - loss of L-alpha-iduronidase function
Excessive ECM deposition?
Liver fibrosis (cirrhosis)
Kidney fibrosis (diabetic nephropathy)
Lung fibrosis (idiopathic pulmonary fibrosis)
Collagen structure?
Layers at right angles to resist tensile strength.
Type I - chains from 2 different genes = [a1(I)]2[a2(I)]
Type II and III - chains from only one chain type so type II = [a1(II)]3 and type III = [a1(III)]3.
Triple helix (gly-x-y, x usually proline and y usually hydroxyproline).
One alpha chain -> 3 alpha chains -> collagen fibril -> collagen fibre.
Collagen synthesis?
in fibroblasts
1. Pro alpha chain post transcriptional modifications - hydroxylation of prolines and lysines and glycosylation of hydroxylysines.
2. Self assembly of 3 pro alpha chains into pro collagen triple helix.
3. Pro collagen non collagenous domains cleaved at N- and C- termini if secreted. Assembly into fibrils then aggregation of fibrils to form collagen fibre.
hydrogen bonds formed between collagen molecules by prolyl and lysyl hydroxylases using Fe2+ and vitamin C as cofactors to form hydroxylysine and hydroxyproline. Low Vitamin C causes underhydroxylated collagens = scurvy.
Ehlers-Danlos Syndrome?
Stretchy skin and joint hyper mobility due to collagen mutations.
Non-fibrillar collagens?
Type IX + XII - fibril associated collagens
Type IV - network-forming collagen, in all basement membrane, has uncleaved N + C termini so can interact with other collagen molecules.
Basement membranes?
Type IV collagen (flexible/bendy as helix interrupted in several regions) + laminin.
Not 90 degree angle like other collagen, instead head-to-tail (rotary shadowing).
essential in kidney (glomerular basement membrane)
Basement membrane disorders?
Diabetic nephropathy - accumulation of ECM leading to highly thickened basement membrane, restricts renal filtration -> renal failure.
Alport syndrome - mutation in type IV collagen, GBM abnormally split and laminated. Progressive loss of kidney function.
Laminins?
Heterotrimeric proteins - alpha, beta and gamma chain. Form cross chained molecules.
Multi adhesive glycoproteins - interact with various receptors (integrins and dystroglycan)
Mutation can cause Epidermolysis bullosa and muscular dystrophy.
Muscular dystrophy - Laminin-alpha 2 deficiency, doesn’t interact with integrin or dystroglycan causing weakness, joint deformity.
Fibronectin?
Insoluble fibrillar or soluble plasma matrix.
Regulate cell adhesion and migration in tissue repair and embryogenesis, disulphide linked, wound healing.
Elastic tissues?
Elastin core surrounded by microfibrils rich in fibrillin.
Elastin - alternate between hydrophobic and hydrophilic, contains lysine which is crosslinked during formation of mature elastin.
Fibrillin - mutation in fibrillin-1 can cause Marfan’s syndrome (thickening of aorta with fragmentation and disarray).
Integrins?
Link ECM with actin cytoskeleton.
Proteoglycans?
Core protein + glycosaminoglycan (GAG) chain
GAG chain - made of repeating disaccharides, 4 categories:
- Hyaluronan - in highly viscous tissues (vitreous humour and synovial fluid), protects cartilaginous surface from damage.
- Chondroitin sulfate and dermatan sulfate
- Heparan sulfate
- Keratan sulfate
Aggrecan?
Major constituent of cartilage, resists compressive forces
Under compression, water given up and regained once load reduces.
In osteoarthritis, aggrecan is cleaved by aggrecanases and metaloproteinases
Can retain water due to sulfated GAGs so negative charge.
Other markers for myocardial damage?
Serum glutamate oxaloacetate transaminase (SGOT) - peaks D2-D5
Lactate dehydrogenase (LDH) - peaks D5-D10
Cardiac troponin - calcium switch in muscle, appears in serum from 48hours-5 days.
Harmartomas
Localised benign overgrowths of one of more mature cell types. Represent architecture but not cytological abnormalities.
Teratomas?
Tumours derived from germ cells and can contain tissue derived from all 3 germ cell layers.
Heterotopias?
Normal tissue found in parts of body where not usually present (e.g. pancreas in wall of large intestine)
Squamous tumours? (Epithelial neoplasms)
Benign - squamous papilloma
Malignant - squamous cell carcinoma
e.g. skin, oesophagus, cervix
Glandular tumours? (Epithelial neoplasms)
Benign - adenoma
Malignant - adenocarcinoma
e.g. breast, colon, pancreas, thyroid
Transitional tumours? (Epithelial neoplasms)
Benign - transitional papilloma
Malignant - transitional cell carcinoma
e.g. bladder
Smooth muscle tumours? (Connective tissue neoplasms)
Benign - leiomyoma
Malignant - leiomyosarcoma
e.g. uterus, colon
Bone tumours? (Connective tissue neoplasms)
Benign - osteoma
Malignant - osteosarcoma
Lymphocyte cancer? (Haematological neoplasm)
Malignant - lymphoma
e.g. gastric lymphoma
Bone marrow cancer? (Haematological neoplasm)
Malignant - leukaemia
Cholesterol synthesis summary?
- Synthesis of isopentenyl pyrophosphate (in cytoplasm)
- Condensation of ipp to squalene (in cytoplasm)
- Cyclisation and demethylation of squalene (by monooxygenase) to for cholesterol (in ER)
HMG CoA -> mevalonate?
HMG CoA reductase - inhibited by mevalonate, cholesterol and bile salts
Mevalonate -> 3-isopentelyl pyrophosphate
Sequential phosphorylation (at 3 and 5) of hydroxyl groups
Decarboxylation
2 farnesyl PP -> squalene?
Condensation to 30C squalene
Cholesterol to pregnelonone?
Desmolase
Bile salts?
Glycocholate and taurocholate
Cholesterol ester synthesis?
phosphatidycholine (+ LCAT)
Statins and resins?
Statins - HMG CoA reductase inhibitors
Resins - bind bile and acid-cholesterol complexes, preventing reabsorption by intestine, so less LDL and more HDL.
Km and Vmax?
Km - concentration of substance at which enzyme works at half of its maximal velocity
competitive inhibition increases Km and vice versa
V Max - maximal velocity a reaction can go
low Vmax = strong binding
H&E staining?
Haemotoxylin and eosin, staining for leukocytes (nuclei and cytoplasmic granules)
Biopsies and frozen specimens?
Biopsies preserved with formalin
Frozen specimens frozen with cryostat machine
CK activity determined with?
Coupled assay, leading to production of NADPH which can be measured on an absorption spectra.
Electrophoresis can determine which dimer is present.
ETC complexes?
I - NADH dehydrogenase
II - succinate dehydrogenase
III - Q cytochrome c oxioreductase
IV - cytochrome c oxidase
carriers ubiquinone and cytochrome C
lower free energy as you move through complexes so higher affinity for electrons
ATP synthase parts?
F0 - bound to inner mitochondrial membrane, rotates and converts mechanical energy into kinetic
F1 - suspended in matrix, synthesises ATP.
Intracellular receptor mechanism?
Cytoplasmic - located in cytosol, associated with chaperone molecules (usually heat shock proteins)
Nuclear - in nucleus and often bound to DNA, binding of hormone ligand leads to transcriptional regulation
Balloon degeneration?
Strands of cytoplasm, from alcohol (in alcoholic fatty liver disease)
