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    Intro Microanatomy 9/29/2011 10:01:00 AM

    1) Name the components that make up cells, tissues and organs.

    Cellsare comprised of three domains:

    Nucleus Intracisternal (inside organelles) Cytosol (Cytoplasm contains organelles Intracisternal space)

    o Contains cytoskeletal elementsTissues: Cells + ECM(Extracellular Matrix)

    4 Types of Tissueo Epithelial:closely linked cells form a liningo Connective Tissue: Supportive tissue can be flexible or rigid

    (cartilage vs bone)

    o Nervous Tissue: Specialized for conductiono Contractile Tissue:Muscles with contractile protein fibers

    Organs:Comprised of Multiple Tissue Types (Usually all 4)

    Complex structureOrgan Systems

    2) List common features of cells

    - Domains(i.e Organelles) have specific functions and are Separated by

    Membranes

    Know:

    (1)nucleolus(2) nucleus

    (3)ribosomes

    (4)vesicle

    (5) roughendoplasmic reticulum(ER)

    (6)Golgi apparatus

    (7)cytoskeleton

    (8) smooth ER

    (9)mitochondria

    (10)vacuole

    (11)cytosol

    - Different from Inclusions which are substances stored in cell (Glycogen,

    Fat)

    http://en.wikipedia.org/wiki/Ribosomeshttp://en.wikipedia.org/wiki/Ribosomeshttp://en.wikipedia.org/wiki/Ribosomeshttp://en.wikipedia.org/wiki/Vesicle_(biology)http://en.wikipedia.org/wiki/Vesicle_(biology)http://en.wikipedia.org/wiki/Vesicle_(biology)http://en.wikipedia.org/wiki/Endoplasmic_reticulumhttp://en.wikipedia.org/wiki/Endoplasmic_reticulumhttp://en.wikipedia.org/wiki/Endoplasmic_reticulumhttp://en.wikipedia.org/wiki/Golgi_apparatushttp://en.wikipedia.org/wiki/Golgi_apparatushttp://en.wikipedia.org/wiki/Golgi_apparatushttp://en.wikipedia.org/wiki/Cytoskeletonhttp://en.wikipedia.org/wiki/Cytoskeletonhttp://en.wikipedia.org/wiki/Cytoskeletonhttp://en.wikipedia.org/wiki/Mitochondrionhttp://en.wikipedia.org/wiki/Mitochondrionhttp://en.wikipedia.org/wiki/Mitochondrionhttp://en.wikipedia.org/wiki/Vacuolehttp://en.wikipedia.org/wiki/Vacuolehttp://en.wikipedia.org/wiki/Vacuolehttp://en.wikipedia.org/wiki/Cytosolhttp://en.wikipedia.org/wiki/Cytosolhttp://en.wikipedia.org/wiki/Cytosolhttp://en.wikipedia.org/wiki/Cytosolhttp://en.wikipedia.org/wiki/Vacuolehttp://en.wikipedia.org/wiki/Mitochondrionhttp://en.wikipedia.org/wiki/Cytoskeletonhttp://en.wikipedia.org/wiki/Golgi_apparatushttp://en.wikipedia.org/wiki/Endoplasmic_reticulumhttp://en.wikipedia.org/wiki/Vesicle_(biology)http://en.wikipedia.org/wiki/Ribosomes
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    Membranes:Phospholipid Bi-layer establishes compartmentalization /

    domains

    Restricts / Regulates movement in and out of cell Fluid-Mosaic Model: Membrane has lipid and protein components

    o Small polar or charged molecules move via Protein MediatedTransport

    o Large Molecules move through vacuoles through Endocytosisand Exocytosis

    o Integral Membrane Proteins: contain hydrophobic domainsare require detergent to remove from membrane

    o Peripheral Membrane Proteins: Do not completelypenetrate bi-layer and are only temporarily associated with

    membrane

    Glycoconjugates:Sugars associated with membrane proteinso Cell-cell interactions

    Recognitiono Cell-matrix interactionso Almost Exclusively on Non-Cytosolic Side

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    Polarity: Non-Random distribution of organelles

    Tight-Junctions cause polarity in Membrane proteins3) Explain the basic processes of tissue preparation and microscopy for

    histological preparations

    1) Fixation: Cross linking of proteins with formaldehyde. Need stronger

    fixatives like glutaraldehyde preserves ultrastructure

    2) Sectioning: Fixed sample is sliced so light can pass through it on a slide

    < 50 microns thick for light microscopy (hair is 100 microns) Electron microscopy needs 60 nanometers

    o Black and White Use Microtome Plane of Section

    3) Staining

    Hematoxylin: Stains basophilic subtances blueo DNA, Rough ER

    Eosin: Stains acidophilic substances pinko Mitochondria, Cytoplasm

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    Protein Structure and Function 9/29/2011 10:01:00 AM

    Prior Review

    1. pH of strong and weak acids

    Stronger acids have a lower pH than weak acids. Weak acids tend to have

    better buffering properties, especially when mixed with weak bases.

    2. pK, buffers (Henderson-Hasselbach), titration curvepKa is the acid dissociation constant, pKb is the base dissociation constant.

    Stronger acids have a larger pKa. As stolen from wikipedia,

    if an acid dissociates like HA

    Then the

    The Henderson-Hasselbalch Equation states that

    Namely, the pH of a solution depends on the extent of dissociation of its

    dissolved acid (a similar equation exists for basic solutions).

    A buffer solution resists change in pH from the addition of acids or bases up

    to a certain point. Its usually a mixture of a weak acid and weak base. As

    more acid is added to the buffer, the weak base dissociates to raise the pH.

    When all of the base has dissociated, the buffer breaks. The reverse is

    true, of course, when the weak acid dissociates to resist an increase in pH.

    3. General Properties of Amino Acids

    An amino acid has a carboxyl (COO-) end, an amino (NH2-) end, and a side

    chain.

    Each side chain has its own pKa. Depending on the pH, this will give the

    chain a charge. The isoelectric pointis the pH at which a specific side

    chain will have no charge. In general, amino acids are assigned a charge

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    based on standard body pH, although there are environments in the body

    with extremely low and high pH.

    4. Structure of the peptide bond

    The peptide bond links together amino acids from carboxyl end to amino endvia dehydration synthesis. Its a covalent bond and difficult to break.

    Learning Objectives

    1. General Properties of proteins

    Functions:Seen in table below.

