M2M+Unit+IV+LOs

Secretory Pathways I + II Monday, November 26, 2007 6:39 AM


 * Secretory Pathways I + II, 11/26/07:**


 * Describe the 3 mechanisms of protein transport
 * Gated transport: requires a specific receptor protein to carry a folded protein from the cytosol into the nucleus through the pore complex.
 * Transmembrane transport: requires a translocator protein or protein complex (ie translocon) to move proteins across membrane.
 * Vesicular transport: membrane-bound transport; requires adaptor and coat proteins.
 * List the major functions of the ER:
 * Synthesis of lipids
 * Control of cholesterol homeostasis
 * Synthesis of proteins on membrane bound ribosomes
 * Co-translational folding of proteins and early post-translational modifications
 * "Quality control": protein degradation and turnover.
 * Describe co-translational translocation:
 * **Translocon** : Protein-conducting aqueous channel that spans the rough ER membrane.
 * Protein sorting signals: most proteins are "tagged" with a region (primary sequence or tertiary structure) that forms a "recognition patch": this tells the cell how to package the protein and where to send it.
 * Notice that there's a specific 5' sequence that signals that the transcript is going to be pulled into the ER; if it's absent, the protein remains soluble in the cytosol.
 * The 5' sequence that signals ER translocations causes the ribosome translating that protein to attach to the membrane of the ER (thus causing rough ER vs smooth ER).
 * **Co-translational translocation** : as mRNA is translated, it's moved through the translocon into the ER lumen to be cleaved by signal peptidases and folded.
 * The 5' signal sequence binds to the SRP (signal receptor particle) in the ER membrane; this opens the translocon and the nascent polypeptide is threaded through the translocon into the lumen as it's translated (the 5' signal sequence is cleaved off almost immediately so that it doesn't make up a part of the final protein).
 * SRPs: have a multi-methionine "pocket" that binds to a wide variety of 5' signal sequences.
 * 5' signal sequences: variable; often mainly nonpolar.
 * Notice that the translocon is regulated on the luminal surface as well by binding protein BiP: it can expel proteins as well as admit them.
 * If the nascent protein is meant to be a membrane protein, the protein contains another region (aside from the 5' signal sequence) that interacts with the translocon: a "stop-transfer sequence" which stops the transfer of the protein into the lumen-- thus the protein remains "stuck" with a transmembrane domain in the ER membrane, one end of the protein in the lumen, and the other end of the protein in the cytosol. The "stuck" protein then translocates out of the translocon for further packaging.
 * Quick note about which end ends up where: the positively charged end of the transmembrane domain (whichever that is) winds up in the cytosol (outside the ER) and the negatively charged end of the domain winds up in the ER lumen.
 * Notice that these transmembrane proteins can span (traverse) the membrane many times, depending on the number and location of start- and stop-transfer domains.
 * Asparagine-linked (N-linked) glycosylation: as a polypeptide is threaded through the translocon, an enzyme (oligosaccharyl transferase) catalyzes the glycosylation of select asparagine residues in the protein ("core glycosylation") with the motif N-X-S/T.
 * Part of N-linked glycosylation involves trimming the attached glycosyl groups to properly package the protein for transport to the Golgi apparatus.
 * The trimming process is what signals the ER that a protein is ready to be transported elsewhere.
 * List the major functions of the Golgi:
 * Synthesis of complex sphingolipids from the ceramide backbone
 * Additional post-translational modifications of proteins and lipids
 * Proteolytic processing (protein cleavage)
 * Sorting of proteins and lipids for Golgi compartments
 * Morphology: notice that the Golgi complex can be bigger or smaller (more or less cisternae) depending on how much packaging and modification needs to happen in that cell.
 * How the Golgi apparatus transports proteins/lipids through itself:
 * Depends on the type of things transported. Sometimes it's by moving them from cisterna to cisterna with small vesicles; sometimes it's by actually moving or modifying the entire cisterna that contains the material ("cisternal progression").
 * Which one used depends on the nature of the material-- if it won't fit inside a vesicle, tend to use cisternal progression.
 * Notice that N-linked glycosylation is finished in the Golgi: this, again, is a signal to move the protein to a new location
 * "Proproteins": proteins that undergo proteolytic cleavage late in processing (Golgi).
 * One package sorting mechanism: by thickness of the package's membrane (thicker -> plasma membrane; thinner stays in the ER membrane, that sort of thing).
 * Other sorting mechanisms:
 * There are some. They're complex. See the handout if you're curious.
 * Apical membrane: tends to be thicker than basolateral membrane.
 * Describe vesicular transport:
 * Vesicular "coats" are assembled at the site of vesicle formation: these "sort" the proteins or lipids to be moved (different coat molecules "select" various types of cargo using adaptor proteins), and aid in pushing the vesicle out the side of the membrane.
 * Notice that there are adaptor binding motifs in cargo: thus it's probably more accurate to say that the cargo "selects" the coating and adaptive molecules.
 * Process: Cargo attracts a certain type of adaptor protein; the adaptor proteins then attract a certain type of coat.
 * Notice that you need SNARE proteins: essentially, the vesicle has to be able to recognize and merge with the target cell membrane. Recall that VAMP SNAREs are found on vesicles and SNAP and syntaxin SNAREs are found on target membranes.
 * Notice that GTP-binding proteins (Rab proteins) determine specificity of docking location with the SNAREs on the outside of the vesicle.
 * These GTP-BPs can also regulate coat assembly (all kinds).
 * Once a vesicle's budded off from the ER, it's moved to the Golgi via the cytoskeletal network. The motor proteins that move them are dynein and kinesin.
 * There's a "vesicular-tubular cluster" that pre-packages the proteins before they get to the Golgi.
 * Name 3 well-studied vesicle coats:
 * Clathrin coats: highly structured and symmetrical, transport from outside of the cell to inside (__endocytosis__) or lysosomal transport to/from Golgi.
 * COPI coats: traffic __from Golgi to ER__.
 * COPII coats: traffic __from ER to Golgi__.


 * [Important notes not in the LO's:]
 * __lumen__ of organelles = interior of organelles; exocytosis from the lumen leads into the cytoplasm.
 * Exocytic pathway: getting proteins to the organelles they're going to.
 * Endocytic pathway: getting proteins, within their target organelles, to their sites of action.
 * Notice that the interiors of lymphocytes are almost entirely ER, like most cells that produce massive amount of proteins (in this case antibodies).
 * ER cisternae: grooves or spaces between folds of the ER.
 * __GPI-linked proteins__: proteins that will eventually end up on the __outside__ of the cell's plasma membrane. Glycosyl-phosphatidyl-inositol link (GPI) links the protein to the cell membrane (the primary sequence of the protein itself doesn't bind to the membrane).
 * __KDEL__ protein: protein for retrieving improperly transported packages.
 * Thing to keep in mind: a protein sequence is essentially __differentiated information__. As such, it contains not only information about its primary function but only information about where it should go and how it should be packaged to get there.

Cytoskeleton Monday, November 26, 2007 6:40 AM


 * Cytoskeleton, 11/26/07:**

[This one has lots of stuff in it and I doubt I really understand it all-- so take with caution.]


 * Discuss the concept of a cytoskeleton.
 * Set of intracellular structures that orders the inside of a cell as well as organizing it in the extracellular environment.
 * Specifically: provides cell shape, mechanical strength, locomotive structures, plasma membrane support, spatial organization of organelles, and intracellular transport "roads".
 * Describe two types of cytoskeletal elements, their properties, their functional roles, and their protein composition.
 * All cytoskeletal elements: polyprotein structures.
 * Three types: microfilaments (listed on slides as "actin filaments"), microtubules, and intermediate filaments. The latter two are discussed here.
 * **Microtubules** : hollow tubular structure, very flexible, don't stretch. Primarily provide the scaffold for spatial organization and movement of organelles, and also the movements of cilia and flagella. Outer diameter 25 nm. Often clustered near the nucleus (used during mitosis).
 * **Intermediate filaments** : ropelike structure (more details below), very strong, primarily provide mechanical strength to the cell. Distributed primarily throughout the plasma membrane.
 * Microfilaments will be discussed later.
 * Microtubules:
 * Can't keep tensile forces (no stretching).
 * Made up of a protein called __tubulin__- specifically alpha and beta tubulin.
 * Tubulin has GTP binding regions, both between alpha and beta subunits and on the outside of the beta subunit.
 * Notice that the GTP between the alpha and beta subunits never gets hydrolyzed, just the one of the outside of the beta.
 * In microtubules, tubulin assumes a linelike, polymerized alpha-beta dimer configuration. Provides a __polarity__ to the microtubule structure (plus end is the end at which polymerization takes place [the end with the free GTP, or the end with the exposed beta subunit], minus end is the other one [the end with the exposed alpha subunit]).
 * This line of tubulin subunits wraps itself into a tube or solenoid to form the microtubule.
 * As the tubulin ages, the GTP in individual dimers at the minus end is hydrolyzed to GDP, and these dimers fall off the end of the tubule, acquire a new PO4 in the cytosol, and are incorporated into the tubule again at the plus end.
 * This means the plus end is more stable (has GTP, dimers can add); the minus end is less stable (has GDP, dimers can fall off). Note that certain cellular processes can destabilize the plus end.
 * Notice that the minus end tends to be around the nucleus; the plus ends grow out from there into the cytosol.
 * Microtubules "hold" the Golgi apparatus in a particular location relative to the nucleus. This stabilizing mechanism also doubles as a transport pathway. (see below, "motor proteins".)
 * Discuss cytoskeletal dynamics and the role of certain proteins and drugs in actin and tubulin polymerization/depolymerization.
 * Certain drugs interact with microtubule structure and, due to microtubules' role in mitosis, __block cell division__ (thus are compounds of interest in cancer treatment).
 * Colchicine and vinblastine (toxic plant extracts) interfere with tubulin polymerization at the plus end.
 * Paclitaxel ("Taxol") binds to and stabilizes the microtubule. The net effect of this is to cause aggregations of microtubules, which shuts down effective function during mitosis.
 * Explain the concept of mechanoenzymes. Explain the mechanisms of tubulin-based movement and intracellular transport.
 * "Motor proteins": dyneins and kinesins. Associated with microtubules; use them as tracks to drag cargo to target organelles.
 * These proteins convert ATP into mechanical energy by conformational changes.
 * Kinesins: 2 head domains and a tail. The heads contain ATPase: effectively the enzyme "walks" its heads down the microtubule and pulls the tail (and cargo) behind it. By hydrolyzing ATP, the enzyme shifts conformation to swing the trailing head group around to attach in front of the leading head group.
 * Notice that the __kinesins__ only ever travel from __minus to plus__ ends of the microtubules.
 * Notice also that the dyneins effectively travel the same way but only ever go from __plus to minus__ ends of the microtubules.
 * Tail domains bind cargo.
 * Discuss the role of microtubules in mitosis.
 * Three types of microtubules in the mitotic spindle: astral microtubules radiating from centrosomes (place centrosomes in center of daughter cell, help pull two centrosome halves apart), kinetochore microtubules connecting chromosomes (plus ends bind to specific site of centromere in chromosomes), and overlap microtubules (plus ends bind to each other on opposites ends from each side of the dividing centrosome).
 * Kinetochore action is driven by "minus-directing motors": the chromosomes are pulled in the minus direction (away from the middle), and the plus ends depolymerize between them as the centromere separates (thus no remnants left over).
 * Bare-bones: Astral and overlap microtubules pull centrosomes apart (plus-directed motors, kinesins); kinetochores separate chromosomes during this process (minus-directed motors, dyneins).
 * Explain the motion of cilia and flagella.
 * Cilia primarily move in a switchlike fashion back and forth; flagella usually move by progressive wavelike contractions.
 * Microtubule-mediated: cilia and flagella contain a microtubule core called an "__axoneme__": two microtubules in center, surrounded by a ring of microtubules connected by mechanoenzymes (dyneins). The dynein-mediated movement is what bends or moves the cilia or flagella.
 * Most cells have a cytoskeletal microtubule network called a __monocilium__ or __primary cilium__: the "non-motile" portion of this network seems to be involved in sensory perception.
 * Describe how intermediate filaments are anchored in the plasma membrane.
 * Effectively you have dimers of filaments; tetramers are formed by antiparallel dimers.
 * This means intermediate filaments have no polarity (there's two plus and two minus ends at each side of the tetramer.
 * Tetramers become polymerized into twisted bands of eight tetramers: ropelike, very strong (32 filaments per intermediate fiber "rope").
 * These anchor to the plasma membrane predominantly by attaching to desmosomes or hemidesmosomes. Both of these are types of junctions, and serve as the basis for cellular mobility/stability. See "Epithelia" notes for more details.
 * Discuss the cytoskeleton in the context of disease processes and as a drug target.
 * __Epidermolysis bullosa simplex__: Keratin mutation that affects intermediate filament stability. IFs can't provide mechanical strength: thus a very slight impact on the skin produces skin lesions and dermal bleeding. Effectively the epidermis is ripped away from the underlying subcutaneous tissue with extreme ease due to abnormal intermediate filament connectivity.
 * __Charcot-Marie-Tooth syndrome__: Neurofilament mutation causes peripheral neuropathy. These mutations are also associated with Lou Gehrig's disease.
 * __Kartagener syndrome__: wide variety of symptoms (respiratory disease, male infertility, left/right symmetry mismatch, etc)
 * Turns out to be a monocilium (cilia/flagella) defect- sperm can't swim, lungs can't clear mucus, cilial direction that establishes left/right symmetry in early gastrulation is disturbed, etc. Problems in outer dyneins of monocilium.
 * Dyneins (minus-end directed motors, can transport into centrosome) often shuttle viruses (which can attach to them) into the nucleus or along peripheral nerves into dorsal root ganglia.

