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3rd Internal Assessment - Biochemistry Paper-2: Complete Answers

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QUESTION 1 (Main)

Types of Mutations

1. Point mutations - single nucleotide change
   • Missense mutation: codon changed codes for a different amino acid (e.g., Sickle cell anaemia - Val instead of Glu in HbS)
   • Nonsense mutation: codon changed to a stop codon (UAA, UAG, UGA) - truncated, non-functional protein
   • Silent/synonymous mutation: codon change codes for the SAME amino acid (due to degeneracy of genetic code) - no phenotypic effect
2. Frameshift mutations - insertion or deletion of nucleotides not divisible by 3 - shifts the reading frame; usually causes a non-functional protein
3. Transition - purine replaced by purine OR pyrimidine by pyrimidine Transversion - purine replaced by pyrimidine or vice versa

• Spontaneous: occur without an external cause (frequency 10⁻⁶ to 10⁻⁸)
• Induced: caused by external mutagens

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Mutagens (Enumerated)

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DNA Repair Mechanisms

1. Photoreactivation (Direct repair) - Photolyase enzyme uses visible light energy to directly break thymine dimers formed by UV radiation (reversal of damage)
2. Base Excision Repair (BER) - DNA glycosylase removes a damaged/abnormal base → AP (apurinic/apyrimidinic) endonuclease cleaves the backbone → DNA polymerase fills the gap → DNA ligase seals
3. Nucleotide Excision Repair (NER) - Recognizes bulky lesions (thymine dimers, adducts); an oligonucleotide of ~12-13 nucleotides is excised around the lesion; defect causes Xeroderma Pigmentosum
4. Mismatch Repair (MMR) - Proofreads newly synthesized strand after replication; distinguishes old from new strand by methylation of adenine in GATC sequences; corrects mispaired bases. Defect leads to HNPCC (Lynch syndrome)
5. SOS Response - A post-replication repair for extensive DNA damage; error-prone; allows DNA replication to bypass lesions when damage is too great; a last-resort system

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Nucleotide Excision Repair - Diagram Description

DNA strand with thymine dimer (T^T):
5'---A---T^T---G---3'
3'---T---A---A---C---5'
         ↓
Endonucleases nick on both sides of the lesion
(~25-30 nucleotides excised in eukaryotes, 12-13 in prokaryotes)
         ↓
5'---A---[gap]---G---3'
         ↓
DNA polymerase fills gap using complementary strand as template
         ↓
DNA ligase seals the nick
5'---A---T---T---G---3' (restored)

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Initiation of Transcription in Prokaryotes

• The enzyme responsible is RNA polymerase holoenzyme (core enzyme α₂ββ'ω + sigma factor σ)
• The sigma (σ) factor directs RNA polymerase to the correct promoter region
• Promoter consists of two conserved sequences upstream of the transcription start site:
  • -10 element (Pribnow box): consensus sequence TATAAT (~10 bp upstream)
  • -35 element: consensus sequence TTGACA (~35 bp upstream)
• The sigma factor recognizes and binds these promoter sequences
• RNA polymerase unwinds ~17 bp of DNA forming a transcription bubble
• Synthesis begins at the +1 site, proceeding 5' → 3' using the template strand
• Once a short RNA chain (~8-9 nt) is synthesized, sigma factor dissociates and elongation proceeds with the core enzyme

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QUESTION 1 (OR)

BMI - Definition and WHO Classification

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Key Nutritional Terms

NPU = (Nitrogen retained / Nitrogen ingested) × 100 = Biological Value × Digestibility

• High NPU: egg (94), milk (82), meat (80)
• Low NPU: gelatin (0), maize (36)

• Low GI (< 55): legumes, oats - slow glucose release, better for diabetics
• High GI (> 70): white bread, watermelon - rapid glucose spike

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Marasmus vs. Kwashiorkor

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Role of Dietary Fibres in Decreasing Disease Risk

1. Cardiovascular disease - Soluble fibre (pectin, beta-glucan) binds bile acids → increases cholesterol conversion to bile acids → lowers LDL cholesterol
2. Type 2 Diabetes - Slows glucose absorption → reduces postprandial glycaemic spikes → improves insulin sensitivity
3. Colorectal cancer - Increases stool bulk → dilutes carcinogens → faster transit time reduces exposure; fermentation produces butyrate (protective for colonocytes)
4. Obesity - Increases satiety by slowing gastric emptying, reducing total caloric intake
5. Constipation & Diverticular disease - Increases faecal bulk and water content → reduces straining
6. Hypertension - Associated with reduced blood pressure (mechanism partly via weight reduction and gut microbiome modulation)

