Lecture Notes, Test Review 2-4 PDF

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Professor Kline; all notes for 2nd exam - Professor Kline; all lecture notes for third test - Professor Kline; all lecture notes from fourth unit (on final) merged files: Test2Notes.docx - BioTest3.docx - BioTest4.docx...

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Biology Test 2 Notes!!!!  Protein: structure and function review o Types: receptors in PM, signal, gene regulatory, motor (dynein, Kinesin), structural (actin, MT, IF), enzymes, transport, storage, special purpose unique o Keratin (main protein in hair, IF): intertwining coils joined by H-bonds= subject to humidity, rain, etc.; blow drying reorganizes these noncovalent bonds—disulfide bonds stabilize filaments in more permanent ways o Folds into 3D shape because AA side-chain R group interactions (folding is determined by weaker non-covalent bonds= easier to break) o Structure: 1. Primary: AA sequence (covalent peptide bond) 2. Secondary: alpha helix (hydrophobic AAs with H-bonding= more interior—hydrophobic vs. hydrophilic) and beta pleated sheet (wrap around parallel, H-bonds interactions) (Interactions of non-covalent bonds between side chains) 3. Tertiary: full 3D structure (interactions of non-covalent bonds between side chains); determined by AA sequence and non-covalent bonds between AA side chains 4. Quaternary: protein composed of >1 polypeptide chain (hemoglobin) o Protein Domain: (functional domain) segment of a polypeptide chain that folds independently into a stable structure (each protein end could be doing something different) o Disulfide Bonds: extra stabilizing covalent cross-linkages some proteins have (not easily broken); doesn’t change confirmation, just reinforces it (doesn’t make 3D, just forms if cysteine side chains are close enough); forms from oxidation of 2 –SH: SH + SH  S-S + 2H+ + 2 e-; usually on external proteins (cytosol has many reducing agents, therefore can be oxidized)  Amino Acids: (building block of protein) (linked by covalent peptide bonds) o Basic Structure: carboxyl, amine, and R group; peptide bond + dehydration synthesis (OH and H from H2N)= polymer (protein) o Terminal NH2 (amine) end assembled 1st on ribosome (Review how ribosomes work; 5 to 3) o There are polar (+, -, or uncharged side chain; hydrophilic= found in exterior of globular protein) and nonpolar AAs (nonpolar side chain; hydrophobic= found in interior) o Acid/Base Chemistry of AAs: charge depends on pH (like enzymes)  Low pH: AA protonated at amine/carboxyl functions; AA carries net +charge= diprotic acid (2 pKa’s)  High pH: carboxyl/amine groups= deprotonated; AA carries net –charge= dibasic  Intermediate pH: AA is a zwitterions= 0net charge= isoelectric point of AA and is designated pHI  Covalent Bonds: strongest chemical bonds, formed by sharing of a pair of electrons  Non-covalent Bonds: Hydrogen, Ionic, Van der Waals (formed between non-polar parts of a molecule)  Hydrophobic Interactions: nonpolar molecules can’t form H-bonds with water  insoluble in water  Denaturation: loss of biological activity; agents= pH, temperature, ionic strength, solubility o Most common observation: precipitation or coagulation of protein o Involves disruption and possible destruction of 2° and 3° (NOT 1°) structures and structural/functional properties of proteins may be permanently altered (egg)  1° not disrupted= AA sequence remains same (denaturation doesn’t break peptide bonds)  Secondary: normal alpha and beta shape disrupted  Tertiary: has types of bonding interactions between “side-chains” that can all be disrupted (H-bonding, salt bridges, disulfide bridges, nonpolar hydrophobic interactions)  Overall: denaturation occurs because bonding interactions responsible for secondary (H-bonds to amines) and tertiary are disrupted  Ex. Eggs are cooked  proteins= denatured= easier for enzymes