    Catalysis

    Regulation

    Transportation

    Contractile elements

    Defense

    Structural elements

    Size: molecular weight ranges from 6000 to 40,000,000

    Shape: Globular

    Fibrous

    Conjugated: Protein and Sugar/Lipid/RNA covalently linkedCharge: Depends on amino acids on surface of protein.

    Solubilitiy: Depends on location in body. Blood proteins are water soluble,

    membrane proteins lipid soluble. Some proteins are amphipathic.

    2. Levels of Protein Structure

    Primary: Amino acid chain derived directly from gene translation. Each unit

    is connected via peptide bonds

    Determines higher orders of foldingSecondary: Smallest possible structure, connection by weak hydrogen

    bonds. Most common structures are the alpha helix and beta pleated sheet.

    H-bonds localized

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    Tertiary: Three-dimensional structure of a single protein chain. Stabilized by

    hydrogen and ionic bonds. Folding usually needs to be done exactly right in

    order for protein to function properly. Chaperoninproteins exist to refold

    damaged proteins correctly, usually by presenting them with a rapidly

    alternating hydrophobic and hydrophilic environment. Ribonucleases always fold correctly after denaturation insulin is irreparable when denatured.

    Quaternary: The conjugation of multiple tertiary protein structures.

    Stabilization by hydrogen, ionic, and hydrophobic interactions. For example,

    hemoglobin, collagen.

    3. Protein folding and denaturation

    Discussed above.

    4. Structure-function relationships

    Protein structure is related to function. Globular proteins usually have

    hydrophobic center and hydrophilic surface. Enzymes have a unique active

    site that binds to a specific substrate.

    Examples given in class:

    A point mutation on a B-chain of hemoglobin results in a long chain insteadof a globular protein. The protein loses its solubility and sickle cell results.

    A mutation in CFTR makes it unable to transport Cl- and cystic fibrosis

    results.

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    Enzymes 9/29/2011 10:01:00 AM

    1. General properties of enzymes:

    -Enzymes are a class of proteins that increase the rate of a chemical

    reaction.

    -Because enzymes control the rates of reactions, they are used to regulate

    the activity of the cell.-Enzymes have a specific distributionwithin subcellular compartments

    and within specific organs.

    *As proteins, enzymes are sensitive to changes in temperature and pH and

    require a relatively stable environment in order to function.

    *Enzymes are often kept in the inactive state, where it is called the

    zymogen orproenzyme. This allows enzyme activity to be strictly

    regulated.

    Example: Many proenzymesrequire s short sequence on the N-terminus to

    be cleaved in order to become active. For example, pepsinogen is translated

    and released by chief cells in the stomach. Trypsin then cleaves the N-

    terminus, converting the proenzymeto its active form pepsin.

    2. Interaction of enzyme with substrate:

    -Substrates bind to a relatively small region of an enzyme called the active

    site. The bound substrate fits in a specific orientation and is fitted through

    ionic bonds, hydrogen bonds, and hydrophobic interactions.

    - No covalent boding to enzyme-The act of the substrate binding to the enzyme can cause a

    conformational changein the enzyme. This is also called induced fit.

    - Enzymes dont affect the Thermodynamics, they do effect the kinetics

    3. Enzyme catalysis; Michaelis-Menten equation

    -Enzymes have no effect on the thermodynamic properties of a given

    reaction and therefore always move the chemical reaction towards

    equilibrium. Instead, enzymes lower the energy of activation, an energy

    barrier required in order for a reaction to proceed, and thereby increase the

    speed of a reaction.

    -Enzymatic reactions can proceed in the forward or backward reactions

    depending on where the chemical equilibrium lies.

    -The Michaelis-Menten equationallows one to predict the rate of reaction

    given a specific amount of substrate.

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    *During catalysis, the enzyme remains unchanged after the reaction has

    taken place. In many cases, the enzyme forms a covalent intermediate.

    However, this covalent bond is not involved in substrate-enzyme binding.

    4. Enzyme inhibition-Competitive inhibitorsdirectly compete with the substrate to bind at the

    active site.

    -A competitive inhibitor will increase the Km, the concentration required for

    half the enzymes to be bound to substrate, because the competitive

    inhibitors will always occupy a specific portion of active enzymes.

    -A competitive inhibitor will leave vmax unchanged because adding an

    infinite amount of substrate will allow the enzyme to bind to the substrate

    more often than to the competitor.

    -Noncompetitive inhibitors, also called allosteric inhibitors, bind to a

    site on the enzyme somewhere other than at the active site.

    -Non-competitive inhibitors will occupy a given portion of enzymes at any

    given time, thereby reducing vmax regardless of substrate concentration.

    *It is hypothesized that the noncompetitive inhibitor binds to the enzyme

    and prevents it from achieving a specific conformational state, thereby

    making the enzyme non-functional.

    5. Mechanism of enzyme reactions

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    -Enzymes can have a high specificity to a given substrate or can be more

    non-specific (digestive enzymes).

    -In a given reaction with an enzyme, two reactants need to bump into each

    other with the proper orientation in order for the reaction to take place. An

    enzyme binds to these substrates, thereby increasing effective proximity andplacing the substrates into the

    proper orientation.

    *An enzyme remains unchanged after performing the appropriate chemical

    reaction.

    6. Regulation of enzyme activity

    -Isozymesare multiple forms of the same enzyme, often with different

    kinetic properties.

    ex/ -Lactate dehydrogenase is given as a specific example where the

    distribution of lactate dehydrogenase is specific to different organs.

    -Phosphorylation can activate or deactivate a given enzyme.

    -Positive and negative modulators can demonstrate cooperativity

    (speeding up reaction) or inhibition in order to alter the kinetics of the

    reaction.