Clinical vignettes Monday, November 26, 2007 6:41 AM


 * Clinical vignettes:**


 * Lung Cancer, 11/26/07:**


 * The students will learn that lung cancer is a major global and national health problem. The epidemiology of lung cancer will be discussed. //You should be able to give an estimate of the incidence of lung cancer and the prognosis.//
 * More than 200,000 new cases in 2007. More than 160,000 deaths the same year. Five-year survival rate is about 16%.
 * What are the symptoms from lung cancer? //You should be able to tell the main symptoms.//
 * …Anyone have any ideas here besides the obvious "hey, you've got this cough and this mass on your X-ray"?…
 * What are the main subtypes of lung cancer? //At least 4 main subtypes should be known.//
 * Notice four stages of lung cancer: I through IV (5-year survival gets a lot better the smaller the stage at diagnosis). Ranked by progression of metastasis.
 * I: local cancer
 * II and III: regional cancer
 * IV: distant cancer
 * __Non-small-cell lung cancer__: 80-85% of total
 * Adenocarcinomas on the rise (60-70%, peripherally located tumors)
 * Arise from glandular structures
 * Squamous-cell carcinomas (centrally located in bronchi)
 * Large-cell carcinomas (undifferentiated, rare)
 * __Small-cell lung cancer__: 15-20% of the total: strongly related to smoking.
 * General principles for diagnosis:
 * Not a lot of good diagnostic or treatment options for lung cancer.
 * Local cancer only (in lung): Stage I
 * Nearby lymph node invasion: Stage II
 * Multiple cancer sites in lung: Stage III
 * Distant cancer sites (liver, bone marrow, adrenal glands, etc): Stage IV
 * Tumor size correlates with poor rates of survival, as does number of invaded lymph nodes and how centrally they are located.
 * What are the general principles in staging and treatment of lung cancer and what is the prognosis related to stages?. //You should know the main differences in treatment depending on stage and know some estimated figures for the different stages as there are significant differences in prognosis dependent on stage.//
 * Treatment depends on the stage of cancer:
 * I and II: Surgery (60-70% or 40-55% cure rates, respectively).
 * IIIA: either chemotherapy or surgery (10-25% or 35% cure rates, respectively).
 * IIIB and IV: Chemotherapy and supportive care (very low cure rates).
 * Adjuvant chemotherapy (given after surgery) seems to lower death rates from lung cancer significantly, by about 10%.
 * Adenocarcinoma of the lung is rising in incidence, especially among women and never-smokers. This subtype have specific characteristics both related to demographics, location, and sensitivity to certain agents. //You should be able to know some specifics about this subtype.//
 * Smokers: lung cancer seems to occur mainly through //Ras// signaling pathway.
 * Non-smokers: lung cancer seems to occur mainly through EGFR (epidermal growth factor receptor) signaling pathway.
 * Newer treatment strategies for cancer is moving away from ”every treatment fits everyone” to” individualized” therapy. What does that mean?
 * Problem is: some patients have no positive response to chemo and have to deal with all the side effects anyway. Molecular profiling should allow targeting of therapy that actually works for individual patients.
 * ie: if there's a mutation in EGFR, treat with EGFR therapy (see below).
 * What is molecular targeted therapy? //Give some examples.//
 * Agents that work against particular biochemical pathways (see Herceptin, probably Gleevac from last unit).
 * What is the role of Epidermal Growth Factor Receptor (EGFR) signaling in lung cancer?
 * EGFR pathway: seems to be significant for carcinogenesis, activated a number of ways; affects angiogenesis (to supply vasculature to tumor), proliferation, anti-apoptosis.
 * Know that inhibition of the EGFR signaling pathway has been successful in the treatment of many cancers, and the students will learn about EGFR inhibition in lung cancer, especially adenocarcinomas.
 * Antibodies can be used to attack the EGF receptor protein, specifically at the receptor binding site and the tyrosine kinase domain.
 * Also can use EGFR tyrosine kinase inhibitors: non-injected; pill form, no bone marrow toxicity.
 * Note that EGFR proteins are members of the HER family (HER1 = EGFR protein).
 * Mutation in EGFR's tyrosine kinase domain commonly results in adenocarcinoma.
 * Can use immunohistochemistry or FISH (copy number) to detect abnormalities with EGFR (thus higher, 'personalized,' rate of response to EGFR therapy).

LO's as stated in class: Magnitude of lung cancer as health problem, types/staging/treatment/prognosis, future cancer therapy: EGFR pathway inhibition (molecular targeted therapy)


 * Cholera, 11/28/07:**
 * Understand the key clinical features and treatment of cholera infection.
 * Features:
 * Sever, watery diarrhea
 * Low-grade fevers and drowsiness
 * Fatigue
 * Decreased urination and dehydration
 * Skin tenting without lesions
 * Tachycardia
 * Muscle cramps
 * Nausea, vomiting
 * Hypovolemic shock
 * Treatment:
 * Oral rehydration therapy. Notice can also use IV rehydration.
 * Rehydrate, maintain hydration, feed early to make sure nutrition doesn't get too poor.
 * Mainly you're just replacing fluids and electrolytes until the body can kick the microorganism out. Notice can use antibiotics (azithromycin) to quicken this process.
 * Just an odd side note: you use azithromycin to treat both cholera (overactivation of CFTR) and cystic fibrosis (inactivation of CFTR). Weird.
 * Understand the actions of the cholera toxin's A and B subunits.
 * Recall: the __bacteria__ isn't the problem, it's the __toxin__ that the bacteria secrete.
 * **A** subunit: active site; **B** subunit: transport/bindng molecule
 * B subunit binds to the GM1 ganglioside receptor on enterocyte surface
 * A subunit cleaved off and endocytosed:
 * Binds to Gs proteins, inactivating their GTPase activity (thus always turned on) -- stimulates production of cAMP.
 * cAMP activates CFTR (cystic fibrosis transport regulator) chloride channel in apical membrane.
 * Notice that the Na-K pump on the basolateral side may also be shut down.
 * Perpetual CFTR activation leads to massive chloride efflux.
 * The efflux of chloride draws water with it, causing diarrhea.
 * Since the tight junctions in apical enteric cells are relatively loose, you can lose a whole lot of water and sodium very quickly paracellularly as they follow the chloride.
 * Understand the physiology behind oral rehydration solutions (ORS).
 * Effectively ORS is just water, sugar, and salt (plus potassium and citrate in WHO formula). Homemade = 8 teaspoons sugar, 1 teaspoon salt in one liter of water.
 * Small amounts can be absorbed quickly; small sips can rehydrate patients even when emesis follows.
 * Glucose helps Na+ uptake (and therefore water retention) in enteric system, also provides nutrition.
 * Notice can replace glucose with rice powder, which may reduce severity of diarrhea by adding amino acids to improve sodium uptake in enterocytes (counteract Cl- and water efflux).
 * Understand why someone with cystic fibrosis may be relatively protected from cholera symptoms.
 * Cystic fibrosis shuts down the CFTR receptors; heterozygotes for cystic fibrosis seem to be partially protected from the effects of cholera toxin.
 * Another odd side note: the notes suggest cystic fibrosis genes arose as a genetic partial defense against cholera-- but CF genes are by far most common in European and Ashkenazi Jewish populations, whereas the cholera bacteria is thought to have originated in India. Something don't sound right there.


 * Cystic Fibrosis, 11/30/07:**
 * Note that a major diagnostic test for CF is the chloride level in sweat ('sweat type'). Large levels of Cl- indicate homozygosity for CF (CF is autosomal recessive).
 * Review the genetics and underlying protein defect in Cystic Fibrosis (CF).
 * Most common lethal genetic disease in Caucasians- median predicted survival is 35 years.
 * CF gene codes for CFTR protein: acts as an ion channel in apical epithelia controlling movement of water and ions in and out.
 * CF is a large gene in chromosome 7. Lots of different known pathological mutations in the gene.
 * Mutations in CF tend to cause either:
 * (1) no protein synthesis
 * (2) misfolding of protein leading to degradation before reaching apical cell surface
 * (3) altered conductance such that the protein can't be conveyed to the apical surface, or
 * (4) partial loss-of-function such that the CFTR proteins don't work as well as normal.
 * Review the multiorgan involvement in persons with cystic fibrosis.
 * (Tends to show up in tubular epithelial systems:)
 * Sinuses (chronic sinusitis)
 * Lungs (main problem: chronic bronchitis and airway infection/inflammation.)
 * Chronic lung problems lead to loss of lung function
 * Pancreas
 * Most CF: pancreatic insufficiency; need to supplement with oral enzymes.
 * ~25% develop cystic fibrosis-related diabetes (requires insulin injections)
 * Intestine
 * Most CF: intestinal closure (can't eat or excrete feces)- "meconium ileus".
 * -tends to be diagnostic for CF
 * Liver
 * Minority of CF patients, but second leading CF cause of death (after lungs).
 * Ductus deferens
 * Male infertility (sperm are viable but can't get out).
 * Notice that male heterozygotes can also have problems with this.
 * Sweat glands
 * Problem with salt-losing dehydration (see below; effectively can't reuptake Cl- through sweat glands).
 * Review current understanding of pathophysiology underlying CF lung disease.
 * Lung disease:
 * Pseudomonas and Staphylococcus aureus are the most common pathogens found in CF lungs (since the mucus is an ideal breeding ground for bacteria).
 * In lung: CFTR normally excretes Cl- to move water into the mucus layer overlaying the lung epithelium (like in the intestine). With defective CFTR, this water secretion doesn't happen.
 * In addition to this, normal CFTR also downregulates epithelial sodium channels. Without CFTR, have elevated levels of import of Na+ into epithelial cell, which further removes water from the lumen (and the mucus) into the epithelial cells, exacerbating the thickness of the mucus.
 * (Notice that in sweat glands, the CFTR defect causes a problem in getting chloride __into__ the epithelial cells, as opposed to in the lung and intestine, where the defect causes a problem in getting them __out__.)
 * Treat (lungs) with airway clearance therapy, antibiotics to keep lungs clear, inhaled mucolytics (break up the mucus), brochodilators, enzyme replacement, vitamin A/D/E/K replacement, azithromycin used chronically.
 * Therapeutic approaches: eventually need lung transplant. Gene therapy not working yet, but "protein rescue" seems promising: in principle, could correct misfolding or get nonconducted CFTR proteins to the cell membrane to work properly.

[Thanks to Andrew Brookens for supplying lecture notes for this one. --jcr]
 * Muscular Dystrophy, 12/6/07:**
 * Describe the genetic basis for, and clinical features of, Duchenne's Muscular Dystrophy:
 * X-linked recessive inheritance
 * Frequency: 1/3500 males
 * “Climb up themselves” (“Gower” sign, showing early, ~1 year)
 * Wheelchair bound by 12; die in 20s due to cardiorespiratory failure
 * 1/3 from spontaneous (non-inherited) mutation
 * 10 year old falls 30-40x/day
 * Genetic defect in DMD**:** dystrophin protein (forms the “steelbelt” supporting the muscle cell membrane) is broken down due to a premature stop codon in its gene.
 * Dystrophin is the largest gene known: on Xp21, 79 exons long, 2.4Mbases.
 * Dystrophin links t-acting component on one side, then to transmembrane complex on plasma membrane that enables force to be conveyed to outside matrix
 * DMD therapy:
 * Surgical tendon releases/scoliosis surgery
 * Corticosteroids prolong independent ambulation (8-24mos), but with side effects
 * Supportive care
 * Future treatments:
 * Clinical trials for exon skipping in Netherlands: the oral agent PTC124 reads through the premature stop codon in dystrophin.
 * Stem cell transplant: replace dystrophin function.
 * Myostatin antibody (Johns Hopkins)
 * AT1 antagonist (Losartan) in mice
 * Method of diagnosis**:** serum creatine kinase recognizes DMD early, without muscle biopsy.

Nuclear Transport Tuesday, November 27, 2007 8:03 AM


 * Nuclear Transport, 11/27/07:**


 * Describe the 3 mechanisms of protein transport.
 * Gated transport: requires a specific receptor protein to carry a protein from the cytosol into the nucleus through the pore complex.
 * Transmembrane transport: requires a translocator protein or protein complex (ie translocon) to move proteins across membrane.
 * Vesicular transport: membrane-bound transport; requires adaptor and coat proteins.
 * List the major functions of the nucleus.
 * Sequester cellular DNA
 * RNA transcription and processing
 * Contains nucleolus: site of ribosomal RNA synthesis, processing, subunit assembly
 * (how big nucleolus is depends on the number of ribosomes needed)
 * On a micrograph, the big dark thing in the nucleus is the nucleolus.
 * Describe the nuclear pore complex.
 * Pore complex: network of large nuclear membrane-spanning structures that control entry to and exit from the nucleus.
 * The nuclear membrane is double-layered; the pore complex penetrates both.
 * Inner layer: binding sites for chromatin and the __nuclear lamina__ (structural supports consisting of intermediate filaments).
 * Outer layer: continuous with the ER membrane.
 * Because this is the case, proteins that are translated into the ER membrane (through the translocon) are only one membrane layer (the inner nuclear membrane layer) away from the inside of the nucleus.
 * Note that the inner and outer nuclear membrane layers are continuous at the pores of the pore complex.
 * 4 structural building blocks of a pore:
 * __Column__ subunits (pore wall)
 * __Annular__ subunits ("spokes" extending in toward the pore's center)
 * __Lumenal__ subunits (transmembrane, hold membrane open and anchor pore to membrane)
 * __Ring__ subunits (cytosolic and nuclear faces of the complex; on the outer and inner surfaces of the nuclear membrane)
 * Very large structures: ~125 million daltons (30X larger than ribosome).
 * Roughly 3000-4000 pores per mammalian cell nucleus
 * 9 nm diameter channel, but can expand to accommodate larger transports.
 * Describe nuclear import/export.
 * Molecules less than 5000 daltons can freely diffuse through the pore complex; everything else has to be gated in by a specific receptor protein.
 * Nucleoporin proteins (lots of hydrophobic repeats) line the central pore transporter channel to interact with import/export receptors.
 * Common imports: histones, DNA/RNA polymerases, gene regulatory proteins, and RNA-processing proteins
 * Common exports: tRNAs and processed mRNAs
 * Export of mRNA: part of mRNA processing is to attach a whole bunch of proteins to it (among others, mRNA exporter proteins). Without all these proteins, it won't exit the nucleus. Note that a lot of the nuclear-exit-signal proteins fall off to be recycled as it leaves the nucleus.
 * Ribosomal proteins: imported into nucleus to be assembled with rRNAs, then exported.
 * Nuclear Localization Signals (NLS): sequences in proteins (primarily positively-charged lysines and arginines) that allow those proteins to be picked up by a nuclear import/export receptor. Notice the sequence doesn't have to be contiguous.
 * Nuclear import/export receptors:
 * Soluble, cytosolic proteins; bind to both NLS on transported protein and to nucleoporins in the pore channel.
 * Exportins export; importins import. Two things: one, they need to be on the right side of the membrane to work, and two, this does take energy (GTP); selective placement of GTPases and GDP phosphorylases on different sides of the nuclear membrane allows a concentration gradient to occur, which helps the proteins to get back to the side that they work on.

Protein Degradation Tuesday, November 27, 2007 9:00 AM


 * Protein Degradation, 11/27/07:**


 * Recall that one of the functions of the ER is "Quality Control"-- that is, protein folding and degradation.
 * If a protein isn't folding correctly, chaperone proteins can help refold it; however, if the protein still can't get it right (due to mutations or what have you), it needs to be chopped into little bits and reassembled (don't try this at home).
 * Basically, if you have a bunch of misfolded proteins, they can aggregate and cause real problems (ie apoptosis) for the cell.
 * About 30% of all new proteins need help from a chaperone to fold right; about 30% of those can't be folded correctly and get sent to the cellular trash can.
 * This trash can is called the proteasome: it chops the proteins up into amino acids so it can be resynthesized.
 * ER quality control:
 * Provides optimal (oxidizing) environment for folding and oligomeric assembly; also has a lot of chaperone proteins, folding enzymes, and folding sensors (proteins that detect misfolding).
 * Note that the proteasomes are primarily in the cytoplasm and not in the ER themselves; therefore proteins marked for degradation need to get out into the cytoplasm again.
 * BiP (binding proteins) can export proteins out of the ER (back through the translocon) to be taken to the proteasome.
 * List 3 mechanisms for endocytosis.
 * Endocytosis (uptake of materials into the cell): major route for cell-specific drug delivery and gene replacement therapy.
 * Clathrin-coated vesicles: use SNARE proteins on vesicles to merge lipid surface of vesicles with plasma membrane.
 * There's effectively a string of lowered-pH steps in the cell to take endocytosed stuff to the lysosome to be degraded into its component parts. Drugs can either be active once degraded by the lysosome or be picked up by their targets somewhere in this process.
 * Caveolae: cholesterol-rich vesicles budding from membrane- evidently have signaling functions. Not a lot of elaboration here.
 * Fluid-phase: stuff can move in and out of the membrane freely. Not a lot of specifics here, either.
 * Describe two types of molecular chaperones.
 * Chaperone protein example: heat shock proteins (Hsp's).
 * __Hsp70__ family: these can bind to exposed hydrophobic domains to reform and prevent aggregation.
 * __Hsp 60__ family: there form large barrels which envelope misfolded proteins to refold them without aggregation.
 * Describe the proteasome and protein degradation.
 * The proteasome subunits are popular proteins: make up about 1% of cellular protein complement. Dispersed throughout the cytosol and the nucleoplasm.
 * Structure: has a central cylinder containing the protease active sites and a cap on either side to detect ubiquitinylated proteins (recall that ubiquitin is the molecular protein tag with which a cell marks proteins for degradation).
 * General idea: proteins are tagged with ubiquitin (four ubiquitin protein attachments are required to degrade a protein) and transported to the proteasome, where it's shoved into the central protease cylinder and degraded.
 * So the entire process looks like: misfolded proteins are detected in the ER, BiP-shoved out the translocon into the cytosol, tagged with ubiquitin, and degraded in the proteasome.
 * Non-proteasomal methods of protein degradation:
 * Lysosome: degrades all cellular components transported to it.
 * The lysosome can degrade pretty much every damn thing in the cell; it's basically a big acid pit.
 * Autophagy: digests entire organelles for energy
 * Cell does autophagy primarily under conditions of extreme starvation or illness, but it also can be used to degrade very long-lived proteins or senescent organelles. Can also be used to remodel cell during differentiation in development.