to digest (egg white shows coagulation of protein) o Breaks non-covalent bonds (H-bonds), not covalent bonds (peptide) o Proteins fold into a confirmation of lowest energy= proteins are stable [Ex. Urea denatures proteins, but removal of urea= original confirmation/shape of protein returns) o Heat can be used to disrupt H-bonds and nonpolar hydrophobic interactions (heat increases Kinetic E  molecules vibrate so rapidly and violently that the bonds are disrupted) o Ex. Medical supplies/instruments are sterilized by heating to denature bacteria’s proteins= destroys bacteria o Alcohol also disrupts Intramolecular H-bonding between “side chains”= new H-bonds form between new alcohol molecule and protein side chain  Ex. 70% alcohol—ethanol—solution is used as a disinfectant on skin  70% = can penetrate bacteria cell walls and denature proteins/enzymes inside, whereas 95% would only coagulate proteins on outside of cell and prevent any alcohol from entering cell  Insulin: 2 protein chains connected by disulfide bonds; 1st protein to be sequenced (Fredrick Sanger) o Glucose transport route: Insulin  insulin receptor  glucose transport  signal transduction  ATP production o Diabetes I: insufficient insulin production in response to glucose levels in blood  requires insulin injection o Past source of insulin= cow or pig pancreas insulin o Today: 1978 biotechnology (genetic information is stored in sequence of DNA bases)  artificial insulin (produced in a non-disease causing strain of E. coli bacteria) o Insulin gene= locate on short arm of human chromosome 11 (genetic engineering processes can make human insulin: human insulin DNA placed into DNA of a 2nd organism  host organism becomes an insulin-producing factory) o Recombinant DNA technology: how make insulin in a micro-organism  Protein Gel Electrophoresis: bio lab study of proteins o Proteins= charged= they move in an electrical field (- charge attracted to anode +) o Proteins= folded (NOT linear) o Migration rate depends on net charge, size, and shape o Larger protein have greater charge but they have more drag= move slower 1. Native or non-denaturing gel: separates proteins based on size (mass) and charge

Biology Test 2 Notes!!!!

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o Ex. Sickle Cell Anemia (2 hemoglobin S genes= SS; S gene has less (-) charge) o Glu (normal gene): carboxylic acid in side chain, hydrophilic, contributes 1 (-) charge to polypeptide chain at a neutral pH o Val and other variations (mutant gene): hydrophobic side chain R groups, uncharged at neutral pH (= less attracted to anode= moves slower) 2. SDS-PAGE polyacrylamide gel (denaturing gel): separates proteins based on size (mass) after masking intrinsic charge (in lab: analyzed fish muscle protein by SDS) o SDS solubilizes protein (3D structure disrupted  linear and more negative—charge masked) o Negative charge binds to hydrophobic regions= protein unfolds o Masks all protein charges= polypeptide moves toward anode o May add Mercaptoethanol to break S-S bonds in order to separate multi-subunit proteins Neuromuscular Junction: Motor neuron and muscle interaction o Synaptic vesicles: docked at membrane awaiting signal ( signal  contraction) o Action Potential: propagation of AP jump starts process of muscle contraction; crucial= Ca2+ entrance= initiates process o NT= acetylcholine (Ach) o Receptor ion channel opened by NTs  inflow of Na+  Muscle contraction o Vesicle docks  entering Ca2+ binds to synaptotagmin  SNARE complexes form to full membranes together  Ca2+-bound synaptotagmin catalyze membrane fusion o SNARE proteins: vesicle docking at PM  V-snare (vesicle): on NT  T-snare (target): on PM  Snap 25 connects 2 SNAREs (V and T come close and interact) Endosome: membrane-enclosed compartment in eukaryotic cells that has distinct structure, macromolecular composition, and functions in uptake of materials