    *indicates relevant information covered in other lectures but not this one

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    DNA Replication 9/29/2011 10:01:00 AM

    DNA REPLICATION

    1. Compare and contrast DNA replication in Prokaryotes and Eukaryotes

    Phase Prokaryotes Eukaryotes

    Initiation DNA Abinds to OriC(only

    origin of replication) and

    melts DNA

    Helicase(?) binds to origin of

    replication (many)

    DNA B(helicase) unwinds

    DNA Helicase (?) unwinds DNA

    Topoisomerase Ineeded to

    nick 1 strand of DNA to

    relieve torsional stress (bc

    continuous circle)

    Single Strand Binding

    Proteins(SSBs) bind to

    prevent DNA from re-binding

    to other parent strand

    RPAs bind to prevent DNA

    from re-binding to parent

    strand

    Priming Primaserecruited to

    replication fork and adds RNA

    primer to leading strand,

    then to lagging strand further

    down DNA

    Primase recruited to

    replication fork and adds RNA

    primer to leading strand, then

    to lagging strand further down

    DNA

    DNA Pol adds a few DNA

    nucleotides to primer

    Elongation DNA pol IIIthen binds

    (tethers with beta clamp) to

    polymerize DNA in 5-3

    direction (leading strand in

    DNA pol then binds

    (tethers w PCNA) to replicate

    in 5'-3' (leading strand in

    continuous manner, and

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    continuous manner, and

    lagging strand in

    discontinuous manner w

    Okazaki fragments)

    lagging strand in

    discontinuous manner w

    Okazaki fragments)

    DNA Pol III can backtrack

    and proofread in 3-5

    direction

    DNA pol can backtrack and

    proofread in 3'-5' direction

    DNA Pol Ireplaces DNA Pol

    III to remove RNA primers

    Fen-1removes primers and

    DNA pol replaces gaps w

    DNA

    DNA ligasejoins strands DNA ligasejoins strands

    Termination Terminator sequences trap

    replication fork near origin

    site and bing TUS proteins

    Telomerasecreates RNA

    template to extend lagging

    strand with junk DNA

    2nd TUS Protein does not

    allow DNA B to pass through,

    and elongation is stopped T-loops formed at ends

    Topoisomerase ivunlinks

    the catenated strands

    2. Examples of diseases that occur due to replication defects

    a. Mutation in RNA telomerase --> Dyskeratosis congenity:

    Developmental delay

    b. Low telomerase levels --> no T-loops = genomic instability =

    increased cancer risk

    c. Fragile X syndrome excess CGG

    d. Muscular dystrophy

    e. Spinocerebellar ataxia

    f. Huntingtons Disease: In coding sequence

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    Mutations in UTR, introns and coding sequence

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    DNA Mutation and Repair 9/29/2011 10:01:00 AM

    1. Describe the relationship between DNA damage, DNA repair, DNA

    replication and mutagenesis

    Mutagenesis: the permanent alteration of DNA

    2. State the major sources of DNA damage and the major types of DNA

    repair

    Sources of DNA Damage

    1. Endogenous Sources DNA Replication Errors (misincorporation, slippage)

    Deamination (cytosine to uracil, 5-methylcytosine to thymine) Depurination

    (creates abasic site) Reactive Oxygen Species (strand breaks, base damage)

    DNA Recombination Errors

    2. Environmental Sources:

    -Ionizing Radiation (IR) increases reactive species (Indirect Mechanism)-Ultraviolet Radiation (UV) generates pyrimidine dimers (Direct Mechanism)

    -Chemical Mutagens (e.g. alkylation by MNNG, MNU O6-methylguanine,

    pairs like A)

    Repair least to most serious

    Proofreading during replication. error rate:10^-4 10^-8

    Post Replication Mismatch excisionrepairafter replication. Error rate

    10^-8 10^10

    Error recognition strand discrimination->excision->resynthesis->ligation

    Base excision repair: DNA glycosylase flips and removes base AP

    endonuclease cuts phosphodiester bond -> DNA polymerase -> ligase

    - can create abasic site that can be premutagenic if not repaired on time

    Direct reversal: MGMT destroys itself to get rid of methylation of guanine

    bases

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    Nucleotide excision repair:Damage recognition Nuclease cleavage

    removal with helicase Pol, Pol DNA ligase

    removes bulky dimmers/unrecognizable bases/

    note: DNA ligaserepairs single-strand breaks > 100,000 / day very efficient

    Homologous Recombinationless errors, only available during mitosis

    when sister chromatid is around. Reliable repair of double strand breaks

    exonuclease cuts to make stick endstrand invasion by sister chromatidDNA

    synthesis/sister chromatid exchangeunwindig/ligation(BLM helicase)

    Non-homologous RecombinationMore error prone, available any time

    of cell cycle. Unreliable possible error in relegation, clean up step is

    wasteful

    synapse formation to hold ends together by Ku70 and Ku80 DNA PKs clean

    up staggered ends Ligation by LIG4 and XRCC4 proteins

    3. Describe the clinical consequences of mutagenesis and of defects in DNA

    repair

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    Nuclear Structure and Function 9/29/2011 10:01:00 AM

    1. Make a simplified schema of the basic structural components of the

    nucleus (nuclear envelope, pores, nucleolus, chromatin, nuclear matrix).

    2. Explain the structure and function of the nuclear envelope and nuclear

    matrix.

    Nuclear Envelope: Is a 2-membrane system which forms a barrier between the

    nucleus and the rest of the cell to:

    - Maintain unique protein and nucleotide environment

    - Sequester mRNA synthesis from Translational machinery

    - Protect / Contain DNA

    Outer Membrane and Inner Membraneare separated by the Perinuclear

    cisterna. It is perforated by Nuclear Porescontaining NPC(Nuclear Pore

    Complexes). The inner membrane is supported by the Lamina

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    Nuclear Matrix: A protein lattice made of fibers (similar to cytoskeleton) which:

    - Anchors DNA replication

    - Anchors transcription complexes

    - Reinforces nuclear envelope --> stability

    3. Explain the importance of the structure of nuclear pore complexes and

    how this plays a role in nucleocytoplasmic exchange.

    Nuclear Pore Complex: (80-100 nm diameter) is the site of selective

    nucleocytoplasmic exchange. It creates a selective barriers for the transport

    of macromolecules across the nuclear envelope (not as selective for smaller

    molecules). Has a 3-ring Structurethat has 8 spokes with a hole in the

    center.

    - Cytoplasmic Ring:(8-subunits) with protusions into cytoplasm formediating import

    - Middle Ring:8 subsunits protrude into Perinuclear spaceo Transport proteins

    Nucleoplasmic Ring: Fibrous proteins protrude into nucleoplasm

    Serves as a dock for importins and exportins.o Importin / exportin mediated transport requires energy

    4. Describe the organization of chromatin and its role in synthesis,

    processing and storage of DNA and RNA.

