B.Sc. Zoology Cheatsheet

Cheatsheet Content

### Enzyme Activity Enzymes are biological catalysts that speed up biochemical reactions without being consumed. #### a) Effect of different factors on enzyme activity - **Temperature:** - Increase in temperature generally increases reaction rate up to an optimum. - Beyond optimum, enzymes denature (lose shape), leading to loss of activity. - Low temperatures decrease activity but do not denature the enzyme. - **pH:** - Each enzyme has an optimal pH range. - Deviations from optimal pH can alter the enzyme's active site, reducing or abolishing activity. - Extreme pH values cause denaturation. - **Substrate Concentration:** - Increase in substrate concentration increases reaction rate up to a saturation point. - At saturation, all active sites are occupied, and the enzyme is working at its maximum capacity ($V_{max}$). - **Enzyme Concentration:** - Reaction rate is directly proportional to enzyme concentration, assuming abundant substrate. - **Presence of Activators/Inhibitors:** - **Activators:** Molecules that increase enzyme activity (e.g., cofactors, coenzymes). - **Inhibitors:** Molecules that decrease enzyme activity (e.g., competitive, non-competitive, uncompetitive inhibitors). #### b) Regulation of enzyme activity - **Allosteric Regulation:** - Binding of effector molecules at a site other than the active site (allosteric site). - Can activate or inhibit enzyme activity by changing the enzyme's conformation. - **Covalent Modification:** - Addition or removal of chemical groups (e.g., phosphorylation, dephosphorylation). - Often reversible and plays a crucial role in cellular signaling. - **Proteolytic Activation:** - Irreversible activation of inactive precursor enzymes (zymogens or proenzymes) by cleavage of specific peptide bonds. - E.g., activation of digestive enzymes like trypsinogen to trypsin. - **Genetic Control (Enzyme Synthesis/Degradation):** - Regulation of the rate of enzyme synthesis (transcription and translation) or degradation. - Allows for long-term control of enzyme levels. - **Feedback Inhibition:** - End-product of a metabolic pathway inhibits an enzyme early in the pathway. - Prevents overproduction of the end-product. #### c) Lock and key and induced fit model of enzyme action - **Lock and Key Model (Emil Fischer, 1894):** - Enzyme's active site has a rigid, pre-formed shape that perfectly complements the substrate, like a lock and its specific key. - Implies a static interaction. - Limited in explaining enzyme flexibility and broad specificity. - **Induced Fit Model (Daniel Koshland, 1958):** - The active site is flexible and undergoes a conformational change upon substrate binding. - The enzyme "induces" a change in the substrate, and the substrate "induces" a change in the enzyme, leading to a tighter fit and optimal catalytic environment. - Explains enzyme-substrate specificity and how enzymes facilitate transition states more effectively. ### Physiological Mechanisms #### a) Positive feedback mechanism with a suitable example - **Definition:** A regulatory mechanism where the output of a system enhances the original stimulus, leading to an amplification of the effect. It drives physiological processes to completion. - **Characteristics:** - Amplifies the response. - Often involved in processes that require rapid, intense completion. - Less common in homeostatic regulation than negative feedback. - **Example: Childbirth (Oxytocin Release):** 1. **Stimulus:** Head of the baby pushes against the cervix. 2. **Receptor:** Nerve impulses from the cervix are sent to the brain. 3. **Control Center:** Brain stimulates the posterior pituitary to secrete oxytocin. 4. **Effector:** Oxytocin travels in the bloodstream to the uterus, causing stronger uterine contractions. 5. **Response:** Contractions push the baby further down the birth canal, increasing cervical stretching, which in turn leads to more oxytocin release, amplifying contractions until birth. #### b) Osmotic and ionic regulation in hypo-osmotic environment - **Hypo-osmotic Environment:** An environment where the external solute concentration is lower than the internal solute concentration of an organism (e.g., freshwater for fish). - **Challenge:** Water tends to rush into the organism by osmosis, and salts tend to diffuse out. - **Adaptations (e.g., Freshwater Fish):** - **Water Balance:** - **Kidneys:** Produce large volumes of dilute urine to excrete excess water. - **Body Surface:** Body surface (skin, scales) is relatively impermeable to water. - **Ion Balance:** - **Gills:** Specialized chloride cells actively absorb ions (Na+, Cl-) from