from ECM Clostridium Botulinum (BoNT) Neurotoxin acts on the Neuromuscular Junction o Receptor-mediated endocytosis: acts on motor neuron (NOT muscle—just place of injection): material  early endosome  late endosome  fuse with lysosome, but toxin not destroyed here (decreased pH in endosome as it gets closer to fusing with lysosome) o Gram-positive, spore forming, anaerobic, bacteria o In soil everywhere (carries out fermentation, turns over CO2 in the environment) o Spores= heat-resistant and require high temperatures to be destroyed o Produces 8 kinds of Botulinum Toxin (3-4 are disease causing—Botulism, can be very potent: 1 pictogram per kg body weight) Botulinum Toxin: 3D structure; amino and carboxyl terminal ends with strong, covalent peptide bonds (Light-chain continuous peptide and Heavy-chain—disulfide bond keeps L and H-chains together); protein domains; 3 parts= L, HN, HC (peptide bond connects parts) o Comes from the bacteria Clostridium Botulinum o Cut when taken up by endosome, cut down S-S bond o Contains: Light-chain Zinc-dependent endopeptidase; Dark-Chain: HN translocates light chain across vesicle membrane; HC binds to neuron 1. L: zinc-dependent endopeptidase (enzyme that cleaves other polypeptide chains) specific for SNARE proteins (which are core components of the NT release apparatus); detaches from dark and is released into cytosol to cleave SNAP 25 in order to prevent Ach from docking, which inhibits muscular contraction 2. HN: H chain’s part composed of a N-terminal domain and involved in translocating the light chain across the vesicle membrane (membrane translocation) 3. HC: H chain’s C-terminal portion responsible for binding the neuron (and for tetanus toxin retrograde transport); formed by 2 independently folded domains (Hcn and Hcc) o Binds to neuron receptors by Hc domain (but in order to become active, toxin must be cleaved into 2 polypeptide chains and the disulfide bond removed—toxin cleaved into light and heavy chains while disulfide bond reduced) 1. Change in pH, disulfide bond broken, releases light chain from endocytotic vesicle via heavy chain transporter 2. Translocated light chain across vesicle membrane (decreasing in endosome pH  changes structure of toxin) o Inactive in vesicles, but as pH decreases: 1. Heavy chain changes confirmation catalytic part of A chain exposed  disulfide bond disrupted (must happen for toxin to become active) 2. Structural changes expose hydrophobic regions heavy chain penetrates membrane and makes pore through which light chain goes into cytosol (light chain released from vesicle using H-chain transporter)  released light chain blocks exocytosis o Blocks NT release by cleaving synaptobrevin (acts on V-SNARE): synaptic vesicle can’t dock so it can’t fuse and no Ach can be released= no muscle contraction (How muscle relaxation caused) [2 cuts involved: BT cut in half and then light chain cuts V-SNARE) o Heat sensitive toxin (not very stable at room T, can be destroyed by boiling/heating) o Food-born botulism: food in which spores have germinated  bacteria grows (ex. in canned food grows anaerobically/ferments) and produces toxin  toxin absorbed by gut  passes into blood system (toxin only acts if enters bloodstream after digestion or through open wound, etc.) o Botulism= an intoxication (NOT an infection, except in infant botulism) from eating uncooked food in which spores have germinated; rare but serious paralytic illness caused by nerve toxin produced by this bacterium o Lethal dose of toxin needed to provoke an immune response= no immunity to toxin o Treatment: toxin can be neutralized by an antitoxin (antibody produced by inactivated toxin in horses or other animals), but only produces passive immunity (short-term); vaccine (no licensed one available for human use, recombinant vaccine composed of HC domains) o Causes muscles to relax

Biology Test 2 Notes!!!!