    Chromatin is highly compacted DNA + Packing Proteins:

    - Heterochromatin: highly coiled and less active

    Genes not transcribed in cell

    - Euchromatin: less coiled, more active DNA

    Composition:

    Nucleosome:(10 nm) DNA wrapped around 8 Histones(166 bp/ histone)

    Forms Beads on a StringCoiled Nucleosome: (30 nm) coiled nucleosomes

    H1 Histone: Activity affects density of nucleosome coil

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    The density of chromatin packing determines the availability of the genes for

    transcription. Histone tails are sites for activating the exposing / coiling of

    local DNA

    5. Have a general concept of the cell cycle and its control as well asapoptosis.

    G1: Cell Growth

    S phase: DNA replication

    G2 phase:Interval before mitosis (valuable for DNA lesion repair)

    Mitosis

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    RNA Transcription 9/29/2011 10:01:00 AM

    Transcription and Control of Transcription Learning Objectives

    1. Describe the basic transcription machinery, the basic structure of

    genes (including promoters) and transcription units, and the basic

    mechanism of transcription in eukaryotes.

    a. Basic machinery neededi. RNA pol(to read your template 3-5)

    ii. Some bases(ATP, GTP, CTP, UTP, all ribonucleotides of course)

    iii. DNA topoisomerasesto unwind the helix

    b. Basic structure of genes, w/promoters & txpn units

    ii. Promoter regiono 1. Where proteins bind to begin transcription. This

    includes:

    o 2. Initiator sequence(which includes the)o 3. TATA boxo 4. A mix of enhancer and silencer sequences

    a. Can be in other places other than right before thetranscribed gene (ex. Behind, in the intron, etc.)

    b. Fxn: assist regulation by allowing a specific txpnfactor to bind to it

    c. This leads to activation/repression of transcription d. Environmental conditions can control the binding of

    txpn factors to these enhancer/silencer elements e. Ultimately, the binding will lead to actions such as

    phosphorylation or binding/dissociation of another

    protein that is related to the txpn factor

    iii. Transcribed gene

    Exon: leaves the nucleus as mature mRNA afte modification

    Intron: Kept inside the nucleus (although problems with the introncould later contribute with mutations and problems with the mature

    mRNA)

    c. Basic mechanism of euk txpn

    - RNA pol transcribes the DNA

    - Depending on the RNA we are attempting to transcribe, we will use a

    corresponding polymerase

    - Basal txpn factors assist pol in recognizing the promoter and initiating txpn

    -NOTE: Mitochondria (have RNA pol that is similar to prok pol, and

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    transcribes their own DNA into their own rRNAs, mRNAs, and tRNAs)

    2.Discuss the roles of transcriptional activator proteins, enhancer elements,

    coactivators, and chromatin in regulation of eukaryotic transcriptiona. Transcriptional activator proteins

    - Bind basal txpn factors associated with RNA pol 2 to get it over to the

    promoter

    - Recruit coactivatorsto perform 2 functions

    Coactivators are proteins that increase gene expression by bindingto an activator or txpn factor which contains a DNA binding domain,

    facilitating the txpn of a desired gene

    Alter chromatinstructure (like unwind it from the histone) to makepromoter region more accessible

    Recruit RNA pol II and its basal transcription factors- Enhancer elements(gene sequences far upstream/downstream for the

    gene or nearby) are brought closer to the gene we want to transcribe

    through complexes of transcriptional activator proteins, coactivators,

    and other transcription factor proteins in preparation for transcription by

    RNA pol II

    3. Describe the cellular response (or signal transduction) pathway used

    by steroid hormones and list the major hormones which interact with

    members of the nuclear rece with the steroid receptor protein family also

    known as the nuclear receptor family

    Glucocorticoids, Mineralcorticoids, Estrogens, Androgens, Progestin Can also interact with steroid-related vitamins, amino acid

    derivatives, and other molecules yet to be discovered

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    b. Pathway

    - Steroid comes into the cell and is bound by a steroid receptor

    - This creates a steroid-protein complex that enters the nucleus, which binds

    to a hormone enhancer element on DNA

    Steroid Receptor can be stabilized as dimmers after binding Steroid- The bound complex + enhancer sequence will fold up/join the promoter

    region, which will now begin to bind txpn factors, coactivators, and Pol II

    onto the promoter region. The TATA box is illustrated in the example above

    to give a frame of reference.

    - Now the desired gene can be transcribed into mRNA

    - The mRNA is then modified and packaged so it can exit the nucleus and be

    translated into protein

    - This protein will in turn create a physiological response

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    4. Explain why agnoists promote gene activation by steroid receptors,

    but antagonists inhibit steroid receptor function

    Agonist binding steps Antagonist binding steps

    1. Agonist molecule binds to a receptor. In

    our notes, the receptor is a steroid

    receptor.

    1. Antagonist molecule binds

    to a receptor

    2.Receptor binds to enhancer sequence on

    DNA

    2.Receptor binds to enhancer

    sequence on DNA

    3.Receptor undergoes a conformational

    change, yielding a new binding spot

    3.Receptor undergoes a

    conformational change, BUT

    there is NO new binding site

    4.A coactivator protein will bind to this new

    spot, and with this binding, will recruit thebinding of other transcriptional factors and

    RNA Pol 2

    4.Coactivator has no place to

    bind, the txpn apparatusnever sets up

    5.Now transcription can occur :) 5.No transcription occurs :(

    Conclusion: promotion of activity Conclusion: inhibited acitivity

    5.Discuss the roles of steroid receptors and their agonist/antagonists in the

    etiology and/or treatment of breast cancer

    Breast tissue development is triggered by estrogen Therefore, estrogen is an agonist for breast tissue/breast cancer

    growth

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    Tamoxifen, an anti-cancer drug, is an antagonist, changingconformation and inhibiting txpn by denying coactivators and txpn

    factors a binding site (see question 4)

    This prevents the growth of breast cancer cells

    6. Explain how the cAMP signaling pathway can regulate txpn of specific

    genes

    a. Ex. Glucagon pathway (which signals that we need to make glucose)

    i. Protein or steroid from outside the cell binds to a receptor.

    ii. The receptor activates a G protein, which activates adenylyl cyclase

    iii. Adenylyl cyclase releases cAMP, which binds to protein kinase A

    iv. Protein kinase A enters the nucleus via nuclear pore, phosphorylating

    CREB (cAMP response element binding) protein. Now this is just like

    question 3!

    v. CREB now binds to its enhancer region, CRE (cAMP reponse element)

    vi. NOT in the notes, but I thought this was helpful: a coactivator called

    CBP (CREB-binding protein) then binds to CRE

    vii. Now txpn is activated

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    9/29/2011 10:01:00 AM

    ---------------------------------------Regulation of Transcription-----------------

