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JHARGRAM GOVT. MEDICAL COLLEGE - BIOCHEMISTRY ANSWERS

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QUESTION 1 (Main)

A. Structural Characteristics of the Watson-Crick Model of B-DNA (5 marks)

• The two strands are held by hydrogen bonds (between bases) and hydrophobic stacking interactions (between adjacent bases).
• Bases are complementary and antiparallel (3'→5' / 5'→3').

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B. How B-DNA Differs from A and Z DNA (5 marks)

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C. How Reverse Transcriptase and Prions Contradict Central Dogma (5 marks)

• Found in retroviruses (e.g., HIV)
• Enzyme: Reverse transcriptase (RNA-dependent DNA polymerase)
• Flow: RNA → DNA (reverse of the dogma)
• Uses viral RNA as template to synthesize complementary DNA (cDNA)
• The cDNA integrates into host genome as a "provirus"
• This violates the rule that DNA is always the template and RNA is always the product
• Discovered by Howard Temin and David Baltimore (Nobel Prize 1975)

• Prions are infectious proteins with no nucleic acid
• Agent of diseases: Creutzfeldt-Jakob disease (CJD), Kuru, Scrapie, BSE ("mad cow")
• Normal prion protein (PrP^C) undergoes conformational change → PrP^Sc (misfolded, protease-resistant)
• Propagation: PrP^Sc acts as a template to misfold normal PrP^C
• This means protein can replicate/propagate without DNA or RNA - completely violates the central dogma
• Proposed by Stanley Prusiner (Nobel Prize 1997)

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QUESTION 1 (OR) - Clinical Case: 26-year-old with muscle spasms + hyperuricemia

a. Name the Condition (1 mark)

• X-linked recessive disorder affecting purine salvage pathway
• Almost exclusively affects males

b. Enzyme Deficiency (2 marks)

• Gene located on X chromosome (Xq26-27)
• Near-complete deficiency causes classic Lesch-Nyhan
• Partial deficiency causes Kelley-Seegmiller syndrome (gout without neurological features)

c. Reactions Catalyzed by HGPRT (4 marks)

Hypoxanthine + PRPP → Inosine monophosphate (IMP) + PPi

Guanine + PRPP → Guanosine monophosphate (GMP) + PPi

d. Why is Uric Acid Raised? (4 marks)

1. No salvage - Hypoxanthine and Guanine cannot be recycled back to IMP and GMP
2. These bases are therefore catabolized to uric acid instead
3. Loss of feedback inhibition - Normally, salvage products (IMP, GMP) inhibit de novo purine synthesis (via PRPP amidotransferase). Without them, de novo synthesis is uninhibited → overproduction of purines
4. All excess purines are ultimately degraded to uric acid, causing hyperuricemia
5. PRPP accumulates (not consumed by salvage) → stimulates de novo synthesis further

e. Why These Reactions Occur Particularly in Liver, Brain, and Bone Marrow (4 marks)

• Liver is the primary site of de novo purine synthesis and purine catabolism
• High metabolic activity, high PRPP availability
• Supplies purines to tissues that cannot synthesize them de novo
• Salvage is critical here to recycle purines and prevent wasteful uric acid production

• Brain has very low capacity for de novo purine synthesis
• It is almost entirely dependent on salvage pathways (HGPRT) to obtain purines for nucleotide synthesis
• HGPRT deficiency severely affects brain function → explains neurological symptoms (choreoathetosis, spasticity, self-mutilation, intellectual disability)
• High metabolic demand for purines (neurotransmitter synthesis, ATP for neurons)

• Rapid cell proliferation requires large amounts of nucleotides for DNA synthesis
• De novo synthesis is energy-expensive; salvage pathways provide a more efficient route
• Erythrocytes lack mitochondria and have very limited de novo synthesis; erythroid precursors in marrow heavily rely on HGPRT
• Without HGPRT, bone marrow cells struggle to meet nucleotide demands

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QUESTION 2 - Explain Any Five Statements

a. Inhibitors and Uncouplers Function Differently

• Complex I inhibitor: Rotenone, Amytal (block NADH dehydrogenase)
• Complex III inhibitor: Antimycin A
• Complex IV inhibitor: Cyanide (CN⁻), CO, H₂S, Azide