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o Used to: stop excess sweating (hyperhidrosis), treats facial paralysis derived from stokes, treats strabismus (effect on extra-ocular eye muscle—helps eye to focus), reduces migraines, relaxes muscle twitching (dystonia), cosmetic, treats bladder dysfunction o Botox: reduces wrinkles temporarily by relaxing facial muscles that underlie and cause muscles (dose= localized so doesn’t enter blood system usually); repeated injections needed every 3-4 months (overtime less may be needed as muscles atrophy and are less contractile) o Chemical warfare: toxin 10,000x more potent than any other biological poison SNARE proteins: target and dock vesicles in all cells (NOT just neurons), involved in vesicular transport in cells (V-SNAREs carry cargo within cell, like ER to Golgi) Clostridium Tetani  Neurotoxin Tetanus Toxin (TeNT) Tetanus Toxin: leads to muscle spasm symptoms; o Blocks GABA and Glycine producing synapses of CNS= toxin causes muscle spasms/ spastic, rigid paralysis neurotoxin that binds to inhibitory interneurons of spinal cords and blocks their release of inhibitory NTs= keeps muscle in constant contraction (if excitatory NTs cant be inhibited then Ach release won’t be stopped and muscles can’t relax  spastic paralysis—all muscles contract) o Stimulus and activation on receptor (input in PNS)  activates sensory neuron  interneuron (some additional interneurons release an inhibitory NT)  information processing in CNS  activation of motor neuron  (Ach released at neuromuscular junction)  muscle (response) o The additional inhibitory interneurons make the neuron less excitable by inhibiting the motor neuron o Transported through neuron to an adjacent neuron in spinal cord and alters NT release in those cells  hyper-excitability of motor neuron  excessive release of Ach at neuromuscular junction  rigid paralysis o Retrograde transport: on MTs; transports TeNT through neuron to an adjacent inhibitory neuron in the spinal cord and alters NT release in those cells [travels up motor neuron into CNS (spinal cord) (by MT and dynein) and are taken up by inhibitory NT (blocks release of NT by cutting V-SNARES in inhibitory interneuron)] o Taken up by vesicle (NOT released from vesicle like BoNT light chain); released in inhibitory interneuron Tetanus, diphtheria, pertussis= can be very serious disease; protection= T-dap vaccine; cause= bacteria o Tetanus (lockjaw)= Risus Sardonicus; causes painful muscle tightening and stiffness; kills 1 in 5 infected o Diphtheria: can cause thick coating to for in back of throat o Pertussis (whooping cough) Receptor-mediated Endocytosis: (how BoNT and TeNT taken up) selective, specific, and efficient o Clathrin: protein coating (3-legged structure) that fit together to form a “Cage”); mediates endocytosis-vesicle formation o Ex. LDL carries cholesterol in to the cell: Cholesterol and LDL  LDL receptors Clathrin coated vesicle (endocytosis)  uncoating  fusion with endosome  transfer to lysosome and budding off of transport vesicles  LDL digested by lysosome and cholesterol released into cytosol AND return of LDL receptors to PM 1. Bind to receptors, adpatin (from inside) helps connect 2. Clathrin-coated vesicle forming  un-coating  fusion with endosome  a. Transfer to lysosome (pH decreases from endosome to lysosome) to free cholesterol from LDL and into cytosol b. Budding off  return to membrane (Pinocytosis= internalization of Clathrin coated pits with EC fluid; membrane turnover may be 100% in an hour!, but less effective than RME) Exocytosis: (secretory pathway) involves ER, Golgi, secretory/transport vesicles o Proteins in ER destined for export then transported by vesicle o Secretion and addition of membrane proteins and lipids Protein synthesis starts on free ribosomes in cytoplasm (NOT RER); where these proteins go: o Nucleus, chloroplast, mitochondria, peroxisome, ER ( Golgi)—enters ER while being synthesized (vs. completely formed before entering chloroplast and mitochondria) o Ways transported:  Gated Transport: through pores into nucleus; has gates because must keep in, allow in, and allow (mRNA) out: proteins  Transmembrane Transport: proteins transported across membranes of chloroplast, mitochondria, peroxisome  Vesicular Transport: packaged into vesicles that’ll move through cell (to ER  Golgi)  Proteins designated to leave the cell begin synthesis in cytoplasm, the