Cell Signaling Overview I Tuesday, November 27, 2007 9:56 AM


 * Cell Signaling Overview I, 11/27/07:**


 * Understand the basic principles of cell-cell signaling. Know all the forms of signaling and their uses in organisms (when organisms uses contact-dependent versus endocrine signaling, etc).
 * Ligand, or signal molecule, gets to target cell, where it binds with a receptor. If no receptors are around, no signal is transmitted.
 * Notice that a single ligand can bind to multiple receptors for different effects, and multiple ligands can bind to a single receptor. Allows for greater variety of responses to stimuli.
 * Forms of signaling:
 * Contact-dependent signaling: signaling molecule never leaves its cell of origin; therefore the signaling cell and the receptor cell need to come into physical proximity to work. An example would be immune response.
 * Contact-independent signaling: signaling molecule leaves its cell of origin.
 * __Autocrine signaling__: one cell type is signaling itself (release ligand, bind the same type of cell)-- example is growth factor.
 * __Paracrine signaling__: the target cell is very nearby. Ligand relies on passive diffusion to get to receptor cell.
 * __Endocrine signaling__: target cell is distant. Ligands are called hormones; travel through bloodstream until it finds receptor cell. More on this under "Epithelia."
 * __Neuronal signaling__: long-range, rapid signaling; can't use endocrines (too slow)-- send APs down axons to synapses.
 * Know all the types of extracellular signaling molecules and how they relate to the forms of signaling.
 * Water-soluble ligands:
 * Usually peptides.
 * Can't cross plasma membrane without transport
 * This means it needs a plasma membrane transporter both from its cell of origin and at its cell of reception; usually bind, at their site of action, to __plasma-membrane-bound receptors__ (see below).
 * Generally faster action.
 * Example: neurotransmitters (neuronal signaling).
 * Water-insoluble (lipid soluble) ligands:
 * Can cross membrane easily, but usually needs to bind to a carrier protein of some kind to be taken through cytosol.
 * Usually bind to __intracellular receptors__ (also called __nuclear receptors__) at their site of action (see below).
 * Generally slower action.
 * Example: steroid hormones (endocrine signaling).
 * Gaseous ligands:
 * Can cross water or lipid boundaries; easily diffuses, no need for transporters.
 * Very short-range (paracrine signaling).
 * Example: nitrous oxide (regulates blood pressure, etc)
 * Understand the different types of receptor molecules and know the types of cellular processes that they regulate. Understand the concept and functions of intracellular signaling molecules.
 * (1) Plasma-membrane receptors: respond to water-soluble signaling. Generally work by activating signal transduction inside the cell. 3 main types:
 * Ion channel receptors:
 * Gated channels-- usually closed until ligand binds, at which point gates open and particular ions travel through.
 * Ion channel receptors largely regulate channels for Ca2+.
 * Notice that one main type of Ca2+ channel takes up calcium into the endoplasmic reticulum (see "Signaling: Calcium" for details).
 * G-protein coupled receptors:
 * Cytosolic side binds to ligand; intracellular side binds to a G protein complex (heterotrimeric G proteins, which bind GTP; more on this under "Signaling: Receptors").
 * This protein complex is normally bound to GDP at its alpha subunit; when the ligand binds, the alpha complex binds GTP instead of GDP and dissociates from beta and gamma subunits.
 * Both the alpha and beta-gamma subunits can serve as signaling agents inside the cell.
 * Notice this allows a variety of responses (different trimeric proteins) to one ligand in a variety of tissues.
 * Receptor kinases:
 * Often growth factor and insulin receptors.
 * Cytosolic side binds to ligand; intracellular side has a kinase domain on it (tyrosine kinase)-- it will PO4 other proteins with particular tyrosine residues.
 * Most receptor kinases act as dimers: once they're both bound to ligand, they will phosphorylate each other, turning on their signaling: once that PO4 is added, it creates a binding site for a set of adaptor proteins, which bind to the receptor kinase dimers and set off a signaling cascade.
 * Lots of variety in receptor action here as well: can PO4 other proteins, can have a lot of different potential adaptor proteins to bind to the binding site, etc.
 * (2) Intracellular/nuclear receptors: respond to lipid-soluble signaling.
 * Generally work by activating transcription of specific genes. It does this largely by activating proteins which activate promoters upstream of transcription start sites.
 * These proteins are also referred to as __transcription factors__.
 * Steroids: bind to receptor proteins, inducing a conformational change which allows the receptor protein to release its inhibitor protein and bind its activator protein (entire complex: receptor-ligand-activating protein); this complex can interact with promoter regions to activate transcription.
 * Understand the concept of signal integration (one signal-many functions and many signals-one function).
 * Example: calcium ligand can bind to a variety of kinase receptors depending on the type of cell.
 * Just remember that no signaling agent or pathway is ever, ever simple - a healthy signal paranoia - and you'll be fine.

Cell Signaling Overview II Tuesday, November 27, 2007 9:56 AM


 * Cell Signaling Overview II, 11/27/07:**


 * Describe principle types of detectors of extracellular signaling molecules.
 * Ligand or voltage-gated ion channels (water-soluble ligands)
 * G-coupled protein receptors (water-soluble ligands)
 * Enzyme-linked receptors (ie receptor kinases) (water-soluble ligands)
 * Nuclear receptors (lipid-soluble ligands)
 * List other "tools" of signaling pathways, including at least three 2nd messengers.
 * Second messengers: small molecules released within the cell in response to the binding of the first ligand; can bind to other intracellular signaling molecules.
 * Calcium: first ligand opens ion channel to admit Ca2+; calcium then binds a variety of other things inside the cell.
 * cAMP (cyclic adenosine monophosphate): generated by adenylate cyclase
 * IP3 (inositol triphosphate)
 * DAG (diacylglycerol): generated by phospholipase C (PLC)
 * NO (nitric oxide): generated by nitric oxide synthase (NOS)
 * (notice that, for example, calcium can trigger the production of NOS, which triggers NO, which triggers.. etc, etc. Thus can have second messengers activating third messengers activating fourth messengers, and so on, though the third and fourth messengers can also act as second messengers. The cell has no idea that it's screwing up all the cool categories we invent for it. Again: develop a healthy distrust of simple signaling pathways.)
 * Other signaling steps:
 * Protein modification (PO4ation, ubiquitinylation, etc)
 * Protein-protein binding/targeting
 * GTP/GDP exchange (G-proteins coupled to receptors, small GTPases like //ras// )
 * Note that GTP does not seem to act as a second messenger: I think it's the fact that GTP changes the configuration of proteins which then act on other proteins that makes the difference. That is, GTP itself is acting as more of a helper than an agent in the signaling.
 * G-proteins: see "Signaling: Receptors."
 * Ras proteins: see "Receptor Tyrosine Kinases."
 * Describe at least three mechanisms for signal termination (including phosphodiesterases).
 * Uptake, breakdown, or diffusion of original signaling molecules
 * Desensitization of receptor
 * Termination initiated by another signal (phosphorylation, dephosphorylation, etc)
 * Termination-dedicated enzymes: ie __phosphodiesterases (PDEs)__ which break down the second messengers cAMP/cGMP.
 * Some kind of feedback inhibition: ie, the bound ligand or receptor acts in such a way as to undermine its ability to stay bound or stay active (G-proteins gradually lose their GTP-bound active state).
 * Evaluate a "pathway" for amplification and termination.
 * Amplification depends on signaling cascades: one signaling molecule can create a large number of signals, which in turn can create an ever larger number of second signals, etc.
 * Positive feedback mechanism (ie. calcium-induced calcium release): very quick but very dangerous signaling pathway (can get out of control rapidly). Usually coupled with a negative feedback mechanism past a certain threshold to keep it under control.
 * Identify "nodes" (such as calcium) and "modules" in a signaling pathway, and evaluate the potential for crosstalk in signal transduction.
 * __Nodes__: points in a network that receive multiple inputs and/or multiple outputs. Calcium is evidently the most extensive node in signaling.
 * __Modules__: groups of components that function together, often physically assembled to form complexes. Ie: G-protein heterotrimeric complex would presumably form a signaling module, as would negative feedback mechanisms.

Mitochondria and Peroxisomes Wednesday, November 28, 2007 8:00 AM


 * Mitochondria and Peroxisomes, 11/28/07:**


 * Know the origin and basic structure of mitochondria.
 * The current theory is that they showed up by way of endocytosis of oxidative-phosphorylating bacteria by our distant ancestor(s).
 * Structure: has a double membrane (only other organelle with a 2x membrane is the nucleus)-
 * Outer membrane: semi-permeable, regular ol' membrane.
 * These have TOMs (Translocases of Outer Membrane: large, non-gated channels allowing protons, etc, to equilibrate with the cytosol).
 * Inner membrane: less permeable, forms the folds or __cristae__ inside the mitochondrion, contains the cellular machinery responsible for ox-phos.
 * These have TIMs (Translocases of Inner Membrane: specific, receptor-based protein channels allowing various required proteins selectively in).
 * Four protein complexes in the cristae: their purpose is to pass electrons from NADPH to various electron acceptors (ending in oxygen to form H2O), generating energy with each transfer.
 * As I understand it, the reason for all these transfers is to minimize the amount of energy wasted through each step- if you just took electrons/protons off NADPH and stuck them on oxygen, there would be a large energy release, most of which would be lost to heat. By breaking it down into a bunch of smaller steps, it's able to keep more of the total energy as ATP. Not necessary, and possibly not even correct, but interesting.
 * Space inside the inner membrane = __matrix__. Contains the mitochondrion's own DNA (recall that the vast majority of proteins that actually function in the mitochondrion are derived from the nucleus, though).
 * As these electrons are transferred, the protons (from NADPH -> NADP+) are pumped out of the matrix, at which point they can leak out of the outer membrane.
 * This creates a chemical gradient on H+ with less concentration inside the matrix than outside it.
 * Notice this also creates an electrical gradient with more negative charge inside the matrix than outside it.
 * Essentially this is a killer drug target: if you disrupt one of these gradients, you can kill cells quickly. The reason for this:
 * The H+ gradient, chemical and electrical, is used by channel proteins (F0) to allow protons to rush through the inner membrane into the matrix and be used by another protein (F1 or ATP synthase) to generate ATP.
 * There's an ATP-ADP exchanger in the inner membrane, run by concentration gradients of both those compounds, that keeps the concentration of ADP in the matrix high.
 * Notice that an alternative method uses creatine kinases. Effectively you keep a phosphate group on creatine (usually in muscles); when required, you can quickly transfer off the PO4 onto ADP to make more ATP in conditions of high energy consumption.
 * Notice that mitochondria also function in targeted apoptosis (as wh.en differentiating your fingers from each other or destroying DNA-mutated cells). When "death receptors" are triggered on the cell's surface (or irreparable DNA damage is detected), the mitochondria release cytochrome C, which releases caspases (real nasty proteases to destroy cell's protein complement).
 * Understand the basic principles of electron transport and ATP production in mitochondria.
 * See above.
 * Know the basic functions of peroxisomes.
 * Small, membrane-bound organelles.
 * Specialized form of lysosome: involved in degradation of fatty acids and amino acids.
 * Peroxisomes generate, and thus also have to deactivate, a lot of H2O2 (hydrogen peroxide). They deactivate it with an enzyme called __catalase__.

Signaling: Receptors Wednesday, November 28, 2007 8:47 AM


 * Signaling: Receptors, 11/28/07:**


 * Draw the membrane topology of a G protein-coupled receptor and identify the basic structural characteristics that mediate ligand binding and coupling to G proteins.
 * Well, I'm not going to draw the topology here, but basically you've got a barrel-shaped receptor protein with a ligand binding domain on the outside of the membrane, then a heterotrimeric G protein complex binding domain on the inside of the membrane, bound to the heterotrimer plus GDP. The G protein complex is made up of three subunits, alpha, beta, and gamma. The beta-gamma subunits are pretty much always stuck together, and in their GDP-bound state they're also stuck to the alpha subunit.
 * [Basic note: "Agonist": ligand that activates receptor. "Antagonist": ligand that inactivates receptor.]
 * Explain how G protein-coupled receptors activate hetero-trimeric G proteins and diagram the GTP-hydrolysis cycle of G protein signaling.
 * When the ligand binds, a conformational change drives the GDP on the alpha-beta-gamma complex to be exchanged for GTP, and the complex becomes "activated".
 * When activated, the alpha subunit (bound to the GTP) and beta-gamma complex break apart, separate from the membrane receptor, and go their separate ways within the cell to serve as signaling molecules ("__transducers__") to other targets ("__effectors__").
 * Notice that you have a lot of different possible responses to this- this is just a basic signaling mechanism and can be used for just about any conceivable purpose.
 * The system is "reset" to get back to GDP-bound trimers by the hydrolysis of GTP on the alpha subunit to GDP; when the GTP becomes GDP, the alpha subunit returns back to the membrane receptor and binds up the beta-gamma complex to reform the inactive state. Obviously this can be helped by particular enzymes that trim phosphates off the GTP on the alpha complex (ie GTPases), but it will also happen spontaneously, providing a built-in time limit for G-protein signaling.
 * Some uses of G protein-coupled receptors:
 * __Adrenergic__ receptors: bind __norepinephrine__. __Muscarinic__ cholinergic receptors: bind __acetylcholine__.
 * Notice that what kind of __ligand__ a receptor binds to has comparatively little impact on what its eventual effect pathway in the cell is. However, what the receptor protein is bound to on the inside of the membrane __does__ have a lot of impact. The reason for this is that the receptor's G-protein binding domain on the inside of the membrane can interact with different G-protein complexes depending on what tissues the receptors are in.
 * That's not to say that the ligand and the eventual effect are unrelated. Generally norepinephrine receptors are involved in sympathetic reactions and muscarinic receptors are involved in parasympathetic reactions. But notice that sympathetic and parasympathetic reactions can have very different effects in different tissues (see below).
 * G proteins: Gs stimulates adenylyl cyclase to form cAMP; Gi inhibits adenylyl cyclase to stop the formation of cAMP; Gq is involved in other methods of calcium transmission.
 * For example, beta-adrenergic receptors in the heart (triggered by norepinephrine) are bound to Gs proteins, which stimulates the formation of cAMP, which eventually leads to calcium influx, which increases heart rate contraction.
 * Beta-blocker drugs: block this effect; decrease heart rate.
 * However, alpha-adrenergic receptors in the periphery (still triggered by norepinephrine) are bound to Gq proteins, which binds phospholipase C to activate a kinase pathway that ends in calcium influx in smooth muscle, causing vasoconstriction, increasing blood pressure and shunting blood away from the periphery towards the body core.
 * Muscarinic cholinergic receptors in the heart (triggered by acetylcholine) are bound to Gi proteins, which inhibit cAMP formation, causing open Ca2+ channels in heart muscle to close (stopping influx) and slowing the heart rate.
 * Other muscarinic cholinergic receptors in the heart are also bound to Gi proteins, but these open potassium channels to regulate the membrane's potential (and thus excitability).
 * As far as Gi is concerned: the reason the level of cAMP goes down when its synthesizing enzyme is turned off is that cAMP is continually degraded to AMP by phosphodiesterases (PDEs).
 * Notice that certain drugs inhibit PDEs (like caffeine)-- this blocks the degradation of cAMP, which keeps cAMP activity (stimulation) high.
 * Viagra and Cialis target PDEs which degrade cGMP, for similar effects in smooth muscle.
 * Notice that beta-adrenergic receptors in the __lung__ (not heart) also act through Gs pathways, but the increased cAMP causes the smooth muscle in the lung to __relax__ (bronchodilation) rather than contract.
 * Notice that muscarinic receptors in the lung act through Gq pathways, causing influx of Ca2+ and causing bronchoconstriction.
 * Describe the function of second messengers in receptor signaling and give two examples for how they are generated by activated G proteins.
 * See above, Gs and Gi. cAMP is a second messenger.
 * Explain how receptor activation leads to signal termination through receptor desensitization.
 * Desensitization: response to overexposure of receptor to ligand.
 * How this works: in the active state, the beta-gamma complex recruits kinases to phosphorylate the receptor protein such that an "arrestin" protein binds to the receptor, not allowing the receptor to bind more inactive G proteins to activate them.
 * In addition, once arrestin binds to the receptor, the receptor is endocytosed, inactivating it until either the phosphorylation is removed to restore the receptor (resensitization) or the receptor is degraded by lysosomes. With long-term exposure, the receptors are more often degraded than resensitized.
 * This means that after an activation, the receptor effectively is taken out of the loop and can't activate any more G-protein signals; with a lot of activation over time, the number of receptors in the membrane goes down as they are degraded.
 * Opiates, for example, over time will reduce the number of receptors available to produce their analgesic effect.
 * Give two examples of drugs that act through modulating different steps in a receptor-G protein-second messenger signaling cascade.
 * See above re caffeine and Viagra, also below re cholera and whooping cough.
 * [Other notes: Pertussis toxin (whooping cough) modifies the Gi protein to prevent the inactive G protein to be able to be activated-- thus inhibits Gi proteins.
 * Whooping cough: no activation of Gi proteins.
 * By contrast, cholera toxin modifies the Gs protein to prevent the active G protein from being able to be deactivated-- thus perpetually keeps Gs proteins activated.
 * Cholera: no inactivation of Gs proteins.