    ---------------------

    Initiation

    Can have multiple promoter and start sites

    Creates diversity by including/excluding exonsAlso changes the UTR length and potential for regulation

    Capping: Guanine cap

    Capped by inverted guanine

    Some groups are methylated

    Done by capping enzymes associated with polymerase as it transcribes

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    - recognized by nuclear pores, necessary for proper export

    - prevents exonuclease degradation

    - promotes circularization and translation

    Polyadenylation- Transcription ends when it recognizes termination sequence AAUAAA

    - Also can have multiple termination sites

    provides 3 end diversityOnce termination sequence is recognized, mRNA is cut 30 bp down and 200

    adenosines are added

    - Necessary for export of the mRNA --> proteins bind poly A tail

    Link to cap to help promote translation Prevents 3 end exonuclease degradation

    Splicing

    Introns out, Exons in

    Alternative splicing creates great biodiversity (when intentional)

    Temporal/spatial regulation

    Consensus site is strong for introns

    Excised structure is termed lariat

    When accidental or mis-spliced, can be harmful to cell Dominant negative forms Protein complexes (snRNPs) remain on mRNA, cells can tell if intron

    is left in --> Tend not to be exported

    Cryptic sites- sites (sometimes mutations) that become splice

    acceptor/donor sites that are not the normal sites

    Ex/ Portuguese family with cystic fibrosis cryptic splice site >> frameshift

    mutation

    - Burkitts Lymphoma

    - chromosomal translocation shortens 3 UTR, removing sequences

    necessary for mRNA downregulation

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    RNA Translation 9/29/2011 10:01:00 AM

    1) Describe the principle of mRNA translation and explain the degeneracy

    of genetic code

    a. mRNA is read in groups of three in two-unit ribosome complexes where

    tRNA match the mRNA and bring the appropriate amino acids. They formpeptide bonds to form the primary structure of the protein

    b. Degeneracy: There are 64 possible combinations of 3 basepairs, and

    only 20 amino acids. Each amino acid is coded by 2-6 codons

    2) Understand and be able to summarize the general steps of translation

    a. Scanning and initiation (2 ATP for aa-tRNA synth; 1 GTP required to

    unwind 5 UTR)

    i. Aminoacyl-tRNA synthetasescouple amino acids to the

    correct tRNA

    ii. 40S ribosomal subunit binds to 5 cap and starts scanning 5-3

    iii. Finds AUG start codon (ACCAUGG = Kozak sequence usually

    start)

    iv. tRNA recognizes codon & brings in Met

    v. 60S ribosomal subunit joins for protein synthesis

    b. Elongation (2 GTP / amino acid)

    i. AAs bind to A site, then P site as new peptide bonds form.

    (Leave via E site)1. This is called Translocation

    ii. N terminus = 5, C = 3

    c. Termination (1 GTP)

    i. Occurs when encounters stop codon

    ii. Requires Release Factor R + GTP

    iii. Polypeptide released

    iv. Likely a physical link bw cap and poly A tail --> ribosome

    recycling

    3) Explain how aberrant translation can play a role in human:

    a. Splicing mutations/ frameshift changes

    i. Occurs if introns not removed or exons are skipped & not

    matched up correctly

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    ii. Frameshift will change all subsequent amino acids & will result in

    incorrect protein

    iii. Only a frameshift of 3 amino acids would be less problematic but

    may still cause a problem w the protein

    b. The role of nonsense-mediated mRNA decayi. A frameshift mutation will likely cause an early stop codon

    ii. If a stop codon is encountered, the ribosome releases a release

    factor that scans for exon/exon junction complexes (EJCs) 50+ bp upstream

    of stop codon

    iii. If a protein is encountered, the mRNA is signaled for destruction

    iv. This is important for cells because dominant negative proteins

    can be very detrimental to cells (eg beta thalassemia = shortened beta

    subunit of hemoglobin ruins all hemoglobin)

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    Protein Secretion and Trafficking 9/29/2011 10:01:00 AM

    1) Describe the general mechanism of protein synthesis, and where

    it takes place in epithelial cells.

    Protein synthesis takes place on ribosomes.

    The ribosome life cycleRibosomal RNA synthesized in nucleolus

    Ribosomal proteins synthesized in cytoplasm

    Ribosomal proteins imported to nucleolus, two subunits assembled

    (The two eukaryotic ribosomal subunits are 40s and 60s, useful to

    remember. Also useful numbers for the CF sweat test: 60mosm is positive.)

    Ribosomal subunits are transported to cytoplasm.

    Small subunits identify and bind to mRNA

    Large subunits join to complex to form the ribosome.

    (The two eukaryotic ribosomal subunits assemble to form an 80s

    protein. Minimum sweat needed for the sweat test is 75 mg.)

    FROM 2011 FIRST AID FOR THE USMLE STEP 1

    Protein Synthesis

    Initiation

    Activated by GTP hydrolysis, initiation factors help assemble the 40s

    ribosomal subunit with the initiator tRNA and are released when the mRNAand the ribosomal subunit assemble with the complex.

    Elongation

    Aminoacyl-tRNA binds to A site (except for initiator methionine)

    Ribosomal rRNA (ribozyme) catalyzes peptide bond formation, transfers

    growing polypeptide to amino acid in A site.

    Ribosome advances 3 nucleotides toward 3 end of RNA, moving peptidyl

    RNA to P site (translocation)

    Termination

    Stop codon is recognized by release factor, and completed protein is

    released from ribosome.

    Mnemonics

    Eukaryotes: 40S+60S=80S (Even)

    prOkaryotes: 30S+50S=70S (Odd)

    ATP = tRNA Activation

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    GTP = tRNA Gripping and Going places (translocation)

    Clinical Relevance

    Many antibiotics act as protein synthesis inhibitors

    Aminoglycosidesinhibit formation of the initiation complex and cause

    misreading of mRNAChloramphenicolinhibits 50S peptidyltransferase

    Macrolides and clindamycinbind 50S, blocking translocation.

    Ribosome location

    Free ribosomes in cytoplasm vs. Bound ribosomes on ER.

    Both form polyribosomes (polysomes).

    2) Diagram and list the basic functions of the RER, SER, and Golgi

    complex.

    Rough Endoplasmic Reticulum

    FROM 2011 FIRST AID FOR THE USMLE STEP 1

    Nissl Bodies (RER in neurons) synthesize enzymes (e.g. ChAT) and peptide

    neurotransmitters.