the surrounding water against a concentration gradient. - **Diet:** Obtain some ions from food. - **Lack of Drinking:** Do not drink water, as this would increase water influx. - **Overall Goal:** Maintain internal osmotic and ionic homeostasis despite the external gradient. #### c) Hibernation - **Definition:** A state of minimal activity and metabolic depression in endotherms, characterized by lower body temperature, slower breathing, and lower metabolic rate. It allows animals to conserve energy during periods of food scarcity and cold temperatures. - **Characteristics:** - **Reduced Body Temperature:** Body temperature drops significantly, sometimes close to ambient temperature. - **Decreased Metabolic Rate:** Metabolism slows down drastically (e.g., by 90-95%). - **Slowed Heart Rate & Respiration:** Heart rate and breathing become very slow and shallow. - **Energy Source:** Animals rely on stored fat reserves for energy. - **Periodic Arousals:** Hibernators periodically wake up for short periods (e.g., to urinate, move to a new location), which is energetically costly but necessary for physiological maintenance. - **Triggers:** Cold temperatures, reduced food availability, shorter day length. - **Examples:** Bears (though often considered "winter sleepers" due to less drastic body temp drop), groundhogs, hamsters, bats. ### Histology & Hormones #### a) Histological structure of Thyroid gland and its functions - **Location:** Located in the neck, anterior to the trachea, below the larynx. - **Gross Structure:** Consists of two lobes connected by an isthmus. - **Histological Structure:** - **Follicles:** The functional unit of the thyroid gland. Spherical structures lined by a single layer of **follicular cells** (cuboidal epithelium). - **Colloid:** The central lumen of each follicle is filled with a proteinaceous substance called colloid, primarily composed of thyroglobulin. This is where thyroid hormones are stored. - **Parafollicular Cells (C-cells):** Located between the follicles (not part of the follicular lining), these cells secrete calcitonin. - **Rich Blood Supply:** The gland is highly vascularized to facilitate hormone transport. - **Functions:** - **Follicular Cells:** Synthesize and secrete thyroid hormones: - **Thyroxine (T4) and Triiodothyronine (T3):** Regulate basal metabolic rate, promote growth and development (especially nervous system), and influence body temperature. - **Parafollicular Cells (C-cells):** - **Calcitonin:** Lowers blood calcium levels by inhibiting osteoclast activity and stimulating calcium excretion by kidneys. #### b) Histological structure of Pancreas and functions of its hormones - **Location:** Posterior to the stomach, extending from the duodenum to the spleen. - **Histological Structure:** - **Exocrine Pancreas (Acinar Cells):** - Majority of the pancreas (80-85%) consists of **acini**, clusters of epithelial cells that secrete digestive enzymes. - These enzymes (e.g., amylase, lipase, proteases) are transported to the duodenum via pancreatic ducts. - **Endocrine Pancreas (Islets of Langerhans):** - Scattered throughout the exocrine tissue are small clusters of endocrine cells called **Islets of Langerhans** (1-2% of pancreatic mass). - Contain different cell types: - **Alpha ($\alpha$) cells:** Secrete Glucagon. - **Beta ($\beta$) cells:** Secrete Insulin. - **Delta ($\delta$) cells:** Secrete Somatostatin. - **PP cells (Gamma cells):** Secrete Pancreatic Polypeptide. - **Functions of its Hormones:** - **Insulin (from $\beta$ cells):** - Lowers blood glucose by promoting glucose uptake by cells, glycogenesis (glucose to glycogen storage), and fat synthesis. - **Glucagon (from $\alpha$ cells):** - Raises blood glucose by promoting glycogenolysis (glycogen to glucose breakdown) and gluconeogenesis (glucose synthesis from non-carbohydrate sources) in the liver. - **Somatostatin (from $\delta$ cells):** - Inhibits the secretion of both insulin and glucagon, as well as growth hormone and TSH. It acts as a paracrine regulator within the islets. - **Pancreatic Polypeptide (from PP cells):** - Regulates pancreatic exocrine secretion and generally inhibits gallbladder contraction and gut motility. #### c) Mechanisms of hormone action - **Definition:** Hormones are chemical messengers that exert their effects on target cells by binding to specific receptors. - **Two Main Mechanisms:** 1. **Steroid/Thyroid Hormone Action (Intracellular Receptors):** - **Hormones:** Lipid-soluble (e.g., steroid hormones like estrogen, testosterone, cortisol; thyroid hormones T3, T4). - **Mechanism:** - These hormones easily diffuse across the cell membrane. - Bind to specific receptor proteins located in the cytoplasm or nucleus of target cells. - The hormone-receptor complex then acts as a transcription factor, binding to specific DNA sequences (hormone response elements, HREs) in the nucleus. - This binding either activates or represses the transcription of specific genes, leading to the synthesis of new proteins (e.g., enzymes, structural proteins). - This mechanism typically results in slower but long-lasting effects. 