Signaling: Calcium Wednesday, November 28, 2007 9:48 AM


 * Signaling: Calcium, 11/28/07:**


 * Understand the functions of cyoplasmic Ca2+ ion buffers and how these buffers affect cytoplasmic Ca2+ signals:
 * These bind free Ca2+, convert to inactive form. They usually involve free carboxyl groups on negatively charged amino acids.
 * They protect against enormous influxes of calcium and also make sure local influxes of calcium are restricted from spreading around. Their other function is to make sure calcium doesn't remain active in the cell for long (inactivated and extruded again)-- limits Ca2+ both spatially and temporally.
 * Buffers are also present in endo/sarcoplasmic reticular lumen: allows greater storage of (temporarily inactivated) Ca2+ in those organelles.
 * Understand the routes by which extracellular Ca2+ enters the cytoplasm. Understand the routes by which Ca2+ moves out of the ER/SR into the cytoplasm. Understand the routes by which Ca2+ is extruded from the cytoplasm (a) into the extracellular space and (b) into the lumen of the ER/SR:
 * Note that calcium doesn't need to be synthesized-- it's already present at high concentrations extracellularly. This means the signaling pathways for calcium are extremely rapid (just need to let it in).
 * Other sources/sinks of calcium (sources: from whence calcium comes. Sinks: to where it shall return.): nuclear envelope, mitochondria, endo/sarcoplasmic reticulum.
 * Most cells: a voltage differential of about -60 mV across the cell membrane. This serves as another driving force to get Ca2+ into the cell.
 * Routes to __enter__ cell cytoplasm:
 * __Passive__ via ion channels:
 * Voltage and ligand-gated Ca2+ channels in the plasma membrane to uptake Ca2+ into cell. Also "store-operated" channels (dependent on existing store of Ca2+).
 * Endo/sarcoplasmic reticulum and nuclear envelope: IP3 receptors and ryonodine receptors moves calcium from E/SR/nuclear membrane to cytoplasm.
 * Mitochondrial pores ('uniporters')- dependent on Ca2+ concentration.
 * Routes to __get out of__ cell cytoplasm:
 * __Active__ transporters against gradients (slower movement of Ca2+):
 * Ca2+ pumps use ATP to move calcium into extracellular space or into lumen of E/SR.
 * Can also use energy from sodium leak into cell to pump calcium out of the cell.
 * Learn what EF hands and C2 domains are. Learn the identity of the archetypical protein that contains EF hands. Learn the identity of the archetypical protein that contains a C2 domain. Learn whether these domains are present in other proteins.
 * See below for greater detail. C2 domains, when bound to calcium, bind to cell membranes; they're contained in __protein kinase C__ and __synaptotagmin__ proteins. EF hand domains are found in calmodulin proteins and cause them to effect calcium-mediated modulation of other proteins. These are, in fact, present in other proteins (parvalbumin and troponin C for EF domains, for example).
 * [Some functions (effectors) of Ca2+ signaling:]
 * Ion movement: changes voltage across membrane (depolarization of neurons and cardiac muscles)
 * Protein Kinase C (PKC) activation:
 * Has a C2 domain: when bound to calcium, this domain tends to fuse with membrane and have activate phosphorylation effects. (not much detail here)
 * Synaptotagmin activation:
 * Also has a C2 domain, which causes vesicles within cell to fuse with membrane and release cargo (like neurotransmitters).
 * Calmodulin activation:
 * Strongly conserved genes in plants, animals.
 * Contain four EF-hand domains that bind calcium.
 * When activated by calcium, sticks to and modulates ion channels, protein kinases, phosphatases, and phosphodiesterases.
 * [Notice that maintained Ca2+ influxes in the cell can activate negative feedback activation that stops the Ca2+ influx: RyR protein channel in ER to allow Ca2+ efflux into the cytoplasm]

Stem cells and cellular diversity Thursday, November 29, 2007 8:01 AM


 * Stem cells and cellular diversity, 11/29/07:**

[general notes; learning objectives are kind of FUBARed.]


 * Differentiation is normally irreversible but exceptions exist, primarily fibroblasts in connective tissue.
 * Differentiation is driven by different combinations of molecular cues.
 * Notice that the same signal combination will have different outcomes in different cell type lineages.
 * __Lineage__: pedigree of a differentiated cell. Describes how related cells are, predicts similarities at level of gene expression, and predicts how easily external cues or mutations may turn one cell type into another.
 * Hematopoeitic lineage (blood cell lineage): a bunch of different blood- and lymph-related cells, all derived from originally quiescent stem cells.
 * __Potency/developmental potential__:
 * Potency (the number of different fates a stem cell can have) is restricted by commitments during differentiation- every time one branch of the 'family tree' is chosen, other branches close off as options.
 * __Totipotent__: have unlimited potential, can give rise to any cell type. Zygote is the only human example of this.
 * __Pluripotent__: can give rise to many, but not all, cell types. Embryonic stem cells are an example: can become just about anything aside from trophoblasts, which aren't found in the adult human body anyway.
 * __Multipotent__: can give rise to a small number of cell types (hematopoietic stem cells, etc). Most adult stem cells are examples.
 * __Commitment__:
 * An irreversible lineage choice has been made and other fates are no longer permitted.
 * Notice that commitment precedes overt differentiation; that is, a stem cell is committed to become a certain cell before it actually becomes that cell.
 * This happens largely because the genes that trigger development along one path inhibit the genes that trigger development along other paths.
 * Underlying mechanisms of stem cell development:
 * (1) __Lineage-specific transcription factors__ (reversible, but not often)
 * "Master regulators"; activate genes necessary for differentiated phenotype, repress genes necessary for alternate lineages, and maintain their own transcription.
 * (2) __Epigenetic chromatin modifications__ (stably transmitted to daughter cells, very difficult to reverse)
 * DNA methylation: CpG dinucleotides near a promoter are methylated to mark that gene for expression or silencing
 * Histone tail modifications: methylation, acetylation, phosphorylation; create docking sites for chromatin modification protein complexes. This does two things:
 * Changes packing of chromatin (opens or closes chromatin loop) to make transcription either more or less accessible.
 * "Template-dependent histone modifications": recognize histone modifications, duplicate them on all new histones. Maintains fidelity of histone modification on all histones in cell.
 * (3) Regulatory microRNAs:
 * Recently discovered; not too much known, seems to be (untestably) important


 * Tissue renewal and stem cells:
 * Adult stem cells are in action all the time: renewing tissues in the intestines, creating sweat gland/hair follicles/skin/sebaceous glands in dermis, etc.
 * Properties of adult stem cells:
 * Can repopulate tissue and self-renew.
 * Generally rare in the body: not a whole helluva lot of them.
 * Give rise to diverse progeny (multipotent), but not very widely diverse (not pluripotent).
 * Self-renewal: generally, during mitosis, one daughter cell will differentiate and one will continue on as a stem cell. However, it can also make two new stem cells or two differentiated cells.
 * Notice that long-term depletion of stem cells contributes to aging.
 * Tend to reside in specialized niches in the body, in contact with regulatory cells which keep them quiescent, self-renewing, and undifferentiated. Without the contact with regulatory cells, they will differentiate and proliferate.


 * Stem cells and disease:
 * Some ideas that leukemia is a stem cell disease that results from the displacement of functional stem cells by abnormal, leukemic stem cells.
 * Aging: aging stem cells are more easily mobilized from their regulatory cells (thus no longer as self-renewing).
 * Difference between adult and embryonic stem cells:
 * Embryonic stem cells are pluripotent: can become any variety of tissue type in the body, unlike adult stem cells. They are also rapid proliferators.

Serine and threonine kinases Thursday, November 29, 2007 9:03 AM


 * Serine and Threonine Kinases, 11/29/07:**


 * [Once again: kinases are proteins that phosphorylate other proteins. Notice that this doesn't say anything one way or the other about what effect this will have on the activity of the PO4'd protein.]
 * Describe a phosphorylation reaction (including which amino acids can be phosphorylated) and explain how it can affect a phosphorylated protein.
 * Possible phosphorylation targets: serine, threonine, tyrosine.
 * Notice that Ser and Thr have very similar structures to each other, but different from Tyr, which is why there's generally two types of kinases (Ser-Thr vs Tyr).
 * This is because they have hydroxyl residues that can be replaced with a phosphate group (OH makes nucleophilic attack on gamma phosphate of ATP)
 * The PO4 is generally donated from ATP; the donation is catalyzed by the kinase.
 * PO4'd proteins can be thus activated, inactivated, made to sing the national anthem, whatever.
 * List at least two other types of secondary protein modification.
 * If I understand the point correctly, this seems to just be asking (once again) to run through the various types of protein modifications that exist: ubiquitinylation, acetylation, glycosylation, etc.
 * Explain the structure of an ATP molecule.
 * Adenine (double loop), ribose, triphosphate. At the risk of stealing Jeff's thunder:
 * Explain how protein kinases can be classified and describe examples.
 * Based on which residue they phosphorylate (Ser-Thr-Asp)
 * Based on their substrate protein
 * Based on their activating stimulus
 * Based on their phylogenic relationship (certain families of related kinases)
 * Describe the structure/function of a protein kinase and principles of their regulation (including: alternation between "open" and "close" conformation in all kinases, and requirement for activation loop phosphorylation in some kinases).
 * Protein kinases have two lobes: one small, one large.
 * ATP binds in cleft between lobes. Substrate mainly binds to large lobe.
 * "**Activation loop** ": region of many kinases that needs to be phosphorylated before the kinase itself is activated. Notice that some kinases can phosphorylate themselves.
 * "**Glycine-rich loop** ": region that presses down on ATP to correctly position the gamma phosphate to bind to substrate. Has two conformational states:
 * "Closed" state = phosphate transfer state. Transfer happens very quickly.
 * "Open" state = ADP-for-ATP exchange state. Happens more slowly.
 * Active conformations of most kinases are very similar (nonspecific)- problem for specific drug interactions. However, the inactive conformations are less similar- better specificity as drug targets.
 * Generally in the inactive state (not the same as the "closed" state of the glycine-rich loop), the activation loop or the ATP binding cleft is characteristically distorted- these can be targeted.
 * Notice also that the substrate binding domain can also be used to bind antagonists to inactive kinase proteins.
 * Some examples:
 * CAMK II and calcineurin: promote long-term potentiation or depression, respectively, of synaptic strength (memory/learning).
 * Notice that rapamycin blocks mTOR (ser/thr kinase) to block T cell proliferation (thus is an immunosuppressant).
 * Cyclosporin (first choice of immunosuppressant in transplants) blocks calcineurin, thus blocking the expression of IL-2 (inflammation cytokine).

Extracellular Matrix and Cell Adhesion Thursday, November 29, 2007 9:42 AM


 * Extracellular Matrix and Cell Adhesion, 11/29/07:**


 * Summary of the general idea here: You've got proteoglycans. They attract water and form a extracellular gel in which fibrous proteins and multidomain proteins are embedded. The fibrous proteins provide a scaffold for cells as well as imparting fibrous/elastic properties to tissue. The multidomain proteins attach to both fibrous proteins and to CAMs (**c** ell **a** dhesion **m** olecules) on the surfaces of cells. The CAMs are transmembrane proteins which attach extracellularly to other CAMs or multidomain proteins and intracellularly to signaling molecules inside the cell (information can flow both ways).
 * Types of fibrous proteins: collagen, elastin
 * Types of multidomain proteins: fibronectin (found all over), laminin (found in basal lamina)
 * Types of CAMs: cadherin (attaches to other cadherins, form calcium-activated 'zippers,' homodimeric), immunoglobulin (attaches to other Igs, form non-calcium-activated 'zippers', monomeric), integrin (attaches to ECM proteins, has intracellular signaling end).