    Mucus-secreting goblet cells of small intestine and antibody-secreting

    plasma cells are rich in RER.

    Smooth Endoplasmic Reticulum

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    Irregular network of membrane-bound tubules. Functions in steroid

    hormone synthesis, drug detoxification, andrelease and recapture of

    calcium ions for muscle contraction.

    FROM 2011 FIRST AID FOR THE USMLE STEP 1

    Liver hepatocytes and syeroid hormone-producing cells of the adrenal cortexare rich in SER.

    Golgi Complex

    FROM 2011 FIRST AID FOR THE USMLE STEP 1

    Golgi apparatus

    Distribution center of proteins and lipids from ER to plasma membrane,

    lysosomes, and secretory vesicles.

    Modifies N-oligosaccharides on asparagine.

    Adds O-oligosaccharides to serine and threonine residues

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    Addition of mannose-6-phosphate to specific lysosomal proteins --> targets

    the protein to the lysosome

    Proteoglycan assembly from core proteins

    Sufation of sugars in proteoglycans and of selected tyrosine on proteins.

    Vesicular trafficking proteins:COPI:Retrograde, Golgi --> ER and more

    Constituitive secretion Intercisternal transport

    COPII:Anterograde, RER -> Golgi

    Clathrin:trans-Golgi --> lysosomes, plasma membrane --> endosomes

    3) Distinguish between regulated and constitutive secretory

    pathways.

    Constitutive Secretion (default pathway): Usually continuous, needs no

    special stimulus. Can be upregulated or downregulated. (cop 1)

    Regulated Secretion: Requires an extracellular stimulus. Until then,

    proteins to be secreted are concentrated into secretory granules.

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    Cytoskeleton 9/29/2011 10:01:00 AM

    Actin

    Microfilaments

    Tubulin

    Microtubules

    Intermediate

    Filaments

    Subunits Made from

    globular (g) actin

    Made from tubulin

    dimers (alpha andbeta)

    Various elongated

    proteins, oftenhelical

    Primary

    Structures

    Form filamentous

    actin (f-actin) in a

    coiled

    microfilament

    Dimers align to

    form

    protofilaments.

    Dimers and

    tetramers

    Higher order

    structures

    F-actin MFs can

    run parallel oranti-parallel,

    branch, be

    capped, anchored,

    or severed (via

    protein partners)

    Protofilaments

    (13) assembleside-side to form

    hollow tubules

    Coiled-coil (likely

    not important)

    Size ~7 nm ~25 nm (hollow) ~10 nm

    Use

    nucleotide?

    ATP GTP Nope.

    Polarity assembly usually

    occurs on (+) end,

    disassembly on

    the (-) end

    (-) is anchored

    centrally to the

    MTOC, or to the

    basal body.

    Assembly usually

    occurs on (+) end

    extending towards

    cell periphery

    None

    Dynamics Fast Fast Very slow

    Motors Myosin Kinesin (+) and

    dynein (-)

    ?

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    Found in: -Cell peripheries,

    for cell shape and

    motility

    (lamellipodia,

    projections)

    -Microvilli

    -Cleavage furrow

    of cytokinesis

    -Sarcomeres

    -Cell interior, for

    rigidity and

    vesicular transport

    -Mitotic spindles

    -Cilia, flagella

    -Nuclear lamins,

    for nuclear

    structure

    -In cytoplasm for

    support

    -ECM (i.e.

    keratin) for

    mechanical

    support

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    Endocytosis and Lysosomes 9/29/2011 10:01:00 AM

    Endocytosis:General phenomenon of cellular uptake via mechanisms

    involving membranes (not channels or pumps)

    Pinocytosis (cell drinking)

    -Macro/microMediated by caveolin, causing cell invaginations

    Non-specific, but not really random; tend to be positive/neutralmolecules

    Phagocytosis (cell eating)

    Can be specific (receptor-mediated) or non-specific Contents go through endosomal system

    Endosomal system

    Consists of variable membranous structures (vesicles, tubules,MVBs), differing in shape and sizes

    Involves progressive acidification of interior , initially by H+ pumps(pH goes down Enzymes are tagged with mannose-6-phosphate (M6P) in cis-Golgi

    - Receptors for M6P (M6PR) binds and sorts these enzymes at the trans-

    Golgi, bud as lysosomes to traffic to endosomes

    - Acidification causes cargo release, and M6PR receptors are recycled- Primary lysosomes are newly minted, have not combined

    - Secondary lysosomes have combined with endocytic cargo

    - Hydrolytic enzymes protected by glycosylation

    *If contents are undegradable, cell removes what it can and then

    condenses/sequesters material into a residual body

    -Progression can be generally categorized as early (near periphery) vs.late

    (near Golgi) but really is a functional continuum

    Ex: LDL receptor-mediated endocytosis

    LDL recognized by receptor (LDLR) and these complexes will startto cluster

    Clathrin, previously free in the cytoplasm, associates with theseclustered receptors at their cytoplasmic ends

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    Coated pits form and pinch off to form vesicles. Clathrindisassociates and is recycled

    Acidification causes LDLR to release LDL, receptors are recycledback to plasma membrane

    Combines with primary lysosome, so that contents can be actedupon

    Vesicle membranes can be recycledHeterophagy: uptake of external materials

    Autophagy:digestion of cellular components for turnover (proteins,

    ribosomes, mitochondria)

    Clinical Correlates:

    Hypercholesterolemia: Defective receptor-mediated endocytosis of LDL

    (at the receptor level or trafficking level) causes extreme fatty deposits in

    various locations of body.

    -Premature atherosclerosis & heart attacks

    Infection

    -Many infectious agents use receptors as cellular entry points- have the

    ability to escape or neutralize lysosomal degradation>i.e. diphtheria toxin, almost every virus, parasites

    Inclusion cell disease

    Defective enzyme responsible for tagging lysosomal enzymes Lysosomal enzymes traffic through constitutive pathway out of the

    cell (secreted)

    Extra (?)

    Secretory lysosomes

    Conventional lysosome whose contents are also secreted

    Immunity

    -Cytotoxic T-cells release cytolytic proteins against cancer cells

    -MHC presentation of degradatory products

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    -Histamine from mast cells

    Albinism

    -Secretion of melanin from melanocytes

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    Peroxisomes and Mitochondria 9/29/2011 10:01:00 AM

    Lecture 6: Peroxisomes and Mitochondria

    Explain the role of peroxisomes in detoxification, catabolism, and

    biosynthesis.