• Examples: 2,4-Dinitrophenol (DNP), Thermogenin (UCP-1) in brown adipose tissue
• They are lipophilic weak acids that carry H⁺ across the membrane, collapsing the electrochemical gradient

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b. hnRNA Undergoes Post-Transcriptional Modification

1. 5' Capping (occurs early, even before transcription ends):
   • Addition of 7-methylguanosine (m⁷G) cap via 5'-5' triphosphate linkage
   • Functions: protects mRNA from 5' exonucleases, aids ribosome binding (translation initiation), aids nuclear export
2. 3' Polyadenylation:
   • Addition of ~200 adenylate residues (poly-A tail) at 3' end after cleavage signal (AAUAAA)
   • Enzyme: Poly-A polymerase
   • Functions: protects mRNA from 3' degradation, aids export and translation
3. Splicing (removal of introns):
   • Introns removed, exons joined by the spliceosome (snRNPs: U1, U2, U4, U5, U6)
   • Lariat intermediate formed
   • Alternative splicing can produce multiple proteins from one gene
4. RNA editing (in some cases): e.g., ApoB mRNA (CAA → UAA creates stop codon in intestine)

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c. Selenium and Vitamin E Act Synergistically as Antioxidants

• Fat-soluble antioxidant
• Located in cell membranes and lipoproteins
• Breaks chain reactions of lipid peroxidation by donating H• to lipid peroxy radicals (LOO•)
• Converts LOO• → LOOH (stable)
• Regenerated by Vitamin C

• Component of Glutathione Peroxidase (GPx) - a selenoprotein
• GPx reduces H₂O₂ and lipid hydroperoxides (LOOH) to water/alcohols
• Reaction: 2 GSH + H₂O₂ → GSSG + 2H₂O (catalyzed by GPx)

• Vitamin E stops new LOOH from being formed
• Selenium (GPx) destroys the LOOH that does form
• If Vitamin E is deficient, more LOOH accumulates → more GPx required
• If selenium is deficient, LOOH builds up even if Vitamin E is present
• Together they cover both prevention AND elimination of lipid peroxides, providing greater protection than either alone

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d. M Band in Serum Electrophoresis is the Diagnostic Feature of Multiple Myeloma

• A sharp, narrow, homogeneous spike appearing most often in the γ (or β) region
• Represents monoclonal immunoglobulin produced by a single clone of plasma cells
• In multiple myeloma, malignant plasma cells proliferate and secrete large amounts of a single immunoglobulin class (usually IgG or IgA) or its fragments (Bence-Jones proteins = free light chains)

• M spike > 3 g/dL (IgG, IgA, or rarely IgM)
• The spike is narrow because it is from ONE clone (monoclonal) vs. polyclonal broad elevation in infection
• Urine shows Bence-Jones protein (free κ or λ light chains)
• Criteria (CRAB): Calcium ↑, Renal failure, Anemia, Bone lesions

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e. DNA Polymerase Has Proofreading Function

• DNA polymerase not only adds nucleotides in the 5'→3' direction (polymerase activity), it also has a built-in 3'→5' exonuclease
• After each nucleotide addition, the enzyme "checks" if the new base is correctly paired
• If a mismatch is detected (wrong base paired), the 3'→5' exonuclease activity removes the incorrect nucleotide
• The correct nucleotide is then re-inserted
• This reduces the error rate from ~1/10⁵ (without proofreading) to ~1/10⁷

• DNA Pol I: Has 3'→5' (proofreading) AND 5'→3' exonuclease; fills Okazaki fragment gaps
• DNA Pol III: Main replicative polymerase; has 3'→5' proofreading exonuclease

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f. E. coli Metabolizes Glucose Only in the Presence of BOTH Glucose and Lactose

• Contains: lacZ (β-galactosidase), lacY (permease), lacA (transacetylase)
• Regulated by negative control (repressor) AND positive control (CAP-cAMP)

• Glucose is metabolized preferentially (catabolite repression)
• Glucose → lowers cAMP levels → CAP (Catabolite Activator Protein) cannot bind promoter → lac operon is NOT fully activated
• Even though lactose removes the repressor, without CAP binding, transcription is minimal
• Therefore, E. coli uses only glucose (not lactose) when both are present