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QUESTION 2 - Explain Why (any five)

i) Free radicals play a beneficial role by killing bacteria in our body

O₂ + NADPH → O₂•⁻ + NADP⁺ + H⁺

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ii) Haemorrhagic disease of newborn is more in breast-fed babies

• The neonatal gut is sterile at birth (intestinal bacteria that produce Vitamin K₂ are not yet established)
• Vitamin K does not cross the placenta well
• Neonatal liver has immature clotting factor synthesis

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iii) P53 is termed the "guardian of the human genome"

1. DNA damage sensor: When DNA is damaged (by radiation, chemicals, oxidative stress), P53 is activated (stabilized by ATM/ATR kinase-mediated phosphorylation preventing its degradation by MDM2)
2. Cell cycle arrest: P53 upregulates p21 (CDKN1A), which inhibits CDK4/CDK6-Cyclin D complex → blocks G1/S transition, giving time for DNA repair
3. DNA repair: Upregulates GADD45 and other DNA repair genes
4. Apoptosis: If damage is irreparable, P53 activates BAX, PUMA, NOXA (pro-apoptotic) and represses BCL-2 → triggers mitochondrial apoptosis pathway, eliminating the damaged cell
5. Senescence induction: Permanently halts the cell cycle of damaged cells
6. Prevents genomic instability: Cells with defective P53 accumulate mutations and can become cancerous

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iv) Genomic DNA library and c-DNA library are not the same

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v) Defective caspases in apoptotic pathway may lead to cancer

• Cells that accumulate DNA damage or oncogenic mutations normally undergo apoptosis via caspase activation
• If caspases are non-functional (due to mutations in caspase genes, overexpression of FLIP which blocks caspase-8, or overexpression of BCL-2/BCL-XL which block caspase-9 activation):
  • Damaged/mutated cells survive instead of dying
  • These cells continue to proliferate
  • Additional mutations accumulate (genomic instability)
  • Eventually leading to malignant transformation (cancer)

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vi) In scurvy collagen loses its tensile strength

• Prolyl hydroxylase (for hydroxyproline)
• Lysyl hydroxylase (for hydroxylysine)

• Prolyl and lysyl hydroxylation is impaired
• Pro-collagen chains cannot form stable triple helix (because hydroxyproline forms inter-chain hydrogen bonds stabilizing the helix)
• Cross-linking of collagen fibrils is also impaired (hydroxylysine is needed for pyridinoline cross-links via lysyl oxidase)
• Result: collagen is structurally weak and lacks tensile strength

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QUESTION 3 - Short Notes (4 × 5 = 20)

i) Mucosal Block Theory of Iron Absorption

• Apoferritin: Iron-free protein in mucosal cells
• Ferritin: Apoferritin + iron (storage form within mucosal cells)
• Transferrin: Plasma transport protein for iron

1. Dietary non-haem iron (Fe³⁺) is reduced to Fe²⁺ by mucosal ferrireductase (Dcytb) and gastric acid
2. Fe²⁺ enters enterocytes via DMT-1 (Divalent Metal Transporter-1) on the apical surface
3. Inside the cell, iron can either:
   • Bind to apoferritin → forms ferritin → stored temporarily in the mucosal cell (this is the "mucosal block")
   • Be transferred to transferrin in plasma via ferroportin on the basolateral surface
4. When body stores are high: apoferritin is abundant → more iron stored as ferritin → mucosal cells are shed into the lumen (with iron trapped inside) → iron is NOT absorbed = mucosal block
5. When body needs iron: less apoferritin synthesis → more iron exported via ferroportin to plasma transferrin
6. Hepcidin (produced by liver) regulates ferroportin - high iron/inflammation → high hepcidin → ferroportin degraded → less iron export

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ii) Post-Translational Modifications (PTMs)

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iii) Type-1 Xenobiotic Reactions (Dehydrogenation)

Drug + O₂ + NADPH → Drug-OH + H₂O + NADP⁺

• Aliphatic hydroxylation: addition of -OH to a carbon chain
• Aromatic hydroxylation: addition of -OH to a ring (e.g., phenytoin → parahydroxyphenytoin)
• N-dealkylation, O-dealkylation, S-dealkylation: removal of alkyl groups
• Desulfuration: S → O substitution (e.g., parathion → paraoxon)
• Deamination: removal of -NH₂ group (e.g., amphetamine)

• Less active (detoxification)
• More active (prodrug activation, e.g., codeine → morphine)
• Toxic/reactive (e.g., paracetamol → NAPQI in overdose)
• More water-soluble for Phase II conjugation

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iv) Inhibitors of Protein Synthesis

• Streptomycin: binds 30S → causes misreading of mRNA → bactericidal
• Tetracyclines: block aminoacyl-tRNA binding to A site → bacteriostatic