 * Discuss the contributions of the ECM to cell and tissue function:
 * Essentially, need an substance to which cells can attach, both for motile purposes and also because non-carcinogenic cells __can't grow__ without something to attach to.
 * Carcinogenic cells can grow without an attaching surface (ie in agar) due to mutations in adhesion signaling pathways.
 * Dr. Pfenninger seems to really be harping on this idea that adhesion is not a simple, glue-like process: cells 'stick' to very specific types of materials, and different cells stick to different things. He describes it as a combination of mechanical adhesion and cellular communication-- that is, the cell 'knows' what it's attaching to and can respond appropriately.
 * Also use the ECM to form functionally distinct compartments in the cell and to allow cells to act in concert with each other.
 * Define the four major classes of ECM components and their properties:
 * (1) **__Glycosaminoglycans__** (GAGs), often linked to proteins to form __proteoglycans:__
 * GAGs: large, unbranched disaccharide polymers. Typically contain an amino sugar and a sugar that's substituted with a carboxy group (acidic sugar), sometimes with a sulfhydryl group. What this means: GAGs are __negatively charged__.
 * **Proteoglycans** are just GAGs linked to a "core protein".
 * Note that these proteoglycans or GAGs can aggregate together: due to their negative charge and solubility, they form a kind of __gel__ as space-filling molecules in the cell.
 * (2) Fibrous proteins (collagen/elastin):
 * **Collagen** is the most abundant (25% of total protein weight in //H. sapiens// ); tough polymers that provide tensile strength in connective tissues.
 * **Elastin**, as the name implies, is elastic protein found in a variety of tissues.
 * Recall that alpha-1-antitrypsin deficiency involves an inability to control the breakdown of this protein by elastases.
 * (3) __Multidomain adaptor proteins__ (fibronectin and laminin):
 * **__Fibronectin__** : dimeric glycoprotein (proteins with saccharides attached to their backbones) linked by disulfide bonds. Can bind a variety of proteins (collagen, heparin, each other, etc).
 * More importantly, can also bind integrins (principal adhesion molecule on cell surfaces).
 * Found all over the body.
 * **__Laminin__** : found predominantly in basal lamina (ECM that provides the "ground" for epithelial cells). Heterotrimeric form in the shape of a cross (proof of intelligent design!). Cross-links with itself pretty good.
 * (4) Water and solutes:
 * Predominantly found in the "gel" between cells: allows easy diffusion of nutrients and growth factors between cells.
 * Notice that the ECM, like anything else, needs to be broken down and remade frequently. This job falls to extracellular proteases:
 * (1) matrix metalloproteases (MMPs)
 * (2) serine proteases
 * Notice these can be used in a targeted manner to allow development of tissues or cell migration or invasion of nearby regions.
 * Define two types of fibrillar proteins and at least two types of multidomain adapter proteins of the ECM:
 * See above (collagen and elastin, fibronectin and laminin).
 * Discuss the role of adhesion in cell function and survival in metazoans
 * As said: need to adhere to each other and not all fall down in a heap; need something to adhere to in order to survive; need to form distinct compartments in cell.
 * Define and describe at least three different types of cell adhesion molecules (CAMs) and their ligands:
 * **__Cadherins__** :
 * Single-pass transmembrane glycoproteins.
 * Found as homodimers.
 * Activated by __calcium__: when activated, cadherins on one surface "zip up" with cadherins on the other surface (homophilic binding reaction)
 * **Ig (immunoglobulins)** :
 * Single-pass transmembrane glycoproteins as well.
 * Unlike cadherins, operate as monomers, and binding doesn't require Ca2+.
 * However, the Ig domains on these proteins do bind to Ig domains of Ig proteins on other surfaces.
 * **Integrins** :
 * Heterodimers (alpha and beta subunits): lots of different types of integrin, both alpha and beta, so can have lots of different combinations for binding to different things.
 * Has a ECM-binding domain at its end: can bind to laminin, fibronectin, collagen, etc.
 * Notice that the intracellular "tails" of integrins attach to various things inside the cell that can respond to the attachment:
 * Can be attached to catenins (tumor suppressor), cytoskeleton, etc.
 * Discuss the role of CAMs in signaling:
 * CAM extracellular portions can interact with signal proteins to tell the cell what it's attached to. (outside-in signaling)
 * This can go the other way: the cell can interact with the CAM to change or get rid of its extracellular adhesion. (inside-out signaling)
 * Describe proteins associated intracellularly with CAMs:
 * Cytoskeletal proteins to anchor the CAMs in the cell membrane (mechanical linkage): often actin-binding proteins.
 * Signaling proteins: often protein kinases or GTPases.
 * mentioned in lecture: (1) tyrosine kinases, (2) //src//, (3) protein kinase C, (4) Rho GTPases.
 * Discuss the ECM and cell adhesion in the context of disease processes
 * Cell adhesion molecules, since they often control apoptosis and proliferation, are frequent targets for cancers and viral oncogenes (ie //v-src// ).
 * Notice that cell adhesion is also intimately involved with immune response (mainly integrins): thus problems with CAMs can lead to compromised immune function.
 * "selectin": CAMs that "catch" leukocytes coming out of the bloodstream. Mutations in this protein can cause obvious problems.

Receptor tyrosine kinases Thursday, November 29, 2007 10:59 AM


 * Receptor tyrosine kinases, 11/29/07:**


 * [Functions: cell growth, motility, metabolism, cell survival, differentiation]
 * Describe the mechanism of receptor tyrosine kinase (RTK) activation.
 * As mentioned by Dr. Prekeris, RTKs are activated by __dimerization__; when two nearby RTKs in the membrane are bound by their ligands, they undergo a conformational change, bind to each other, and co-activate by __phosphorylating__ each other.
 * Ie: for these RTKs, their substrates (PO4 targets) are other RTKs.
 * Once activated, they can also act as catalysts for other reactions.
 * Notice that this doesn't have to be homodimerization: two different kinds of RTKs can dimerize to generate a different signal than a dimer formed of two of either kind.
 * RTKs generally trigger complex kinase cascades that result in changes in gene expression.
 * Explain the molecular mechanism of stimulation of ras GTPase by RTKs.
 * **All** RTKs activate //ras// signaling.
 * Ras proteins: membrane-bound switches regulated by GAPs and GEFs (see below). **Involved with cell proliferation.**
 * In the "off" state, it's bound to GDP; in the active state, it's bound to GTP.
 * In GTP-bound (active) form, Ras proteins sets off proliferative cascade.
 * Generally, Ras proteins can switch themselves off (intrinsic GTP->GDP activity)
 * **GAP** : GTP activating protein (increases its ability to switch itself off). Inactivating mutations in GAPs can cause cancer since //ras// proliferation is unchecked.
 * GAPs: turn Ras proteins off (tumor suppressor genes).
 * **GEF** : Guanine nucleotide exchange factors: helps exchange "spent" GDP for fresh GTP (thus activating Ras proteins). Increase-of-function mutations in GEFs can lead to cancer since Ras protein is constantly being activated again after it inactivates itself.
 * GEFs: turn Ras proteins on (oncogenes).
 * **Mechanism** :
 * Dimerized RTKs bind to and activate __Grb2__, an adaptor protein; this has a SH3 binding domain which binds to __Sos__, which is a GEF (Ras activator).
 * Sos being bound, it's brought into proximity with Ras proteins, which are located in the cell membrane like the RTKs.
 * Essentially __RTKs activate Ras proteins by changing the location of GEFs, not by catalytic activity__. (Movement of proteins into proximity causes proliferation).
 * [Epidermal growth factor receptor (EGFR): overexpressed in a variety of tumors; good drug target for cancer treatment.]
 * Describe mechanism of action of two main classes of RTK-targeted anti-cancer agents (antibodies and TKI's).
 * Antibodies: Recognize extracellular domain of RTKs.
 * Effectively act as competitive antagonists: prevent ligand from being able to bind to the extracellular receptor domain.
 * Nicely specific to particular RTKs.
 * TKIs (tyrosine kinase inhibitors): inhibit tyrosine kinase activity of RTKs.
 * This works by competitively binding to the ATP binding pockets of specific RTKs. No ATP, no phosphorylation, no activation of RTK dimers.
 * Recall that the "closed" or un-ATP-bound TK has a specific conformation that allows more specific drug targeting-- however, there's still enough similarity that TKIs will inevitably have multiple TK targets.
 * List tumor cell characteristics that predict clinical response to EGFR-targeted therapeutics.
 * Patients with non-small-cell lung cancer show only a 20% response to TKIs.
 * The 20%: usually non-smokers, women, Asian descent.
 * When you've had a mutation in EGFR to make the activation domain much more active, the response to TKIs tends to get better.
 * Why this is: it's thought that with an improved or more efficient EGFR proliferation pathway, the cell may be "addicted" to that pathway-- which would make the cell much more vulnerable to TKIs, which target that pathway.
 * Notice this also applies to patients with abnormally high levels of EGFR in their tumor cells: again, may be "addicted" to this pathway.
 * Describe mechanism of resistance to TKI's caused by EGFR somatic mutations.
 * There are secondary (after first treatment) mutations in EGFR that cause decreased response after an initial improvement when treated with the TKI.
 * Essentially, seem to be selecting for EGFR proteins that have mutations that block TKI binding.
 * Note that there are drugs that can inhibit these mutant EGFR proteins-- since we know what the mutations are, we can use the sequence to target inhibitory drugs.


 * Important note: Dr. Thorburn kindly filled out his own LO's in his handouts; might check them out for a slimmer version of all this verbiage.

Epithelia I, II, + III Friday, November 30, 2007 7:55 AM


 * Epithelia I, II, +III, 11/30/07:**


 * State the various structural arrangements, classifications, and functions of epithelial tissues, and state their general structural relationships (orientation) to connective tissue, blood vessels, muscle, and neurons (peripheral nervous tissue).
 * Make up "business ends" of many organs.
 * Most cancers derived from epithelia.
 * Epithelia: tissues that line all surfaces of body, internal (gut, glands, tubes, ducts, etc) and external (skin). Note this includes a variety of internal organ linings.
 * Functions: (1) barrier to microorganisms and toxins; (2) selective transport into and out of body; (3) biochemical modification of molecules and metabolites (detoxification in liver); (4) specialized reception of stimuli (ie taste receptors); (5) self-renewal.
 * Properties:
 * (1) epithelial cells are highly adherent to each other and form 'sheets,' often wrapped to form tubes;
 * (2) most epithelial cells have polarity: the surface that faces the lumen is called the __apical__ surface, and the surface that faces opposite the lumen (towards the connective tissue) is called the __basal__ surface.
 * Note that polarity allows unidirectional transport (in either direction) through epithelial cells for specific substances.
 * (3) Structure found in all epithelia, underlying the basal surface: the __basal lamina__ layer.
 * (4) All epithelia are attached on the basal side to __connective tissue beneath the basal lamina__.
 * Inside the connective tissue is vasculature. This is important because __epithelial cells have no inbuilt vascular supply__. Blood needs to diffuse through the connective tissue to reach the epithelia. This will be important when we get to epithelial self-renewal.
 * Notice that the cells lining the vascular system are not called epithelia; they're called __endothelia__. Just a side note.
 * Also within the connective tissue: muscles and nerves.
 * During development, primitive epithelial cells become mesenchymal cells and migrate through the body to form new regions of epithelia (__epithelial-to-mesenchymal transition__).
 * The reason this is significant is that this is also what some tumors do-- reactivate the mesenchymal transition, migrate through the body, and metastasize their little evil hearts out.
 * Types of epithelial cells:
 * Single sheet of cells (__simple__) vs. multiple stacked sheets of cells (__stratified__).
 * Notice an exception: certain columnar stacks are called "pseudostratified" (cell nuclei are in all kinds of places, not in rows)
 * Also a "transitional" stack type, which seems to be more or less like regular ol' stratified. Evidently when you stretch it, it gets wonky.
 * Also classified based on cell shape: __cuboidal__ (like cubes), columnar (like columns), squamous (just kind of flat and messed-up looking).
 * Notice that stratified cell shapes are classified based on the __outermost__ layer of epithelia.
 * Describe the cellular basis for apical-basal polarity of epithelial cells and describe the functions of epithelial polarity.
 * Molecular/protein composition on apical side is different from that on the basal side.
 * On the face of it this seems weird-- if membrane proteins can run around throughout the lipid membrane, why don't they equal out on both sides? Answer is that there are __tight junctions__ between epithelial cells that prevent apical membrane components to getting to the basal side, and vice versa.
 * Interestingly, the cytoskeletons of epithelial cells are also polarized, which make the organelles inside epithelia polarized as well (have directionality).
 * State the different cell junctions that connect epithelial cells to one another and to the basal lamina, and describe their key components and functions.
 * Tight junctions: hold adjacent epithelial cells together. Made of transmembrane proteins; most common such proteins are called "occludens".
 * Tight junctions wrap all around the cell; prevent flow of molecules from the apical to the basal side.
 * Basically the tight junctions are the basis of impermeability of epithelia: it forces substances in the lumen (water, ions, etc) to go __through__ the cells as opposed to __between__ them to get into the basal surfaces.
 * As noted last unit, there are epithelia that are 'looser' than others; ie, their tight junctions are a little less tight (need to hit the gym).
 * Looser epithelia are found primarily in the intestines (as noted by Betz): allows for quick, massive transport of water and ions between cells (__paracellular transport__).
 * Important note: tight junctions can be loosened or tightened depending on what substances are being transported (the intestinal epithelia gets a lot looser in the presence of glucose, for example).
 * Notice also that when tight junctions are loosened, substances on the basal side of epithelia can leak __out__ into the lumen as well (simple concentration-driven).
 * Notice that there are punctiliar, specific regions between cells that are also strongly adherent. These are called either __desmosomes__ or __adherence junctions__ and are caused by a variety of specialized proteins, including __cadherins__.
 * Cadherins, recall ("Extracellular Matrix" lecture), bind with each other in the presence of calcium.
 * These provide a way to regulate the tightness of specific cell junctions.
 * Notice that desmosomes have no barrier function: they're just there to bind the adjacent cells together.
 * Third type of cell junctions: __gap junctions__. Essentially a small "tunnel" or channel between intracellular regions of two adjacent cells. Function is to selectively allow the flow of small molecules (like signaling molecules) between cells (speed up broad response to stimuli).
 * State the types and functions of the different cell surface modifications on epithelial cells.
 * Microvillae: "pouches" on the apical side of epithelial cells: increase surface area of apical surface. Filled with actin, generally.
 * Cilia: "hairlike" structures, move substances by rhythmic motion. Made up of microtubules powered by dynein motors.
 * Stereocilia: found in cochlea of ear; seem to be important in stimulus reception.
 * Describe basal laminae by stating their basic components, the basis of their diversity, their functions, and their structural relationship to epithelia and other tissues.
 * Structure: a thin sheet made up of interlocking proteins. Some proteins are common to most basal laminae (ie type IV collagen). Some proteins are unique to particular laminae in particular tissues.
 * Functions of the basal lamina:
 * (1) promote attachment of the epithelia to the underlying connective tissue.
 * Two kinds of attachment:
 * __Hemidesmosomes__ (no relation to desmosomes, irritatingly), which contain __integrins__ that provide membrane attachments to the epithelial cells and also other protein complexes that link the connective tissue to the lamina. Effectively hemidesmosomes are a kind of protein 'hitch' that hooks the lamina to both the epithelium and also the connective tissue.
 * Notice that in some tissue, you have specialized hemidesmosomes called __focal adhesions__ which connect the basal lamina to intracellular components inside the epithelial cells. This allows signaling communication between the lamina and the epithelium, which allows the lamina to influence the development or regulation of the epithelium.
 * (2) Regulate epithelial cell biology (through focal adhesion signaling).
 * (3) Barrier function: barrier to movement from epithelial layer to connective tissue. Generally not very good at this.
 * (4) Sort of a subset of (3)- specialized types of basal lamina can act to filter specific molecules, particularly in the glomerulus of the kidney.
 * Compare and contrast exocrine and endocrine glands in terms of their development, general structure, and functions. For both types of glands, trace the path that a secreted molecule must take from its synthesis to its destination, and describe all the barriers/structures the molecule must cross en route.
 * Exocrine glands secrete agents molecules onto epithelial surfaces (inside or outside the body).
 * Developed as simple invaginations of the epithelium (still in contact with epithelial surface).
 * Synthesize a variety of substances.
 * Notice that exocrine secretions have to cross the __apical__ membrane to get to the 'outside' of the epithelial cell.
 * "Secretory units" = invaginated pouches that function as exocrine glands. These make the secretions and transport them across the apical membrane, from which they flow down the narrow part of the invagination ("channels") to reach the outside of the epithelium.
 * Notice that sometimes you don't get all this invagination junk and you just have single cells within the epithelial layer that produce secretions and transport them out the apical membrane. The mucus-producing cells in the intestine are a well-populated example of this.
 * Types of exocrine secretions:
 * (1) mucus secretions: mucusy. You know.
 * (2) serous secretions: watery. Sweat, saliva, etc.
 * Endocrine glands always secrete agents into blood.
 * Developed as invaginations of the epithelium that are pinched off from the epithelial surface; surrounded by blood vessels in the connective tissue, the secretions from endocrine glands go out into the vascular system.
 * Synthesize hormones.
 * Notice that endocrine secretions (generally) have to cross the __basal__ membrane, as well as getting through connective tissues and blood vessel surfaces to make it into the bloodstream.
 * Exception: thyroid endocrine hormones secrete through the apical membrane, mature in the lumen of the gland, and then are transported across both membranes out again.
 * Describe the epithelial to mesenchymal transition during development.
 * See above (epithelial cells transition to mesenchyme, travel in the body, and transition back to epithelium to set up epithelial regions in other locations).
 * Describe how epithelial tissues are maintained and regulated, and describe the properties, functions, regulation and development of epithelial stem cells.
 * Most epithelia have self-renewing potential via specialized stem cells.
 * These are stored in "crypts" or pouches at the bottom of epithelial sheets.
 * Stem cells are more or less as described by Dr. Hooper on Thursday.
 * Tightly regulated: few of them, spread out, slowly dividing.
 * Notice that there is a polarity of cell proliferation (in skin, towards the apical "outside")-- new cells push up old cells and old cells migrate towards the apical direction. Cells become further differentiated as they migrate.
 * Regulatory proteins of epithelial stem cells: **Wnt** proteins.
 * Wnt proteins in colon inhibit differentiation by binding at the cell surface and setting off a signaling cascade. Notice that they also promote cell division (which goes along with a general stem cell principle: more differentiation, less division).
 * Normally, **APC** protein inhibits the Wnt signaling pathway in the colon.
 * If APC is knocked out, differentiation stops and cell proliferation is activated.
 * APC mutations -> colon cancers
 * Similar proteins in lung, but opposite pathway: in lung, Wnt promotes differentiation and inhibits division.
 * State the general terms for epithelial-derived cancer, and describe how defects in epithelial cell regulation can contribute to cancer.
 * **Carcinoma** (general term for all cancer of epithelial origin)
 * **Adenocarcinoma** (term for cancers derived from glandular epithelia)
 * Interestingly, high levels of cadherin activity correlate with higher survival rates in cancer, and low levels of cadherin with lower survival rates-- this could be from (a) tighter connections between epithelial cells lead to less metastasis of epithelial cancer cells, or maybe (b) cadherins can act as signaling molecules to sense cancer and respond appropriately.
 * Describe how tissue sections are made and visualized for histological (microscope) examination, both for general staining and for staining with antibodies (immunohistology). Distinguish what general stains visualize from what immunohistological staining techniques visualize. (NOTE: info on this objective is found in the Intro/Epithilia lab PDF file, and will be discussed in class.)
 * Tissue sample is cut out, put in saline buffer, and "fixed" (solutions that preserve large cell structure). Notice that carbohydrates and lipids tend to leak out in this process.
 * The sample is sliced very thinly and, usually, stained to create a two-dimensional image.
 * Stains can be very general (bind to lots of different macromolecules), or very specific. Notice that anything that doesn't bind to the stain will not show up dark on the stained image.
 * Evans' tip: Find nuclei. These tend to be big dark blotches. They will tell you where individual cells are.
 * Immunohistochemistry/immunofluorescence: methods of detecting presence of particular proteins in pathology slices- information about quantity and location of protein.
 * Use antibodies: antibodies have high affinity (bind tightly) and specificity (just bind target) for certain types of proteins, or antigens.
 * Method: after fixing and staining, apply "primary antibody" (antibody that sticks to the protein you want to look at). Wash off the excess, then add "secondary antibody" (antibody that sticks to the primary antibody, which is also linked to either a fluorescent tag or an enzyme that produces a colored product). Then you wash off the unbound secondary enzyme, and look at either the fluorescence or the color against the background of the cells.
 * Secondary antibodies that fluoresce: immunofluorescence. Secondary antibodies that use enzymes to produce color: immunohistochemistry.