• High cAMP → CAP-cAMP binds → positive activation of operon
• Lactose induces operon (allolactose removes repressor)
• β-galactosidase produced → lactose metabolized

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QUESTION 3 - Short Notes

a. Antioxidant Paradox

• Reactive oxygen species (ROS) - superoxide (O₂•⁻), H₂O₂, OH• - damage DNA, proteins, and lipids
• Epidemiological studies showed people with high dietary antioxidants (Vit E, C, β-carotene) have lower rates of cardiovascular disease and cancer

• Large RCTs (e.g., ATBC trial, CARET trial) showed that β-carotene supplementation increased lung cancer in smokers
• Vitamin E supplements showed no benefit or even slight harm in some trials
• Despite oxidative stress being clearly linked to disease, antioxidant pills don't mirror the benefits of dietary antioxidants

1. Dose-dependent pro-oxidant effect: At high doses, antioxidants become pro-oxidants (e.g., Vitamin C can reduce Fe³⁺ → Fe²⁺, generating •OH via Fenton reaction)
2. ROS have physiological roles: They are signaling molecules (e.g., NF-κB activation, immune defense - neutrophil oxidative burst). Excessive antioxidants may impair these functions.
3. Antioxidant networks are complex: Single-supplement interventions miss synergistic interactions found in whole foods
4. Timing and context: Antioxidants during cancer treatment may protect tumor cells
5. Dietary antioxidants have non-antioxidant benefits (anti-inflammatory polyphenols, fiber, etc.)

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b. Interphase of the Cell Cycle

• Cell grows in size
• RNA and protein synthesis increases
• Organelles duplicate
• Cell prepares for DNA replication
• Restriction point (R point): checkpoint that determines if conditions are favorable for division; regulated by cyclins D/E + CDK4/6
• Cells that exit cycle (permanently or temporarily) enter G₀ from G₁

• DNA replication occurs
• Each chromosome is duplicated → sister chromatids joined at centromere
• DNA content: 2N → 4N
• Histone synthesis peaks during S phase
• Lasts ~6-8 hours in human cells
• Licensed by cyclin E-CDK2

• Cell continues growing
• Enzymes and structural proteins for mitosis are synthesized
• G₂ checkpoint: verifies DNA replication is complete and damage-free
• Cyclin B-CDK1 (MPF - Maturation Promoting Factor) accumulates
• Lasts ~4-6 hours

• G₁/S checkpoint: checks for DNA damage, cell size (p53, Rb)
• S-phase checkpoint: checks replication fidelity
• G₂/M checkpoint: checks for unreplicated DNA

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c. Nucleosomes

• A nucleosome consists of:
  • Histone octamer core: 2 copies each of H2A, H2B, H3, H4 (total 8 histones)
  • ~147 base pairs of DNA wrapped 1.65 times around the octamer
  • Linker DNA: ~20-60 bp between nucleosomes
  • Histone H1: binds at entry/exit point of DNA; stabilizes linker DNA ("seals" the nucleosome)

• Small, basic proteins; rich in lysine and arginine (positively charged, interact with negatively charged DNA)
• H3 and H4: most conserved evolutionarily
• H1: least conserved, varies between species

• Nucleosomes on linker DNA → "beads on a string" (11 nm fiber)
• Further coiling with H1 → 30 nm fiber (solenoid)
• Higher-order folding → 300 nm, 700 nm → metaphase chromosome (2400 nm)

• Compact DNA (~6 feet of DNA into nucleus of ~6 μm)
• Regulate gene expression: Nucleosome positioning controls access to DNA
• Histone modifications: acetylation (relaxes chromatin, activates genes), methylation, phosphorylation, ubiquitination - the histone code
• Histone acetyltransferases (HAT) add acetyl groups; HDACs remove them

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QUESTION 4 - Applied Aspects

a. Mucosal Block Theory

• Proposed by Hahn et al. (1943) and expanded by Granick
• The intestinal mucosal cell acts as a "gatekeeper" regulating iron uptake into the body
• The amount of iron absorbed is inversely related to the body's iron stores