• Chloramphenicol: inhibits peptidyl transferase → blocks peptide bond formation → bacteriostatic (also inhibits mitochondrial ribosomes in eukaryotes → aplastic anaemia)
• Erythromycin (Macrolides): blocks translocation (inhibits movement of ribosome along mRNA) → bacteriostatic
• Linezolid: blocks formation of initiation complex at 50S
• Clindamycin: inhibits peptide bond formation (50S)

• Cycloheximide: inhibits peptidyl transferase of 60S subunit → useful in research
• Diphtheria toxin: ADP-ribosylates EF-2 (elongation factor 2) → blocks translocation in eukaryotes
• Ricin: inactivates 60S ribosomal subunit (depurination)

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QUESTION 4 - Short Notes (3 × 6 = 18)

i) Mechanism of Action of Glucagon, Norepinephrine, and Epinephrine

1. Hormone binds to Gs-protein coupled receptor (GPCR) on cell membrane
2. Gs protein activates Adenylate cyclase (AC)
3. AC converts ATP → cAMP
4. cAMP activates Protein Kinase A (PKA)
5. PKA phosphorylates target enzymes (activates glycogen phosphorylase kinase, inhibits glycogen synthase, activates hormone-sensitive lipase)

• Released during fasting/hypoglycaemia
• Acts mainly on liver (and adipose)
• Effects: ↑ glycogenolysis, ↑ gluconeogenesis, ↑ lipolysis, ↑ ketogenesis
• Net effect: raises blood glucose

• Acts via β₁/β₂ adrenergic receptors (cAMP pathway) AND α₁ receptors (IP₃/DAG pathway)
• Acts on liver, muscle, adipose, heart, blood vessels
• Effects: ↑ glycogenolysis (liver AND muscle), ↑ gluconeogenesis, ↑ lipolysis, ↑ heart rate & contractility, bronchodilation
• Note: muscle lacks glucagon receptors but responds to epinephrine

• Acts mainly via α₁ and α₂ adrenergic receptors; weaker β effect than epinephrine
• Metabolic effects similar but less potent; stronger vasoconstriction (α₁ >> β₂)
• α₂ acts via Gi protein → ↓ cAMP (inhibitory)

↑ cAMP → ↑ PKA → phosphorylation of phosphorylase kinase → activation of glycogen phosphorylase → glycogenolysis → ↑ blood glucose

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ii) Proto-Oncogenes, Oncogenes, and Tumour Suppressor Genes

• Normal genes that regulate cell growth, proliferation, and differentiation
• Encode: growth factors (e.g., SIS/PDGF), growth factor receptors (e.g., ERBB2/HER2), signal transduction proteins (RAS), transcription factors (MYC, FOS), cell cycle regulators (cyclin D)
• They are the normal, non-mutated counterparts of oncogenes

• Mutated/overexpressed proto-oncogenes that cause uncontrolled cell proliferation
• Require only ONE mutant allele to exert their effect (dominant gain-of-function)

1. Point mutation: RAS mutation (Gly→Val at codon 12) locks RAS in permanently active state - found in 30% of cancers
2. Gene amplification: Multiple copies of the gene (e.g., c-MYC amplification in neuroblastoma, HER2 amplification in breast cancer)
3. Chromosomal translocation: bcr-abl fusion (t(9;22) - Philadelphia chromosome in CML); c-MYC near IgH promoter in Burkitt's lymphoma
4. Promoter insertion: Viral promoter inserted near proto-oncogene

• Genes that normally inhibit cell division or promote apoptosis
• Require BOTH alleles to be inactivated (loss-of-function, recessive - "Two-hit hypothesis" by Knudson)

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iii) Changes in Serum LFT (Liver Function Tests) in Different Types of Jaundice

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QUESTION 5 - Short Notes (4 × 5 = 20)

ii) Renal Defence in Acid-Base Imbalance

• H⁺ secreted into lumen (via Na⁺-H⁺ exchanger)
• H⁺ + HCO₃⁻ → H₂CO₃ → H₂O + CO₂ (via carbonic anhydrase on brush border)
• CO₂ diffuses into cell → CO₂ + H₂O → H₂CO₃ → H⁺ + HCO₃⁻ (intracellular carbonic anhydrase)
• HCO₃⁻ exits basolaterally into blood
• Net effect: HCO₃⁻ is "reclaimed"

• Secreted H⁺ combines with HPO₄²⁻ → H₂PO₄⁻ (titratable acid)
• This "traps" H⁺ in the tubular lumen for excretion
• Contributes ~30-40 mEq H⁺/day