2. **Peptide/Protein Hormone Action (Cell Surface Receptors):** - **Hormones:** Water-soluble (e.g., insulin, growth hormone, adrenaline, oxytocin). - **Mechanism:** - These hormones cannot pass through the lipid bilayer of the cell membrane. - Bind to specific receptor proteins located on the outer surface of the target cell membrane. - This binding activates intracellular signaling pathways, often involving **second messengers** (e.g., cAMP, cGMP, IP3, DAG, Ca2+). - The second messengers then trigger a cascade of events, leading to a rapid change in cell function (e.g., enzyme activation/inhibition, altered ion channel activity, protein phosphorylation). - This mechanism typically results in rapid but short-lived effects. ### Cell Culture & Sterilization #### a) Method used for isolation and preparation of cells for culture - **Primary Cell Culture:** Cells directly isolated from tissues. - **Steps:** 1. **Tissue Collection:** Obtain tissue sample (e.g., biopsy, organ explant) under sterile conditions. 2. **Mincing:** Mechanically cut the tissue into small fragments to increase surface area. 3. **Enzymatic Digestion (Disaggregation):** - Treat tissue fragments with enzymes (e.g., trypsin, collagenase, dispase) to break down the extracellular matrix and dissociate cells. - Incubation at 37°C for a specific duration. 4. **Inactivation of Enzyme:** Add serum (e.g., Fetal Bovine Serum - FBS) to neutralize residual enzyme activity. 5. **Filtration/Centrifugation:** - Filter the cell suspension to remove undigested tissue fragments. - Centrifuge to pellet the cells and remove enzyme solution and debris. 6. **Washing and Resuspension:** Wash cells multiple times with balanced salt solution (BSS) to remove residual enzymes and debris, then resuspend in complete culture medium. 7. **Cell Counting & Viability:** Count cells using a hemocytometer and assess viability (e.g., trypan blue exclusion). 8. **Plating:** Seed cells into culture vessels (flasks, plates) at an appropriate density. 9. **Incubation:** Place cultures in a CO2 incubator (37°C, 5% CO2, humidified atmosphere). #### b) Types of media used for animal tissue culture - **Basic Components of Culture Media:** - **Inorganic Salts:** Maintain osmotic balance, regulate membrane potential, provide essential ions (Na+, K+, Ca2+, Mg2+, Cl-, PO43-). - **Amino Acids:** Building blocks for proteins. - **Vitamins:** Cofactors for enzymatic reactions. - **Glucose:** Primary energy source. - **Buffers:** Maintain pH (e.g., bicarbonate buffer system with CO2). - **Antibiotics:** Prevent bacterial contamination (e.g., penicillin, streptomycin). - **Phenol Red:** pH indicator (turns yellow if acidic, purple if basic). - **Supplements:** - **Serum (e.g., FBS):** Most common supplement. Provides growth factors, hormones, attachment factors, protease inhibitors, trace elements. (Can be serum-free for specific applications). - **Growth Factors:** Specific proteins that stimulate cell proliferation and differentiation. - **Hormones:** (e.g., insulin, hydrocortisone) to promote specific cell functions. - **Trace Elements:** Essential micronutrients. - **Common Media Types:** - **Dulbecco's Modified Eagle Medium (DMEM):** High glucose, rich in amino acids and vitamins. Widely used. - **RPMI-1640:** Developed for lymphocyte culture, suitable for many hematopoietic cells. - **Minimum Essential Medium (MEM):** Simpler, used for a wide range of adherent cells. - **Ham's F10/F12:** Used for clonal growth of various cells, often in serum-free conditions. #### c) Basic principles of sterilization - **Definition:** The complete destruction or removal of all viable microorganisms (bacteria, viruses, fungi, spores) from an object or surface. - **Methods:** 1. **Heat Sterilization:** - **Autoclaving (Moist Heat):** Most common and effective method. Uses saturated steam under pressure (e.g., 121°C, 15 psi for 15-20 min). Coagulates and denatures proteins. Used for media, glassware, instruments. - **Dry Heat (Hot Air Oven):** Used for heat-stable materials (e.g., glassware, metal instruments) that cannot withstand moisture. Requires higher temperatures and longer times (e.g., 160-170°C for 1-2 hours). Oxidizes cellular components. 