Connective Tissues I + II Monday, December 03, 2007 8:01 AM


 * Connective Tissues I + II, 12/3/07:**


 * State the types, origins, and functions of the different cell types found within connective tissues.
 * Immune system function: connective tissue provides a leukocyte-filled barrier to pathogens coming through the epithelium.
 * Cells that make ECM:
 * fibroblasts, chondrocytes, osteoprogenitors (all derived from mesenchymal or connective stem cells).
 * Fibroblasts differentiate into adipocytes or smooth muscles, or can shift into osteoprogenitors or chondrocytes.
 * Chondrocytes make cartilage, osteoprogenitors make osteoblasts (which eventually turn into osteocytes).
 * Notice fibroblasts are capable of division and can interconvert into other kinds of cells as needed.
 * Obviously, fibroblasts need to be highly regulated due to their potential for uncontrolled division.
 * Cells not made in connective tissues but present in ECM:
 * lymphocytes (make antibodies when activated; activated lymphocytes are called 'plasma cells'.)
 * neutrophils/eosinophils (undetailed immune cells)
 * mast cells (start out as 'basophils' in blood, involved in inflammatory response)
 * macrophages (start out as 'monocytes' in blood, phagocytic 'scavenger' cells)
 * These can break down and restructure tissues by engulfing other cells.
 * Notice also that they can act as signaling centers: synthesize and secrete signaling molecules that direct restructuring of tissue.
 * Can trigger blood vessel formation (angiogenesis).
 * This is why tumor growth is aided by macrophages- promote new blood supply to tumors.
 * Describe the components of the extracellular matrix, their functions, and how they are organized in different connective tissues. Describe the structural relationship between connective tissue and epithelia, blood vessels, muscles and nerves (this will be best understood after you have studied all of these tissues).
 * General structure: Extracellular fibers embedded in gel-like mix called the "ground substance."
 * Fibers: collagens and elastins.
 * Collagens:
 * __Fibrillar__ collagens: make long, thick composite fibers, or fibrils (primarily Type I collagen). Lots of tensile strength.
 * __Fibril-associated__ collagens: make thin fibers connecting basal lamina to fibrillar collagens and fibrillar collagens to each other.
 * __Networking__ collagens: form thin sheets that provide the scaffold for the basal lamina (Type 4).
 * General unit of collagen is the triple helix; these helices pack together (for both width and length) to form fibrils.
 * The assembly into triple helices takes place outside the cell; proteases cleave both N- and C-ends of the collagen molecules, which enable them to form helices.
 * The N-cleaved ends can be detected in blood and urine-- an indicator of metabolic activity in bone and other connective tissues.
 * N-cleaved ends are called "N-telo peptides."
 * Recall that non-hydroxylation of proline residues of collagens (caused by deficiency of vitamin C) leads to scurvy.
 * Elastins:
 * As you'd expect, these are elastic fibers, cross-linked to form "rubber bands."
 * These fibers are supported by proteins called fibrillins; it's mutations in fibrillins that cause Marfan's Syndrome.
 * Marfan's susceptibility to cardiac problems has to do with the reduced elasticity of their major vessels.
 * Ground substance:
 * Composed largely of proteoglycans (proteins + glycosaminoglycans, or GAGs-- see "Extracellular Matrix and Cell Adhesion"):
 * Essentially a protein core and massive carbohydrate side chains.
 * The carbohydrate chains are negatively charged, attracting water and hydrating the ground substance.
 * Because ground substance is hydrated, solutes can move through it.
 * Notice that GAGs are also involved in storing and regulating polypeptides, like growth factors, that diffuse near them.
 * Also composed of GAGs unlinked to proteins (most common kind is __hyaluronic acid__) and a lot of proteins (signaling proteins, proteases, etc).
 * For the proteins that form extracellular fibers, describe their types, their properties, and how they are made and assembled in the extracellular matrix.
 * See just discussed-- collagen, elastin, fibrillin. Note that the N-telo ends are cleaved outside the cell (thus detected in blood and urine).
 * Describe the basis and functional consequences of connective tissue diversity.
 * Basis: stem cells that can differentiate into many kinds of basic cells, difference in the function and ECM secretions of those differentiated cells.
 * Functional consequences: more sketchy. "We have working connective tissue," I guess. Could also mean that from one type of cell (mesenchymal stem cells) we get a massive diversity of connective tissue, dependent on differentiating signaling and signals that tell those post-differentiated cells to produce different proteins.
 * Describe how connective tissues are regulated upon tissue growth, use or injury. Describe the events that occur following wounding or during edema.
 * Tissue growth, or use: not sure what he means here. They can be remodeled at just about any point by proteases and stem cells. Injury is covered under the next point.
 * Wound response: generally signaled by platelets from torn blood vessels.
 * Inflammation phase: __mast cells__ activated to secrete various molecules, among them **histamines** and **chemoattractants** (polypeptides that attract migratory cells).
 * These go into the blood vessels and cause an influx of normally vascularly-located immune cells (leukocytes and macrophages) into the ECM at the site of the wound.
 * Macrophages, in addition to 'eating' damaged tissue and bacteria, trigger angiogenesis in the wound (though this often doesn't transpire until the tissue actually begins to heal in the next phase); also triggered are __cytokines__, which attract the cells involved with the next phase.
 * Proliferation/development phase: Fibroblasts are attracted by cytokines and other signals; these divide and increase their rate of ECM secretion.
 * Fibroblasts in area begin to repopulate damaged epithelium and connective tissue (if basal lamina is damaged extensively, end up with scar tissue).
 * Fibroblasts also differentiate into __myofibroblasts__: exert pressure on area to restrict blood loss and close the wound. These generally die off after the wound has healed.
 * Maturation/remodeling phase: Effectively a continuation of the development phase, in which the fibroblasts replace temporary collagen laid down to close the wound with more permanent collagen oriented for maximal tensile strength.
 * [Bone functions: structural support, calcium/PO4 homeostasis, houses hematopoeitic system]
 * For cartilage, describe its cellular and extracellular composition, its structural properties, and how it is organized. State the functions of cartilage tissue.
 * Cartilage: serve as template for bone or as resilient/pliant support system.
 * Mesenchymal stem cells form chondrocytes, which secrete cartilaginous ECM.
 * These chondrocytes become cocooned in "lacunae" of their own secretions. Notice that they can still divide in lacunae (matrix is flexible) and grow out the cartilage (interstitial growth, see below).
 * The chondrocytic matrix is covered with a hard outer layer called the "perichondrium", which contains the mesenchymal stem cells that gave rise to the chondrocytes in the interior.
 * This means that cartilage can grow in two different ways: __appositional__ growth, in which the perichondrium lays down new layers of cartilage on the surface, and __interstitial__ growth, in which the cocooned chondrocytes in lacunae continue to divide and secrete (growing cartilage from within the existing cartilage).
 * This is significant largely by contrast: bone matrix is much less flexible and cannot grow interstitially (bone lacunae can't shift apart to accommodate internal growth like cartilaginous lacunae).
 * Note that cartilage is **avascular** -- it has to derive nutrients and oxygen from peripheral vessels.
 * Describe how cartilage grows during fetal and child development.
 * All I can figure from the notes (I don't think he mentioned this in class) is that chondrocytes arise from mesenchymal stem cells during fetal development and do what they always do (interstitial and appositional growth, with chondrocytes secreting cartilaginous ECM and becoming encased in lacunae) from that point on.
 * State the characteristics that distinguish the three basic types of cartilage.
 * __Hyaline__ cartilage: full of __hyaluronic__ acid (recall, that's the GAG type that isn't bound to proteins). Fairly sparse and irregular collagen fiber matrix.
 * Hyaline is the type of collagen that will ossify in long bones (see next section).
 * In particular locations, hyaline also has a lot of elastic tissue (earlobes, etc). This is called elastic cartilage.
 * Fibrocartilage: often found where tendons attach to bone or in joints. Much more densely packed with fibrous collagen; tougher.
 * State the different cell types found in bone. For each cell type, describe their functions, their origins, and describe how they are organized in bone tissue.
 * Bone cells: also formed (except for osteoclasts, see below) from mesenchymal stem cells, which form __osteoprogenitor__ stem cells which can then self-renew and also produce __osteoblast__ secretory cells.
 * Osteoblasts assemble on the surface of forming bone; they secrete a watery, loose ECM characteristic of bone called __osteoid__ (contains collagen, but not dense).
 * Like chondrocytes, osteoblasts become cocooned within their own secretions; when they're fully enclosed, osteoblasts transform into __osteocytes__, which have vastly reduced secretory activity but which send out processes that contact their osteocyte and osteoblast neighbors to form gap junctions (signaling function).
 * These long processes form __canaliculi__ (sing. canaliculus) through bone matrix.
 * This seems to be involved in sensing the current status of the bone matrix and regulating osteoblast activity depending on that status.
 * Notice that osteoblasts also trigger the __mineralization__ of the loose original bone ECM: they secrete Ca2+- and PO4-filled __matrix vesicles__, which break open in the ECM as their contents form crystallized calcium phosphate ("hydroxyapatite"). The released hydroxyapatite gives the bone ECM its extreme hardness; it also serves as the main storage site of calcium in the human body.
 * As mentioned, due to this hardness, there is no interstitial growth in bone (matrix is too inflexible to allow it)-- bone only grows by appositional growth at the surface due to division and secretion of osteoblasts and osteoprogenitors.
 * Completely different lineage of osteo- cells: __osteoclasts__, derived from monocytes in blood vessels (as are macrophages).
 * Osteoclasts are effectively bone-matrix-specific macrophages: when directed, they 'chew up' and degrade bone matrix and release liberated calcium into newly loosened ECM, where it can get back into the bloodstream.
 * Functions of osteoclasts:
 * Degrade cartilage (for ossification of 'template' cartilage) as well as bone (for remodeling and calcium mobilization)
 * Stimulate angiogenesis (like macrophages).
 * As these tunnel through bone, they bring blood vessels with them (note that bone //is// vascularized).
 * Innervation: Nerves follow osteoclast-formed channels in bone matrix.
 * Describe the composition of bone extracellular matrix. What are the functions of the different components (only those discussed in class and in the lecture notes)? Describe where these extracellular matrix components are made, and how they are deposited to form bone matrix.
 * See above. Loose collagenous bone ECM is secreted from osteoblasts, which also secrete the matrix vesicles that contain the hydroxyapatite that will mineralize the original, soft ECM. Osteoclasts resorb the mineral matrix and excrete the inorganic ions inside into the reloosened ECM, from where they can get back to the bloodstream. Note that bone ECM is vascularized and innervated due to osteoclast action.
 * Not real specific, but about as specific as the lecture and notes.