    Peroxisomes (aka microbodies)producing and breaking down hydrogen peroxide

    Detoxification

    Alcohol in liver and purines in kidney (abundant in organs)Catabolism

    Oxidase breaks down long chain fatty acids, H2O2 side productCatalase degrades H2O2H2O + O2

    Biosynthesis

    Gets membranes from the ER Myelin precursor, bile acids, cholesterol

    Other notes:

    Replicate by fission

    Peroxins transport proteins from cytosol (receptor for PTS), important for

    biogenesis

    PTS (peroxisome targeting signals) at the C and N terminus on these

    proteins

    Diseases:

    Long chain fatty acid accumulation and low myelinPeroxisome biogenesis disorder (more severe) (eg: Zellweger syndrome:

    severe, peroxin mutations that lead to virtually no peroxisomes, fatal w/I 1st

    yr of life)

    Demyelination and accumulation of long-chained fatty acid

    Very flaccid and no muscle contraction

    Peroxisomal enzyme deficiencies (eg: X linked adrenoleukodystrophy, gene

    mutation of fatty acid transport into peroxisomes, death 1-10yrs after onset)

    Demyelination and fatty acid accumulation

    Lorenzos oil

    2. Explain the role of mitochondria in ATP production, catabolism, calcium

    homeostasis, and apoptosis.

    Mitochondria

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    ATP production: electron transport chain makes intermembrane acidic to

    drive ATP synthas

    (I) NADH dehydrogenase: H+intermebrane, e-ubiquinone

    (II) FADH2 dehydrogenase (succinate): FADH22H + FAD-+e-(ubiquinone)

    (III) Cytochrome C reductase: H+ intermembrane, e- (ubiquinone)cytochrome c

    (IV) Cytochrome c oxidase: H+ intermembrane, e- used to reduce O2 to

    water

    ATP synthase: H+ returns to matrix

    Catabolism: long chain fatty acid oxidation, pyruvateacetyl CoAoxidation

    Calcium homeostasis: sequesters excess cytosolic Ca (refer to muscles)

    Apoptosis: mito releases cytochrome c and activates a caspase cascade to

    induce apoptosis

    *NOT the same as necrosis (inflammation, uncontrolled release of cell

    contents)

    Other:

    Inner membrane is impermeable to small ions

    Tubular and can form networks

    Acetyl CoA Citric Acid cycle ETC (ATP synthase)

    Mt protein import (general): proteins contain mt targeting sequence

    Proteins bind the TOM and TIM (transfer protein of outer/inner mitochondrial

    membrane) and binding allows import into mitoThere are also secondary sequences which target inner membrane,

    intermembrane space, outer membrane

    Identify peroxisomes and mitochondria in electron micrographs.

    Compare and contrast the structure, function, and genetics of peroxisomes

    and mitochondria.

    Peroxisomes Mitochondria

    Structure 0.5-1m

    Lipid bilayer; single membraneGranular matrix; oxidase,

    catalase

    Nonhumans have a nucleoid

    structure in the middle (big black

    spot)

    0.5-10m

    Inner membrane (cristae e-transport)

    Outer membrane

    Intermembrane space (high

    H+)

    Matrix: mtDNA, ribosome,

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    tRNA, TCA enzymes

    Function Biosynthesis (bile acid,

    cholesterol, myelin)

    Detoxification of alcohol and

    purines

    oxidation of fatty acids (very

    long chain)

    breakdown H2O2

    ATP synthesis

    Ca homeostasis

    Regulation of apoptosis

    oxidation of fatty acids

    Genetics Binary fission replication

    Membranes from the ER

    No endogenous DNA

    Binary fission replication

    Inherited from egg (gamete)

    mtDNA (can have defects)

    autosomal mtprotein defects

    Disease biogenesis disorders are moresevere

    Neurological myelination

    diseases

    Zellweger syndrome (no

    peroxisome; buildup of very long

    chain fatty acids)

    X linked adrenoleukodystrophy

    (no fatty acid transport into

    peroxisomes)

    Mt enzyme deficiencyMitochondrial DNA defects

    Autosomal DNA defects for mt

    proteins

    Lebers hereditary optic

    neuropathy (loss of central

    vision, affects mostly males,

    optic nerve degeneration)

    Identify maternal inheritance by pedigree analysis; explain how

    mitochondrial diseases can be maternally or autosomally transmitted;

    explain heteroplasmy.

    Maternal inheritance: Mitochondrial DNA defects (13 mt proteins, 22 tRNA,

    2rRNA)

    Autosomal inheritance: nuclear DNA defects that code for mt proteins

    Heteroplasmy: mixture of mutated and normal mitochondria redistributeunevenly during development, so this is the occurrence of cells having

    different mitochondria and may occur within one individual

    Eg: Lebers hereditary optic neuropathy (lossof central vision, affects mostly

    males, optic nerve degeneration because not enough functional mito

    produce energy to maintain cells)

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    Microanatomy 7: Cell Differentiation

    1. Describe the main characteristics of differentiated cells and

    understand that it is a result of differential gene expression, not

    gross changes in the genome itself.

    Differentiated cells are cells that have become

    morphologically/structurally, biochemically, and functionally

    specialized. Since nearly all cells of the body have genomic equivalence

    i.e., they all possess the same full genome cell differentiation typically

    results from changes in gene expression, not any changes in the DNA itself.

    Exceptions:Gametes (sperm and egg cells); immune system (B cells, T

    cells splicing of immunoglobulin genes in order to generate antibody

    diversity)

    Once a cell is differentiated, the changes are often permanent and

    heritable, such that the cells produced through divisions of this

    differentiated cell have the same specialized characteristics that it does.Terminally differentiated cells (ex. neurons) lose their capacity for mitotic

    division entirely.

    In short, differentiation = restriction of cell fate

    2. Describe the general properties of stem cells and compare them to

    differentiated cells.

    Properties of stem cells:

    - Capable of self-renewal or differentiation

    - May give rise to transit amplifying cells with limited division capacity

    - Often lack specialized organelles, and show high nucleus/cytoplasm ratio

    - Long-lived express telomerase

    - Slow to divide, few in number

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    - May be restricted spatially to specific zones or niches

    - Respond to signals that will regulate their growth and proliferation

    Stem cells vs. differentiated cells seems pretty obvious just by extrapolating

    from the above properties, so a finer distinction to make might be stem cellsvs. progenitor cells:

    Stem cellscan divide an unlimited number of times, and can give rise to

    more stem cells (self-renewal) as well as differentiate into specialized cells of

    any of the three germ layers (pluripotency).