2. **Filtration Sterilization:** - Used for heat-sensitive liquids (e.g., serum, antibiotics, vitamins, growth factors). - Passes liquid through membranes with very small pore sizes (e.g., 0.22 µm) to physically remove microorganisms. 3. **Chemical Sterilization (Cold Sterilization):** - Uses liquid or gaseous chemicals (e.g., ethylene oxide, hydrogen peroxide, glutaraldehyde). - Used for heat-sensitive, moisture-sensitive items (e.g., plastics, medical devices). - Requires careful handling due to toxicity. 4. **Radiation Sterilization:** - **Ionizing Radiation (Gamma Rays, X-rays, Electron Beams):** Used for pre-packaged disposable items (e.g., plastic petri dishes, syringes). Damages DNA and cellular structures. - **Non-ionizing Radiation (UV Light):** Used for surface sterilization (e.g., inside laminar flow hoods) and air purification. Limited penetration. - **Key Principles:** - **Aseptic Technique:** Practices used to prevent contamination during handling of sterile materials. - **Validation:** Ensuring the sterilization process consistently achieves sterility. - **Sterility Assurance Level (SAL):** Probability of a single viable microorganism remaining after sterilization. ### Short Notes #### a) Lineweaver Burk plot and its significance - **Definition:** A double reciprocal plot of enzyme kinetics, where the reciprocal of the initial reaction velocity ($1/V_0$) is plotted against the reciprocal of the substrate concentration ($1/[S]$). It is derived from the Michaelis-Menten equation. - **Equation:** $1/V_0 = (K_m/V_{max}) \cdot (1/[S]) + 1/V_{max}$ - **Plot Characteristics:** - A straight line. - **Y-intercept:** $1/V_{max}$ - **X-intercept:** $-1/K_m$ - **Slope:** $K_m/V_{max}$ - **Significance:** - **Accurate Determination of $K_m$ and $V_{max}$:** Provides a more precise way to determine these kinetic parameters compared to the hyperbolic Michaelis-Menten plot, especially at high substrate concentrations. - **Analysis of Enzyme Inhibition:** - **Competitive Inhibitor:** $V_{max}$ remains unchanged (same Y-intercept), $K_m$ increases (X-intercept shifts closer to zero). - **Non-Competitive Inhibitor:** $V_{max}$ decreases (Y-intercept increases), $K_m$ remains unchanged (same X-intercept). - **Uncompetitive Inhibitor:** Both $V_{max}$ and $K_m$ decrease (parallel lines, both intercepts shift). - **Visualization of Kinetic Data:** Helps in understanding enzyme mechanisms and the effects of various factors. - **Disadvantage:** Can distort experimental error, especially at low substrate concentrations (where $1/[S]$ is large). #### b) Ectothermy versus Endothermy - **Ectothermy (Poikilothermy / Cold-blooded):** - **Definition:** Animals that primarily rely on external environmental sources of heat to regulate their body temperature. - **Heat Source:** Absorbs heat from the sun, warm surfaces, or warm air/water. - **Metabolic Rate:** Generally lower metabolic rates. - **Energy Cost:** Lower energy expenditure for temperature regulation. - **Behavioral Regulation:** Use behavioral strategies like basking in the sun, seeking shade, burrowing, or changing orientation to control body temperature. - **Examples:** Reptiles (lizards, snakes), amphibians (frogs, salamanders), fish, invertebrates. - **Endothermy (Homeothermy / Warm-blooded):** - **Definition:** Animals that primarily generate their own heat metabolically to maintain a relatively constant internal body temperature, regardless of external temperature fluctuations. - **Heat Source:** Internal metabolic processes (e.g., cellular respiration, muscle activity, shivering). - **Metabolic Rate:** Generally higher metabolic rates. - **Energy Cost:** High energy expenditure for temperature regulation, requiring more food intake. - **Physiological Regulation:** Use physiological mechanisms like shivering, sweating/panting, vasoconstriction/vasodilation, insulation (fur, feathers, fat) to control body temperature. - **Examples:** Mammals, birds. #### c) Circadian rhythms - **Definition:** Biological rhythms that oscillate with a periodicity of approximately 24 hours. They are endogenous (internally generated) but can be entrained (synchronized) by external cues, primarily light-dark cycles. - **"Biological Clock":** Controlled by a master clock, the **Suprachiasmatic Nucleus (SCN)** in mammals, located in the hypothalamus. - **Characteristics:** - **Endogenous:** Persist even in constant environmental conditions (e.g., constant darkness). - **Entrainable:** Can be reset by external cues (zeitgebers), most importantly light. - **Temperature