1. Dietary iron enters the duodenal enterocyte via DMT-1 (Divalent Metal Transporter-1) after reduction of Fe³⁺ → Fe²⁺ by Dcytb (ferrireductase)
2. Once inside the cell, iron is either:
   • Transferred to blood via ferroportin (with hephaestin oxidizing Fe²⁺ → Fe³⁺), then bound to transferrin
   • Stored in the cell as ferritin (apoferritin + iron)
3. When body iron stores are full (high ferritin):
   • Liver secretes hepcidin (the master regulator)
   • Hepcidin binds ferroportin → internalization and degradation of ferroportin
   • Iron is trapped inside mucosal cells as ferritin
   • When the mucosal cell is shed (~3-day lifespan), iron is lost in feces
   • This is the "mucosal block" - the cell physically blocks iron from entering circulation

• Iron deficiency anemia: hepcidin is low → ferroportin is upregulated → maximum absorption
• Hemochromatosis (hereditary): hepcidin mutations → no block → excessive iron absorption
• Anemia of chronic disease: high hepcidin → mucosal block → functional iron deficiency
• Explains why oral iron supplements are less effective in inflammation (high hepcidin state)

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b. Role of CYP450 Enzymes in Drug Metabolism

• Family of heme-containing monooxygenases located primarily in hepatic endoplasmic reticulum (also gut, lung, adrenal, kidney)
• Named because the CO-bound reduced form absorbs at 450 nm
• ~57 CYP genes in humans; key drug-metabolizing enzymes: CYP3A4 (metabolizes ~50% of drugs), CYP2D6, CYP2C9, CYP2C19, CYP1A2

Drug + O₂ + NADPH → Hydroxylated Drug + H₂O + NADP⁺

• Phase I (Functionalization): CYP450 introduces/unmasks polar groups (-OH, -NH₂, -SH) via hydroxylation, oxidation, reduction, hydrolysis → increases reactivity
• Phase II (Conjugation): Glucuronidation, sulfation, acetylation → increases water solubility → excretion in bile/urine

1. Drug induction: Some drugs induce CYP450 → increased metabolism → therapeutic failure
   • Inducers: Rifampicin, phenytoin, carbamazepine, phenobarbital, St. John's Wort
   • Example: Rifampicin + warfarin → rapid warfarin metabolism → INR drops → thrombosis
2. Drug inhibition: Some drugs inhibit CYP450 → reduced metabolism → toxicity
   • Inhibitors: Ketoconazole (CYP3A4), erythromycin, grapefruit juice (furanocoumarins)
   • Example: Azithromycin + statins → statin accumulation → rhabdomyolysis
3. Genetic polymorphism (Pharmacogenomics):
   • Poor metabolizers (PM) vs. Extensive metabolizers (EM) vs. Ultrarapid metabolizers
   • CYP2D6 PM: codeine not converted to morphine (no analgesia)
   • CYP2C19 PM: clopidogrel not converted to active form (no antiplatelet effect)
4. Prodrug activation: Codeine → Morphine (CYP2D6), Cyclophosphamide → active alkylating agent (CYP2B6)

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c. Applications of Polymerase Chain Reaction (PCR)

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d. Frameshift Mutation

• The genetic code is read in triplets (codons) from a fixed starting point (AUG)
• If 1 or 2 (or 4, 5, 7...) nucleotides are inserted or deleted, all downstream codons are shifted
• This changes the amino acid sequence from the point of mutation onward
• Usually results in a premature stop codon (nonsense) downstream → truncated, non-functional protein

• Loss of protein function (most cases)
• If stop codon is not generated, abnormal elongated protein

• Duchenne Muscular Dystrophy (DMD): Frameshift deletions in dystrophin gene → no dystrophin protein → severe myopathy (Becker MD = in-frame deletion → milder)
• Insertion of 4 bases in lac operon: Shifts reading frame → no functional β-galactosidase
• BRCA1 mutations: Many are frameshift → truncated protein → breast/ovarian cancer risk

• In-frame insertion/deletion (multiple of 3): adds/removes amino acids but preserves reading frame → milder effect
• Point mutation: missense, nonsense, or silent - only one codon changed

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QUESTION 5 - Short Notes

a. Telomerase

• Protein component: TERT (Telomerase Reverse Transcriptase)
• RNA component: TERC (Telomerase RNA Component) - serves as template
• Contains the repeat sequence TTAGGG (template 3'-AAUCCC-5')