enter ER Signal Sequences: sorting signal that consists of a short continuous sequence of AAs (usually on amino terminal end), determines where a protein goes; usually, but not always removed from final protein; for import into transport locations Signal Patches: protein sorting signal that consists of a specific 3D structure of atoms on the folded protein surface (not one end, within structure—can be multiple patches) Signal AA recognized by SRP (Signal-receptor protein): o SRP attaches to signal AA  connects to SRP receptor (reason ribosomes are on RER)  SRP released  as protein is made it’s inserted through membrane by protein translocator and it finished synthesis at ER  automatically folds up into 3D protein shape o ER signal sequence and SRP direct ribosome to ER (signal sequence is for import into ER or retention in ER) (proteins destined for secretion=imported into ER lumen then vesicular transport) o Protein threaded through then released, but not all released—some remain in the membrane due to hydrophobic stop sequence, phobic vs. philic o (Proteins enter ER while being synthesized) Functions of RER: only rough when ribosome brings protein to be finished o Produces proteins that’ll go to PM or Golgi or secretory/transport vesicles, ER itself vacuoles, lysosome, endosomes o Sugar groups added to form glycoproteins o Lumen  secretion (protein folding) protein quality control o Proteins now carried in vesicles (NOT free) SER: ER= one continuous tubular network o Makes lipids (in some cases: metabolizes lipids)

Biology Test 2 Notes!!!! Makes steroid hormones (uses cholesterol) in adrenal cortex and endocrine glands Detox function (like in liver): organic chemicals  safer soluble productions (liver cells: detoxify metabolic products like ethanol and barbiturates) Golgi: modifies proteins delivered from ER; glycoproteins (sugars added to protein here) o Enzymes within cisternae  modify proteins by adding carbs (glycosylation) and phosphates (phosphorylation) o Transport of lipids o Formation of lysosome (proteins destined for lysosomes are labeled with mannose-6-phsophate) Gene Regulation Fertilization: (46 chromosomes); implantation is necessary o Zona Pelusada: EC coat layer sperm must go through to enter egg o Sperm enters (n+n= 2n zygote)  nucleus fusion  exocytosis of molecules to keep other sperms from entering (blocks polysemy) o Fertilized egg (has 2 pro-nuclei—male and female)  1 nucleus zygote  cell division (mitosis) in oviduct  morula (collection of cells)  (some differentiation to produce inner cell mass and trophectoderm)  blastocyst formation (hollow ball with layer of cells on inside and outside)  implant in uterus  placenta creation encouraged  inner cell mass of blastocyst eventually forms embryo, while outer cells (trophectoderm) makes embryonic placenta (NOT involved in embryo creation) Twins: Identical= Monozygotic (totipotent cells can produce entire embryo; splitting of early 2 cell stage= 2 blastocysts) vs. Fraternal (Dizygotic Twins)= non-identical fertilized eggs, 2 blastocysts Undifferentiated Cells: unspecialized; can grow, divide, and reproduce o Inner cell mass: (developed by day 7-9) potential to form any cell in the body (pluripotent), but can NOT make an individual on its own (can’t implant) Differentiated Cells: specific function; same genetic combination as undifferentiated but produce different set of proteins that turn on/off genes Stem Cells: 1. Unspecialized (undifferentiated) 2. Capable of dividing and renewing for longs periods of time without differentiating 3. Can give rise to specialized cell types Embryonic Stem Cells: source= blastocyst inner cell mass (can also come from IVF clinic—leftover lab embryos); advantage= can be cultured for a long time o Has all properties of stem cells (pluripotent) o Potential uses= many (drug research, gene control, tissues/cells differentiated for therapy—bone marrow, nerve cells, heart muscle cells, pancreatic islet cells, can treat disease) o Problem in culture: can form embryoid bodies (if cells allowed to clump together they begin to differentiate spontaneously) that can form all 3 germ layers (form of teratoma in lab) Adult Stem Cells: restriction= doesn’t grow and proliferate well in lab (and multipotent) o All properties of stem cells, BUT restriction= multipotent (differentiate to yield all specialized cell types of tissue from which it originated; variety of cells can b...