Connective Tissue III Tuesday, December 04, 2007 9:04 AM


 * Connective Tissue III, 12/4/07:**


 * Describe the two different processes that lead to bone formation. Describe how long bones grow in length and in width.
 * **Intramembranous ossification** :
 * During development: mesenchymal stem cells condense (come together and proliferate) and form layers with surrounding connective tissue. Some of the stem cells differentiate into osteoprogenitors and then to osteoblasts, which secrete bone matrix that's vascularized/innervated/remodeled by osteoclasts and mineralized by osteoblasts.
 * Effectively this is the simple route of bone formation: direct formation of bone from mesenchymal stem cells without a cartilaginous intermediate.
 * Most of the flat bones form this way.
 * **Endochondral ossification** :
 * Initial, 'template' cartilaginous 'bones' are ossified and replaced with actual bone.
 * Recall that the perichondrium of cartilage contains mesenchymal stem cells which continue to create new chondrocytes.
 * At some point, a signal is received at around the middle of the diaphysis (center of long bone) which tells the mesenchymal cells to begin producing osteoprogenitors instead of chondrocytes. This begins a transitional wave that transforms the perichondrium to a periosteum.
 * Formation of periosteum induces two events: __calcification of cartilage__, and the __destruction of chondrocytes__ embedded in the cartilaginous matrix.
 * Chondrocytes tend to get very large before they're killed off (apoptosed)-- this is called hypertrophy, which is an indicator that chondrocytes are becoming apoptotic prior to ossification.
 * Osteoprogenitors/osteoblasts send signals to bring osteoclasts to the cartilage-- the osteoclasts show up, see the calcified cartilage, and begin to degrade it and engulf the apoptosed chondrocytes.
 * Note that osteoclasts bring blood vessels and nerves with them while this is going on.
 * After the osteoclasts have done their thing, the osteoblasts set in to lay down bone matrix and calcification around the vessels that have just invaded the nascent bone.
 * Notice that the cartilage is still growing (appositional and interstitially) wherever it can while this is going on.
 * The secondary ossification centers, one at each end (epiphysis) of the bone, form while the primary ossification center is still being vascularized and mineralized. These progress in the same manner as the primary.
 * Note that there's a layer of cartilage left behind (the epiphyseal or growth plate) between the diaphysis and epiphysis.
 * This cartilage continues to grow interstitially in the direction of the epiphysis (towards the ends of the bone); the primary ossification center 'chases' this cartilaginous growth, ossifying the new growth as it occurs.
 * If you're looking at a histology slide of ossifying cartilage, can always tell which layer is the cartilage by looking at 'lines' of proliferating chondrocytes pushing out in the direction of the end of the bone.
 * Interstitial growth of cartilage is what drives the __length__ of long bones; __width__ of bone is driven by appositional growth of the periosteum (which completely replaces the perichondrium along the edge of the bone).
 * Most of the skeleton (including all the long bones) are formed this way.
 * Describe the sequence of events that occur in bone remodeling.
 * Periosteum forms on the surface of cartilage.
 * Osteoblasts calcify the cartilage; chondrocytes undergo hypertrophy and apoptose.
 * Osteoclasts degrade the cartilage, engulf chondrocytes, lay down new blood vessel and nerve pathways.
 * Osteoblasts make bone matrix and mineralize it around vessels and nerves.
 * Describe how bone formation and remodeling is regulated.
 * [**Endosteum** : inside surface of bone (inner surfaces of osteoclast-derived tunnels).]
 * Bone remodeling (osteoclast activity and subsequent osteoblast secretions) occurs immediately upon formation of new bone, and also recurs almost continuously throughout life.
 * Notice that the activation of osteoclasts is tightly linked to the activation (ie production and secretions) of osteoblasts.
 * In early bone, the coordination of vessels/nerves with matrix is disorganized and random; over time, the constant remodeling leads to a particular division between __compact__ and __spongy__ bone.
 * Compact bone: On the edge of the bone; dense, multi-layered, no trabeculae.
 * Spongy (or 'cancellous' or 'trabecular') bone: Deeper inside the bone, form honeycombed networks of endosteal surface, filled with bone marrow.
 * Notice that both flat and long bones show this pattern (spongy vs compact).
 * Mechanisms of bone-remodeling-signaling:
 * Short-range: Bone morphogenetic proteins (BMPs) released at bone site.
 * Don't travel through bloodstream.
 * Effectively instruct other cells to lay down and remodel bone.
 * Significant pathophysiologically due to FOP disease (//Fibrodysplasia ossificans// ) that causes ossification to occur in loose connective tissue. Caused by the BMP4 gene being fused to an abnormal promoter; this causes it to be expressed in lymphocytes (from where it acts on fibroblasts/mesenchymal stem cells to form osteoprogenitors and bone).
 * Can also use Wnt and Notch proteins to do short-range signaling (no details needed).
 * Long-range: Endocrine hormones, particularly __calcitonin__ (lay down more calcified matrix: Ca2+ out of bloodstream, build up bone) and __parathyroid hormone__ (reabsorb more calcified matrix: Ca2+ into bloodstream, break down bone), as well as steroid hormones (no details).
 * Mechanical stress: as per anatomy, putting stress on bones leads to different cell activity in bone remodeling-- thickens bone (greater appositional growth) in particular directions.
 * Neuronal stimulation: CNS regulates bone remodeling. A "who knows" category.
 * Describe how defects in bone remodeling leads to disease.
 * See FOP disease above.
 * Describe how calcium is deposited and resorbed from bone matrix, and how regulation of bone cells controls the levels of blood calcium.
 * See calcitonin and parathyroid hormone, above.

Cytoskeleton II Tuesday, December 04, 2007 5:48 PM

[Notice that I wasn't present for some of this lecture and have tried to fill in those LO's from the notes. Dr. Pfenninger's notes being what they are, the following may not be particularly complete or comprehensible.]
 * Cytoskeleton II, 12/5/07:**


 * Describe (reviewing material from Cytoskeleton I) the three types of cytoskeletal elements, their properties, their functional roles, and their protein composition.
 * (1) Microtubules: long, flexible, __polarized__ polydimers (alpha and beta tubulin subunits). Involved in mitosis as well as in ciliar and flagellar movement and transport inside the cell. Added to at plus end (end with exposed beta subunit); partially GTP-regulated. Little tensile strength. Express mechanical activity through motor proteins (dyneins and kinesins) that can shuttle cargo down them or use them as levers (as in mitosis). Note that microtubule polymerization requires GTP.
 * (2) Intermediate filaments: long, thick fibers, assembled in __nonpolar__ bundles of 32 filaments per bundle. Assist in mechanical support and strength. Found both inside the nucleus (nuclear lamins) and outside (eg. keratin). Lots of different possible protein sources.
 * (3) Microfilaments: mainly, __polarized__ polymers of actin. Polymerized G (globular) actin strands dimerize to form F (filament) actin. These have lots of actin-binding proteins associated with them to assist in polymerization (both of G-actin with other G-actins and of F-actin with other F-actins), which usually takes place at the plasma membrane and at the anchored minus end (plus end pushes out with polymerization). Note that microfilament polymerization ("nucleation") requires ATP.
 * Discuss cytoskeletal dynamics and the role of certain proteins in actin polymerization/depolymerization.
 * As mentioned, F-actin polymers dimerize to form the basic unit of microfilaments. Notice that certain drugs stabilize F-actin, causing abnormal aggregation.
 * __ARP proteins__: catalyze actin polymerization at the minus-end (elongating the plus-end). Effectively these act as "growth bases"-- plug into nuclear membrane and elongate actin into the cytosol.
 * __Cofilins__: catalyze actin depolymerization, reversing ARP protein action.
 * Rho family of proteins: signals having to do with particular assemblages of actin.
 * Rho: involved with stress fiber formation (contractile bundles).
 * Rac: involved with "veil" formation at the border of the cell (important in cell locomotion, see below).
 * Cdc42: involved with forming filopodia (fingerlike extensions of the cell).
 * Basically, the cool thing about actin is that it's a scaffold that can be assembled and taken down again very quickly, all over the cell. Want a protrusion of the cell towards a food source? Use actin. Figured out a food source isn't friendly and need to retract the protrusion? Actin all the way. Feel like rolling your way down the lamina to the nearest single-celled pub? Actin's your man.
 * Explain the concept of mechanoenzymes. Explain the mechanism of actin-based movement and contrast it to tubulin-based movement.
 * Mechanoenzymes: enzymes that convert chemical stored energy into motion.
 * Actin-based movement: actin-myosin contraction, described here as "sliding-filament" motion; it involves "winding a spring" as the target of ATP use (more on this under "Muscle I, II, + III").
 * Tubulin-based movement: two-headed dyneins and kinesins "walk" down the tubulin chain; ATP is used to swing the lagging head around in front of the leading head.
 * Discuss the actin cytoskeleton in the context of disease processes.
 * Actin makes up the core of microvilli (thus problems result when no actin).
 * Essential to amoeboid movement (see below): needed for axonal development, leukocyte migration; required for cancer metastasis.
 * Describe two types of locomotion of mammalian cells.
 * Swimming motion propelled by cilia or flagella (ie sperm).
 * Crawling movement over a surface (__amoeboid__ motion, much more common).
 * Discuss the concept and the key steps of amoeboid locomotion.
 * Essentially: the leading edge of the cell "pouches" forward, driven by polymerization of actin filaments branching off from other actin filaments (ARP proteins bind to filaments instead of nuclear membranes and drive elongation of new microtubules from them), and adheres the pouch to a new section of surface. Then it pulls the bulk of its cell body towards the pouch (possibly with actin-myosin) and un-adheres and retracts its lagging end (by depolymerizing the branched actin filaments there).
 * Steps called:
 * Protrusion (pouching forward)
 * Attachment (adhering to new surface)
 * Traction (pulling forward)
 * Detachment (retracting of lagging surface)
 * Discuss the relationship between cell adhesion and the cytoskeleton in amoeboid locomotion.
 * Well, if the thing can't adhere, it can't pull itself forward, can it?
 * Describe some of the molecules critical for amoeboid locomotion.
 * Actin, obviously. Then there's __ARPs__ to polymerize actin and __cofilins__ to depolymerize it again at the lagging edge. Adhesion proteins (cadherins, Igs, integrins) also critical. Notice the Rho family of proteins for signaling particular actin formations (like the filopodia or outpouching) and the WASp proteins (among others) involved in pushing those filopodia forward in protrusion.
 * Discuss cell motility in the context of developmental and disease processes.
 * 2 diseases for this:
 * //Wiskott-Aldrich Syndrome// : X-linked non-clotting disease-- caused by problems with protrusion of the leading edge of amoeboid cells by WAS protein (WASp). (Platelets are formed from castoffs of these protrusions.)
 * //Lissencephaly// : neuronal disease-- non-migration of neuronal axons leads to a smooth cortical surface and mental retardation.

Muscle I, II, + III Wednesday, December 05, 2007 3:39 PM

[I missed all four lectures on this day (family). So, again, I'm going mainly by notes here. Though I have to say that I'll go by Caldwell/Tseng notes before Pfenninger notes any day of the week and twice on Sundays.]
 * Muscle I, II, + III:**

[Thanks to Andrew Brookens for supplying lecture notes on this one too. --jcr]