• Normal cells lose ~50-200 bp of telomere per division (end-replication problem)
• After ~50 divisions (Hayflick limit), critically short telomeres trigger senescence or apoptosis
• Telomerase adds TTAGGG repeats to 3' ends, extending telomeres

• High in: germ cells, stem cells, most cancer cells (~85% of cancers)
• Low/absent in: most somatic cells

1. Cancer: Telomerase reactivation allows unlimited cell division (replicative immortality - Hallmark of Cancer)
   • Target for anticancer therapy (telomerase inhibitors, e.g., imetelstat)
2. Dyskeratosis congenita: Mutations in TERT/TERC → short telomeres → premature aging, bone marrow failure
3. Aging: Telomere shortening = biological clock; shorter telomeres linked to age-related diseases
4. Regenerative medicine: Telomerase activation to rejuvenate stem cells

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b. Activation of Proto-Oncogenes

1. Point Mutation:
   • Single nucleotide change → constitutively active protein
   • Example: RAS mutation (codon 12, 13, or 61) → RAS locked in GTP-bound active state → constant proliferation signal (found in 30% of all cancers)
2. Gene Amplification:
   • Multiple copies of proto-oncogene → overexpression
   • Examples: HER2/neu amplification (breast cancer), N-MYC amplification (neuroblastoma), EGFR amplification (glioblastoma)
3. Chromosomal Translocation:
   • Gene placed under a strong promoter of another gene
   • Example: t(9;22) Philadelphia chromosome → BCR-ABL fusion protein (CML) → constitutively active tyrosine kinase
   • t(8;14) → MYC placed under IgH promoter (Burkitt lymphoma)
4. Insertional Mutagenesis:
   • Retroviral insertion near a proto-oncogene → activation by viral promoter/enhancer
5. Epigenetic Activation:
   • Promoter demethylation → overexpression

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c. Role of Free Radicals in Atherosclerosis

1. LDL Oxidation:
   • LDL enters the intima of arteries and is oxidized by ROS → Oxidized LDL (ox-LDL)
   • Ox-LDL is pro-inflammatory and cytotoxic to endothelium
   • Ox-LDL is not recognized by normal LDL receptors → taken up by scavenger receptors (SR-A, CD36) on macrophages → no feedback inhibition → foam cell formation
2. Endothelial Dysfunction:
   • ROS (especially O₂•⁻) react with nitric oxide (NO) → peroxynitrite (ONOO⁻)
   • Reduced NO → impaired vasodilation, increased adhesion molecule expression (VCAM-1, ICAM-1, E-selectin)
   • Monocytes adhere to endothelium and migrate to intima
3. Foam Cell Formation:
   • Macrophages engulf ox-LDL via scavenger receptors → lipid-laden foam cells → fatty streak (earliest atherosclerotic lesion)
4. Inflammation Amplification:
   • Ox-LDL activates NF-κB → inflammatory cytokines (IL-1β, TNF-α, IL-6) → recruit more monocytes
   • ROS cause smooth muscle cell proliferation → fibrous plaque
5. Plaque Destabilization:
   • ROS activate matrix metalloproteinases (MMPs) → degrade fibrous cap → vulnerable plaque → rupture → thrombosis → MI/stroke

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d. Doctor-Patient Relationship

1. Paternalistic (Priestly) model: Doctor decides what is best; patient is passive
2. Informative (Technical) model: Doctor provides information; patient decides (autonomy dominant)
3. Interpretive model: Doctor interprets patient values and helps make decisions aligned with them
4. Deliberative model: Doctor acts as a teacher/friend, guiding patient toward best choice

• Autonomy: Respect patient's right to make informed decisions; informed consent
• Beneficence: Act in the patient's best interest
• Non-maleficence: "First, do no harm"
• Justice: Fair treatment; equitable distribution of resources

• Trust and confidentiality (fiduciary relationship)
• Effective communication (active listening, empathy, non-judgmental)
• Informed consent (disclosure, comprehension, voluntariness, competence, decision)
• Shared decision-making: Patient participates in treatment planning
• Continuity of care

• Duty to treat
• Maintain patient confidentiality
• Not abandon a patient
• Avoid undue influence or exploitation
• Maintain professional boundaries

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