• Glutamine → glutamate + NH₃ (glutaminase)
• NH₃ diffuses into lumen + H⁺ → NH₄⁺ (non-diffusible, trapped and excreted)
• Most important mechanism during chronic acidosis - can increase 10-fold
• Accounts for ~50-60% of net acid excretion

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iii) Lac Operon

• lacI gene: codes for the lac repressor protein
• P (promoter): RNA polymerase binding site
• O (operator): repressor binding site (overlaps promoter)
• lacZ: encodes β-galactosidase (cleaves lactose → glucose + galactose)
• lacY: encodes lactose permease (brings lactose into cell)
• lacA: encodes transacetylase

• Lac repressor (product of lacI) binds to operator → blocks RNA polymerase from transcribing structural genes → NO β-galactosidase produced

• Lactose → allolactose (by traces of β-galactosidase) → allolactose is the actual inducer
• Allolactose binds lac repressor → conformational change → repressor CANNOT bind operator
• RNA polymerase transcribes lacZ, lacY, lacA → β-galactosidase produced

• When glucose is present: cAMP is LOW → CAP (Catabolite Activator Protein / CRP) is inactive → low transcription even if lactose present
• When glucose is absent: cAMP is HIGH → cAMP binds CAP → CAP binds CRE site upstream of promoter → RNA polymerase binding enhanced → maximal transcription
• Lac operon is maximally active when: glucose absent + lactose present

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iv) Different Renal Clearance Tests in Chronic Kidney Disease (CKD)

• All clearances progressively fall as nephrons are lost
• Creatinine clearance < 60 mL/min/1.73m² for >3 months = CKD
• CKD stages by GFR: G1 (≥90), G2 (60-89), G3a (45-59), G3b (30-44), G4 (15-29), G5 (<15 = kidney failure)

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v) Types of Hypersensitivity Reactions (Gell and Coombs Classification)

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QUESTION 6 - MCQs (Mark the Correct Response)

i) In obstructive jaundice, which enzyme is NOT significantly elevated?

• ALP: markedly elevated (hallmark of cholestasis - synthesized in bile duct epithelium when bile backs up)
• GGT: markedly elevated (most sensitive marker of biliary obstruction and cholestasis)
• 5'NT (5'-nucleotidase): elevated (confirms ALP is of hepatic/biliary origin)
• ALT (Alanine aminotransferase): This is an enzyme of hepatocyte damage (hepatitis), not biliary obstruction. In pure obstructive jaundice (e.g., gallstones, carcinoma head of pancreas), ALT is NOT significantly elevated unless there is secondary hepatocyte damage.

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ii) A 10-year-old girl with neurological symptoms, pellagra-like skin rash, increased urinary excretion of Indole Acetic Acid and Indole Pyruvic Acid - Most probably suffering from:

• Hartnup disease is an autosomal recessive disorder caused by a defect in the SLC6A19 gene encoding a neutral amino acid transporter in the intestinal epithelium and renal tubules
• There is impaired intestinal absorption and renal reabsorption of neutral amino acids, particularly tryptophan
• Unabsorbed tryptophan in the gut is converted by bacteria to indole compounds → Indole acetic acid, Indole pyruvic acid, Indole lactic acid → excreted in urine (indicanuria)
• Tryptophan deficiency → nicotinamide (niacin) deficiency → pellagra-like skin rash (photosensitive dermatitis)
• Neurological symptoms: cerebellar ataxia, intellectual disability (due to CNS effects of tryptophan deficiency and toxic indole metabolites)

• Phenylketonuria (PKU): excess phenylalanine in blood/urine, musty odour, NO pellagra
• Alkaptonuria: deficiency of homogentisate oxidase, ochronosis, dark urine on standing, NO pellagra
• Homocystinuria: elevated homocysteine/methionine, lens dislocation, thrombosis, NO indole excretion

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iii) ABG Analysis - Young girl hyperventilating (pH 7.3, pCO₂ 20 mmHg, serum bicarbonate 23 mEq/L)

• pH 7.3 → Acidosis (normal 7.35-7.45)
• pCO₂ 20 mmHg → LOW (normal 35-45) → indicates hyperventilation (CO₂ being blown off)
• HCO₃⁻ 23 mEq/L → Normal (normal 22-26)

• Low pH = acidosis
• Low pCO₂ (not high as expected in respiratory acidosis) = respiratory system is compensating by hyperventilating
• Normal HCO₃⁻ suggests NO metabolic compensation yet (acute process)

• Respiratory alkalosis from hyperventilation would show: HIGH pH + LOW pCO₂
• But pH is 7.3 (acidotic) with low pCO₂ (compensatory hyperventilation) and normal HCO₃⁻

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