Compensation:** The period of the rhythm is relatively stable across a range of physiological temperatures. - **Physiological Processes Regulated:** - Sleep-wake cycles - Hormone secretion (e.g., cortisol, melatonin) - Body temperature fluctuations - Feeding and digestive activity - Cell regeneration - **Significance:** Essential for maintaining internal physiological balance and adapting to environmental cycles. Disruption can lead to health issues (e.g., jet lag, shift work disorders). #### d) Hormones of Neurohypophysis and their functions - **Neurohypophysis (Posterior Pituitary):** - Not a true gland, but an extension of the hypothalamus. - Stores and releases two hormones produced by neurosecretory cells in the hypothalamus (supraoptic and paraventricular nuclei). - These hormones are transported down axons to the neurohypophysis. - **Hormones and Functions:** 1. **Antidiuretic Hormone (ADH) / Vasopressin:** - **Production:** Primarily by supraoptic nucleus. - **Function:** Increases water reabsorption in the collecting ducts and distal convoluted tubules of the kidneys. This reduces urine volume and increases blood volume/pressure. Also causes vasoconstriction at high concentrations. - **Regulation:** Released in response to increased plasma osmolarity (dehydration) or decreased blood volume/pressure. 2. **Oxytocin:** - **Production:** Primarily by paraventricular nucleus. - **Function:** - **Uterine Contractions:** Stimulates strong contractions of the smooth muscle of the uterus during childbirth (positive feedback). - **Milk Ejection (Let-down) Reflex:** Stimulates contraction of myoepithelial cells in the mammary glands, leading to milk release during suckling. - **Social Bonding:** Plays a role in social recognition, bonding, and sexual reproduction. - **Regulation:** Released in response to sensory input from cervical stretching during labor and suckling. #### e) Complete media - **Definition:** In cell culture, "complete media" refers to a basal cell culture medium (e.g., DMEM, RPMI-1640) that has been supplemented with all necessary additives to support optimal cell growth, proliferation, and survival for a specific cell type. - **Components:** - **Basal Medium:** Provides essential inorganic salts, amino acids, vitamins, glucose, and a pH buffer. - **Serum (e.g., Fetal Bovine Serum - FBS):** Most common supplement, providing growth factors, attachment factors, hormones, and protease inhibitors. Typically added at 5-20%. - **Antibiotics:** Prevent bacterial contamination (e.g., penicillin-streptomycin, gentamicin). - **Antimycotics:** Prevent fungal contamination (e.g., amphotericin B). - **L-Glutamine:** An essential amino acid for cell metabolism, often added freshly as it degrades over time. - **Other Supplements (cell-type specific):** - **Growth Factors:** Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Insulin-like Growth Factor (IGF). - **Hormones:** Insulin, hydrocortisone, thyroid hormones. - **Trace Elements:** Selenium, zinc. - **Pyruvate, Non-Essential Amino Acids (NEAA):** For enhanced growth. - **Significance:** Ensures that cells receive all the necessary nutrients and growth signals to thrive *in vitro*. The exact composition varies depending on the cell line and research objectives. #### f) Coverslip culture - **Definition:** A method of cell culture where cells are grown on small, thin pieces of glass or plastic (coverslips) placed within culture dishes or multi-well plates. - **Purpose:** Primarily used when cells need to be observed under a microscope, stained, or processed for immunofluorescence, immunohistochemistry, or electron microscopy. - **Procedure:** 1. **Sterilization:** Coverslips are sterilized (e.g., by autoclaving or UV light). 2. **Coating (Optional):** Coverslips may be coated with extracellular matrix proteins (e.g., collagen, poly-L-lysine, fibronectin) to enhance cell adhesion, especially for fastidious cells. 3. **Placement:** Sterilized coverslips are placed into sterile culture dishes or multi-well plates. 4. **Cell Seeding:** Cells are seeded onto the coverslips in complete culture medium and incubated. 5. **Processing:** After desired growth, cells on the coverslips can be easily removed, fixed, stained, mounted onto microscope slides, and observed. - **Advantages:** - Facilitates direct microscopic observation and imaging. - Allows for various staining techniques without disrupting the cell monolayer. - Useful for morphological studies, co-localization experiments, and electron microscopy. - **Disadvantages:** - More labor-intensive than standard flask culture. - Limited surface area for large-scale cell expansion.