 * Be able to explain the structural basis of skeletal muscle contraction by constructing a sarcomere:
 * Ok. Draw two lines. These are Z lines. The actin (thin) fibers poke out from them. Draw a line in the middle. This is the M line. The myosin (thick) fibers poke out from it between the thin fibers (which don't extend all the way to the M line, but do extend enough to have some cross-over with the myosin). Ta-da, a sarcomere. Line up a whole bunch of them in a row. Now you have a myofibril. Repeat that a lot in an orderly fashion. Now you have a striated muscle.
 * -What is the molecular structure of the sarcomere and the arrangement of contractile and linker proteins? How does this structural organization relate to contraction?
 * Basics: Thin filaments are made up of actin chains with tropomyosin stuck to them and troponin stuck to the end of tropomyosin. Thick filaments are made up of myosin chains that have big globular loops ('heads') on them that interact with actin.
 * -What is a myofilament? Define a myofibril. What is the relationship between myofibrils and the sarcoplasmic reticulum?
 * A myofilament is a polymerized strand made up of either myosin or actin/tropomyosin/troponin.
 * A myofibril is a bunch of sarcomeres placed end to end. Each myofibril is covered with its own sarcoplasmic reticulum (see below).
 * -How are connections of muscle contractile proteins made to surrounding connective tissues, and how do they contribute to contractile force/movement?
 * __Dystrophin__ connects actin to the surface membrane. __Titin__ links myosin to the Z line (centering the thick filaments). __Nebulin__ does something similar for the thin filaments. __Alpha-actinin__ crosslinks actin fibers.
 * The idea is that there's "passive" tension in a muscle fiber, presumably generated by attaching everything in its original configuration, that gives the cell something to return to after contraction. These proteins help maintain that configuration.
 * Be able to discuss the physiological and biochemical basis of skeletal muscle contraction:
 * (1) Influx of calcium binds to __troponin__.
 * (2) Bound troponin changes the configuration of __tropomyosin__.
 * (3) The shifted tropomyosin exposes binding sites for myosin on the actin filament.
 * (4) The actin filament binds to the myosin and the "spring" tension pre-loaded into the myosin releases, shortening the sarcomere by about 10 nm.
 * (5) ATP binds to myosin, allowing actin to be released.
 * (6) ATP is hydrolyzed to ADP + Pi, pre-loading the "spring" tension into the myosin fiber by shifting its configuration a little.
 * (7) Repeat steps 4-7 as long as there's Ca2+ and ATP around.
 * (8) Once the calcium supply runs out (Ca2+ is being pumped out while this is going on), troponin goes back to its original configuration, as does tropomyosin, causing actin to no longer be able to bind to myosin.
 * - How do contractile proteins work and how are they regulated in skeletal muscle?
 * More or less discussed this already. The myosin heads binds to and ratchets down actin with ATP activity.
 * -Describe the regulatory proteins (where are they located and how do they respond to changes in calcium concentration).
 * Regulatory proteins: means troponin and tropomyosin.
 * As mentioned, troponin sits at the end of tropomyosin and binds calcium.
 * Tropomyosin sits on actin and covers the myosin binding sites until troponin is triggered by Ca2+.
 * - What is the length of a sarcomere in resting muscle, contractile muscle, and muscle that is stretched almost to the point of injury (tearing)? How does this clarify the ambiguity of the question – “what is the length of a sarcomere?”
 * Length of a sarcomere in resting muscle: around 2.4 µm.
 * Length of a sarcomere in stretched muscle: around 3.6 µ m.
 * Length of a sarcomere in contracting muscle: unspecified. Less than 2.4 µ m.
 * Sarcomere is constantly stretching and contracting as it's used, thus length shifts.
 * What is the molecular basis of skeletal muscle diversity (fast and slow fibers) and what is the value of having this additional complexity?
 * Molecular basis: the different skeletal muscle types have different mixes of oxidative and glycolytic energy-producing frameworks.
 * Fast-twitch muscles tend to be centered around glycolytic energy production (quick bursts of energy, less sustainable). Used for things you need quick bursts of energy for (ie. catching the last beer can after you knock it off the table).
 * Slow-twitch muscles tends to be red in color due to their high myoglobin content (high oxygen load); they have all that oxygen because they primarily use oxidative phosphorylation to generate ATP for contraction. Tend to be used for sustained or postural movements (ie. assuming a relaxed and attractive mien with that beer can).
 * Usefulness: Well, obviously, if you can catch the beer can but not relax with it, you won't attract a mate on account of no one wants to date a spaz. If you're too slow to catch the beer can in the first place, you won't attract a mate because catching beer cans is just one of those intrinsic abilities that's very attractive. Only with the right mix do your slightly intoxicated genes get a chance to propagate.
 * Describe the key structural and physiological features of cardiac muscle and how they are similar to and different from skeletal muscle (sarcomere, regulatory proteins, events involved in a single contraction and relaxation, response to injury).
 * Intercalated disks: only found in cardiac muscle. Two functions: hold adjacent muscle cells together and allow gap junctions (see next point).
 * Mononucleated cells vs multinucleated in skeletal muscle.
 * Actin-myosin contraction and relaxation is the same as skeletal muscle (though note (a) that smooth muscle contraction is quite different and (b) that cardiac AP excitation pathways are subtly different from their skeletal counterparts, see below).
 * Cardiac cells cannot regenerate damaged tissue like skeletal muscle does (no satellite cells). So be careful with that double cheeseburger, bucko.
 * What is the role of gap junctions in each of the three muscle types (skeletal, cardiac and smooth)?
 * Gap junctions only occur in cardiac muscle fibers and some types of smooth muscle. Their role in cardiac muscle is to ensure that cardiac contractions occur rhythmically throughout the heart muscle by allowing selective passive of signaling molecules; they may also form the basis for synchronous contractions of gastroenteric smooth muscle (ie. peristalsis). Also link adjacent cells together, though this is usually associated less with gap junctions and more in the intercalated discs that they're found in.
 * What is the molecular basis of familial hypertrophic cardiac myopathy? What proteins are mutated? Where are the mutations most likely to occur in the proteins?
 * Familial hypertrophic cardiomyopathy (FHC) occurs as a result of mutations in cardiac muscle tissue. Usually the mutations are located in the cardiac myosin heavy chain, where it interacts with actin and ATP; occasionally a mutation will occur in troponin instead.
 * Why does smooth muscle appear smooth? What are the key structural and physiological features of smooth muscle and how do they compare to skeletal and cardiac muscle (contractile proteins, regulation of contraction, cell organization)?
 * Smooth: because there's no striations (ie. the sarcomeres aren't nicely lined up like cardiac and skeletal muscle).
 * Structural: smooth muscle cells are extremely thin-diameter, spindle-shaped cells. Classically these are the "involuntary" muscles of the body (ie. digestive muscles).
 * Contractions: a little different from the other two:
 * Instead of having calcium bind troponin as the catalytic step for actin to bind myosin, in smooth muscle calcium binds calmodulin (remember this one? With the EF hand domains?), which binds calmodulin kinase (CAMK), which phosphorylates the myosin chains, causing actin and myosin to bind and the ratchet action to begin.
 * This is a lot slower than skeletal/cardiac contractions.
 * Notice that smooth muscle cells are mononucleated, like cardiac cells and unlike skeletal cells.
 * How does skeletal muscle develop (single cells going to multinucleate cells)?
 * You start out with individual __myoblast__ cells (muscle precursors). These merge together into long chains during development. Predictably, the reason you get multinucleate cells from single cells is that the single cells decide to cohabitate.
 * Explain the reason for the transverse-tubule system (t-system) in skeletal and cardiac muscle.
 * Essentially you need a way to get the membrane surface signal (the AP) to get to the sarcoplasmic reticulum so it can trigger the release of calcium and cause contractions. Having the AP be transmitted throughout the SR would be too electrically laborious, so those wacky cells decided t-tubules were the way to do it.
 * How is excitation-contraction coupling accomplished in skeletal muscle?
 * Ok. The AP's coming down the membrane, continuing down the t-tubule. When it gets down to the end of t-tubule (which sits down on the edge of the sarcoplasmic reticulum of the muscle cell), it runs into a particular kind of receptor system, consisting of two parts:
 * **DHP receptor** : voltage-gated receptor
 * **RyR (ryanodine receptor)** : Calcium channel in the sarcoplasmic reticulum.
 * Current theory: voltage hits DHP receptor, causes it to shift in such a way as to cause the RyR channels to open and allow calcium to flow out into the muscle cell, causing contraction.
 * How is excitation-contraction coupling accomplished for cardiac and smooth muscle? Why is the t-system not required in smooth muscle?
 * Cardiac:
 * Similar to skeletal, but here the difference is that the voltage seems to release calcium before the RyR-equivalent channels will open-- the cardiac calcium release channels (to trigger contraction) open in response to a calcium signal. I think they're not entirely sure how this works.
 * Smooth:
 * Don't need t-tubules at all: calcium released at the cell surface can just go ahead and diffuse through the whole cell very quickly. The reason this works for smooth muscle (but not for the others) is that smooth muscle cells are so small and thin. If you tried this with skeletal muscle, you'd get weird asymmetrical contractions as the parts of the muscle that got calcium first would contract first and the other only later, as the calcium diffused. Obviously you don't want that, and you really don't want it in cardiac muscle-- thus t-tubules.
 * What is the molecular basis of malignant hyperthermia? - What protein contains the mutation? - Why does the temperature rise? - Why is the disease not noticed until surgery? - What type of compound would you use to prevent the temperature rise? Why wouldn’t induced muscle paralysis solve the problem?
 * Wow, that was a mouthful. Break it down:
 * Basis is a mutation in the calcium release channel proteins in the sarcoplasmic reticulum.
 * This mutation causes inhaled anesthetic to trigger the release of calcium from the SR; this causes ATPases to pump calcium back out, and a vicious cycle is initiated in which the heat generated by the pump just keeps rising, causing hyperthermia.
 * Patient's muscles also become rigid during this time (muscles firing at speed).
 * It's not noticed until surgery because most of us don't jaunt around inhaling halothane all day.
 * **Dantrolene** blocks calcium release from the SR; thus, administered as a prophylactic, it prevents malignant hyperthermia.
 * Muscular paralysis (by which is meant blocking APs) would have nothing to do with this because this isn't AP-generated Ca2+ release-- it's triggered by inhaled anesthetic.
 * -Starting with an action potential in a motor neuron, explain the processes required to have a skeletal muscle undergo a single contraction and relaxation (a twitch).
 * (1) AP in motor neuron travels down to the synaptic area.
 * (2) AP causes a release of acetylcholine (neurotransmitter) at the synapse.
 * (3) ACh binds to ACh receptors in the muscle fiber, opening ion channels in the muscle fiber and causing depolarization (muscular AP).
 * (4) Muscle AP propagates down the fiber.
 * (5) AP goes down into t-tubules on its way.
 * (6) DHP receptors at ends of t-tubules sense voltage change and open the RyR channels in the SR, releasing calcium into the cytosol of the muscle.
 * (7) Calcium binds to troponin, causing tropomyosin to shift and actin to bind to myosin and contract.
 * (8) Calcium and ATP drive contraction as long as signal persists and calcium/ATP are present.
 * (9) Calcium pumps (using ATP) move calcium back into the sarcoplasmic reticulum; troponin, released from calcium, relapses and causes tropomyosin to cover up the myosin binding sites on actin. The muscle relaxes.
 * -Where do motor nerve terminals associate with skeletal muscle fibers and what is the distribution of cells innervated by one motor neuron in a muscle?
 * Motor nerve terminals (synapses) are located around the center of skeletal muscle fibers; the AP propagates in both directions from there. (note that the __contraction__ of the muscle is not the same as the __AP__ to the muscle-- the former depends on t-tubule-triggering of calcium release and occurs more or less at the same time throughout the muscle, as you'd like for maximum contraction.)
 * The distribution of innervated cells varies greatly depending on the particular neuron and the particular muscle, but is generally called the __motor unit__ whatever its size.
 * Fine motor actions tend to have small-size motor units (you're triggering less muscle contractions with one AP); coarse motor actions tend to have larger motor units.
 * Notice that a single "muscle" (ie thigh muscle, triceps, etc) may have lots of different motor neurons innervating it, all attached to differently-sized motor units.
 * What is a muscle motor unit and what does the average size of a motor unit in a muscle tell you about the function of that muscle?
 * Pretty much covered this already. If the average size of a motor unit is small, that muscle is probably used largely for fine movement; if larger motor units, probably mainly gross movements.
 * - If you add the motor unit sizes of all the motor neurons innervating a single skeletal muscle, is this sum greater than, less than, or equal to the number of muscle fibers in the muscle?
 * Should be equal-- normally, no muscle fiber is innervated by more than one neuron (though see next point) and no muscle fiber isn't innervated by any neuron at all. (yes, it's a double negative, all you English majors out there. All two of you.)
 * - Following nerve injury and muscle reinnervation or in someone in the early stages of ALS (amytrophic lateral sclerosis: motor neurons are dying), the sum of motor unit sizes can be greater than the number of muscle fibers – how would you explain this?
 * Probably has something to do with muscle fibers being innervated by more than one neuron during the regenerative process.
 * How is tension graded in cardiac and smooth muscle (two mechanisms)? How is this different from the gradation of tension for skeletal muscle?
 * Cardiac and smooth muscle: predominantly graded by __cell length__ and __neurotransmitter/hormone__ receptor activity.
 * Skeletal muscle: Doesn't change length (anchored at both ends)-- thus its tension is graded differently (see below).
 * What is the role of “satellite cells” in skeletal muscle development and repair? What are the physiological and structural responses to exercise (or lack of exercise) on skeletal muscle (number of cells and size of cells)?
 * Satellite cells: specialized stem cells that produce new skeletal muscle cells. These new cells, instead of replacing the muscle cell that's there, will fuse with it to form a larger muscle fiber. This is useful in both development of muscle during exercise and repair of damaged cells.
 * Exercise:
 * Exercise __does not__ add more muscle fibers. What it does is increase the size of the muscle fibers you have.
 * Atrophy, likewise, reduces the size of the fibers, not their numbers.
 * - How is skeletal muscle tension graded and regulated (the two major mechanisms), and what is the basis of muscle fatigue?
 * Skeletal muscle is tension-graded by the __frequency of action potentials__ fired and the __number of motor units recruited__ for the contraction.
 * Note 'tetanic' tension at which the muscle can't contract any farther.
 * Muscle fatigue: involves four different parts of the contractile reaction:
 * (1) K+ builds up and Na+ is reduced in the t-tubular network; this reduces its ability to propagate APs and trigger calcium release from the SR. This problem is corrected quickly (K-Na balance restores in seconds).
 * (2) - (4) Involves buildup of Pi (from ATP hydrolysis) and a drop in pH (6.5 from 7- pH drops due to lactate production after glycolysis under conditions of insufficient oxygenation). Notice that we don't really know why these happen, just how.
 * (2) Pi and H+ buildup inhibits calcium release from the sarcoplasmic reticulum.
 * (3) Likewise, Pi and H+ inhibit the binding of calcium to troponin.
 * (4) They also reduce the contractile force exerted by the myosin-actin binding.

Vasculature Friday, December 07, 2007 7:59 AM


 * Vasculature, 12/7/07:**


 * Describe the structure, organization, and function of the basic layers of blood vessel walls:
 * Three layers surrounding lumen:
 * **Tunica intima** : endothelial layer closest to the lumen of the vessel.
 * **Tunica media** : middle layer, composed of elastic tissue, smooth muscle, or collagen.
 * **Tunica adventitia** : outer layer, composed of collagen/collagenous tissue.
 * In large arteries, these often contain smaller vessels running through them; these are called **vasa vasorum** (vessels of vessels).
 * Discuss the morphological characteristics that distinguish the different types of blood vessels:
 * [In largest arterial vessels, like the aorta ("**elastic arteries** "):]
 * Tunica intima:
 * endothelial cells (at the interface with the lumen)
 * Stuff just outside the endothelial layer but still in the intima:
 * fibroblasts
 * connective tissue
 * myointimal cells (responsible for laying down fibrous plaque in atherosclerosis)
 * Tunica media:
 * Lots and lots of smooth muscle cells and, particularly, elastic fibers
 * Tunica adventitia:
 * Vasa vasorum (supplies blood and nutrients to outer parts of larger vessels)
 * Loose collagenous connective tissue, elastin
 * This is loose so that leukocytes (white blood cells) can exit the blood vessels and get out into the connective tissue.
 * [In more distal but still large arteries ("**muscular arteries** "):]
 * Less tunica intima volume
 * Shows characteristic "inner" and "outer" **elastic laminae** that form the boundary of the tunica media, containing smooth muscle cells (note less elasticity as you get farther from the aorta on account of there's less arterial pressure farther on).
 * Adventitia doesn't show vasa vasorum once you get to a sufficiently small size-- the blood from the lumen can diffuse well enough on its own.
 * [In smaller muscular arteries:]
 * Small (3-4 layers) tunica intima
 * In the tunica media, the __outer__ elastic lamina disappears, but the __inner__ remains.
 * Notice the smooth muscle remains thick-- dilation and constriction extremely important by this point.
 * Adventitia: quite thin, can blend into surrounding tissue.
 * [In smallest arteries immediately adjacent to capillaries ("**arterioles** "):]
 * Tiny tunica intima, a few layers of smooth muscle, and a little collagenous tissue on the outide rim.
 * "Gatekeepers" for the capillaries (can constrict and shut off blood flow to them).
 * [In veins:]
 * Still have a tunica intima, media and adventitia. Media is much, much smaller than those of similarly sized arteries.
 * Note that you can find valves or "flaps" in both veins and lymphatic vessels.
 * Occasionally a few smooth muscle cells in the media, but not many.
 * Notice that the shape of the lumen often looks "collapsed" (acircular).
 * [In lymphatics:]
 * Also have irregular lumen (acircular), but have extremely thin walls (just a thin endothelial layer, no media or adventitia) compared to veins.
 * **[Important note** : the endothelial cells of the tunica intima are pretty much universally __stratified squamous__ in shape (need to have things diffuse through them quickly, need to be thin). If it's cuboidal or columnar, odds are good it's not a vessel endothelium.]
 * [Another note: size of red blood cells is always about 7-8 microns.]
 * Explain the structure and function of the different types of capillaries:
 * All capillaries have relatively wide lumens and, thus, slower blood flow.
 * This means that regulation of diameter is particularly important, since it's easier to close off or open up capillaries completely than larger vessels.
 * Capillary structure:
 * Small endothelial layer
 * Specialized layer wrapped around endothelium: **pericytes**.
 * If tissues are damaged, pericytes can generate smooth muscle cells; mechanism not clearly understood.
 * Three different types of endothelial layers:
 * **Continuous** (standard) endothelium: continuous wall of lumen; can have pinocytosis (vesicle-bound transport) across it, but no free flow.
 * **Fenestrated** endothelium: small windows in lumen, allows plasma to freely diffuse through endothelium.
 * In spleen and to some extent in the liver, can see **discontinuous** endothelium: large holes in lumen or between adjacent endothelial cells, allows entire red blood cells to diffuse out of endothelium.
 * Outline the unique functions of post-capillary venules
 * Post-capillary venules: Where leukocytes interact with the endothelial walls (actually, they break them selectively down, a process called **diapodesis** ) and leave the blood; also the action of histamines regulating permeabilities of blood vessels occurs at the post-capillary venules. Slow blood flow.
 * Describe how blood flow is regulated in capillary beds
 * As mentioned: smooth muscle "gateways" in arterioles.
 * Discuss the general structure and functional significance of arterio-venous shunts, portal systems, pampiniform plexus, anastomoses, and end arteries:
 * Arterio-venous shunts: connecting vascular passages between arterioles and post-capillary veins (controlled by smooth muscle sphincters).
 * [Metarteriole: System of vessels leading from arteriole through capillary beds to the post-capillary veins. Also controlled by smooth muscle sphincters.]
 * Portal systems: From a capillary bed to a capillary bed, ie. hepatic portal system. If that system is any indication, these probably aren't primarily used for oxygenation of the second capillary system in the chain.
 * Pampiniform plexus: Countercurrent heat exchange between arteries and veins. Say you're a scrotum. (did you say it?) You're hanging around, so to speak, outside the body and you're a little cold for a tissue system. What you don't want is for all that good warm blood coming out to you to get cold and carry that cold back into the body, cooling down the core. So you run your arteries right next to your veins on the following philosophy: if the veins coming in are cold, and the arteries going out are warm, then the incoming blood in the veins will be warmed up (thus preventing core cooling) and the outgoing blood will be cooled down (thus minimizing its heat loss). That arrangement is a pampiniform plexus.
 * End arteries: arteries that supply blood to a region that isn't supplied by any other artery-- eg. kidney/lung arteries.
 * Anastomoses: come on, seriously, you better know what anastomoses are. How did you pass anatomy?

Vocabulary Artery, elastic; Artery, muscular; Arteriole; Aorta; Capillary; Capillary, continuous; Capillary, fenestrated; Capillary, discontinuous; Tunica intima; Tunica media; Tunica Adventitia; Post-capillary venule; Diapedesis; Muscular vein; Venous valves