    Progenitor cellshave a limited number of divisions and do not maintain

    their ability to self-renew, are considered to be at a further stage of

    differentiation than stem cells, and are more limited in the variety of cell

    types into which they can differentiate (multipotency or oligopotency).

    *Note: These two terms are sometimes used interchangeably, and the

    distinction between the two is still contested, since some stems cells (ex.

    adult stem cells) are also only multipotent, oligopotent, or unipotent. The

    best criterion to go by for distinguishing the two seems to be the self-

    renewalaspect, which is exclusive to stem cells.

    3. Distinguish determination and differentiation.

    Differentiation= the process by which different, specialized cells

    (morphologically, biochemically and functionally) arise from a homogeneous

    group of unspecialized cells.

    Determination= the commitment of a cell to differentiate into a certain

    cell type at some later time. Determination generally occurs as a step (or a

    series of steps) that is prior to, and separate from, differentiation. Cells can

    be determined a long time before they differentiate (at least by histological

    criteria). Two major ways that cells can become committed are by: 1)

    possession of special cytoplasmic determinants localized within the egg or

    early embryo, and 2) interaction with other cells or the factors secreted by

    other cells. Cytoplasmic determinants are components localized to a

    particular region of the egg or embryo that, when segregated to a cell or cell

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    lineage cause those cells to become determined, i.e., to adopt a particular

    cell fate.

    First column: Normal pattern of differentiation; cells in region A differentiate

    into one type of cell, cells in region B into another typeSecond column: Sample of region B cells labeled and transplanted into

    region A early on, before they have become determined transplanted cells

    differentiate into region A type cells

    Third column: Sample of region B cells labeled and transplanted into region

    A later on, when they are already determined transplanted cells

    differentiate into region B type cells

    [Fourth column is not relevant to this objective, but is basically

    demonstrating that region B cells that are specified (specification = early,

    reversible/changeable step of determination, so not fullydetermined) will

    still differentiate into region B type cells in an isolated environment.]

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    4. Explain the potency of stem cells and understand it in the context

    of differentiation stages.

    Cell potency = amount of differentiation potential; the following are listed

    from least differentiated stage (have the most differentiation potential) tomost differentiated stage (have the least differentiation potential)

    Definition Example

    Totipotent

    Can differentiate into

    any cell type (ectoderm,

    mesoderm, and

    endoderm) as well as

    extra-embryonic tissue

    such as the placenta

    Human zygote is

    totipotent up through

    the 8-cell stage.

    Pluripotent

    Can differentiate into

    any cell type (ectoderm,

    mesoderm, and

    endoderm), but cannot

    produce full embryo

    Embryonic stem cells

    (which originate from

    the inner cell mass of

    the human blastocyst)

    are pluripotent.

    Multipotent

    Can differentiate into a

    limited number of cell

    types

    Progenitor cells; also,

    hematopoietic stem

    cells (a type of adult

    stem cell) can

    differentiate into various

    blood cells (RBCs, white

    blood cells, platelets,

    etc.) but not other

    mesodermal cell types.

    OligopotentCan differentiate into

    very few cell types

    Progenitor cells; some

    adult stem cells

    Unipotent

    Can differentiate into

    only one cell type;

    however, unipotent

    stem cells can still self-

    renew

    Skin cells

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    Terminally

    differentiated

    Have fully differentiated

    and can no longer divide

    via mitosis

    Neurons

    5. Explain inductive interactions and epigenetic controls in cell

    differentiation.

    Induction= process by which surrounding cell or environment guide the

    cell differentiate process; can be mediated through cell-cell, cell-matrix, or

    diffusible growth factor interactions; can be instructive(signal itself causes

    target cell to differentiate) or permissive(cell already committed, induction

    creates conducive environment for differentiation); timing of induction is

    crucial (see objective #6 on temporal specificity).

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    Epigenetic controlscontribute to maintenanceof differentiation through

    heritable alterations in chromatin structure (ex. DNA methylation) and

    transcription factor expression.

    6. Draw a diagram that demonstrates the principle of temporalspecificity in induction.

    7. List the sources of stem cells for use in research and medicine.

    Type Pros Cons

    Adult stem cells Easily obtained; no

    controversy

    Multipotent or

    unipotent, so have

    limited usefulness

    Embryonic stem cells Pluripotent Political/ethical

    controversy; potential

    for aberrations leading

    to tumor formation

    during culturing stageCord blood stem cells Mostly multipotent

    (peripheral blood stem

    cells, or PBSCs), but

    also some pluripotent

    stem cells; less

    (None mentioned in

    lecture)

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    controversial

    Induced pluripotent

    stem cells (iPSCs)

    Derived from non-

    pluripotent cell, ex.

    adult skin cells, so also

    no controversy; can be

    reprogrammed to regain

    pluripotency and even

    totipotency

    Chances of aberrations

    tumor formation in

    origin cells, and during

    reprogramming and

    culturing stages; require

    more research

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    Intro to Tissues 9/29/2011 10:01:00 AM

    Intro to Tissues

    Dr. Wood 9.1.2011

    Generalities:

    All cells have:-Nuclei,EXCEPT RBCs or mitotic cells

    -Cytoplasm

    -Plasma membrane

    **They may be hard to see, due to abundance or plane or section

    Common cell shapes:

    Squamous

    Cuboidal

    Columnar

    Spherical

    Spindle

    Stellate

    When talking about tissues, dont forget to think about the ECM associated

    with the cells

    Epithelial tissue

    -Lines free surfaces, both externally and internally (i.e. skin, sweat glands,

    GI, kidney tubules)

    -Supported by connective tissue

    -Serves as barriers

    -Cells tend to be tightly packed (look for high density of nuclei) with

    little ECM

    -Avascular

    -Tend to be polarized

    Connective tissue

    -Provide structural support for many other types of tissue

    -Tend to have few cells (sparse density of nuclei) with lots of ECM

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    -Fibroblasts are the most common type of connective cell

    >Stellate/spindle shape, constitutively secreting ECM (collagen)

    -Classified as loose (more cells/water, less protein/fibers) or dense (more

    protein/fibers, less water/cells)

    -Blood is connective tissue-Also fat, bone, tendon, cartilage

    Contractile tissue

    -Specialized for rapid movement, responsive to electrical signals

    -Striated (cardiac, skeletal) vs. smooth muscle

    -Voluntary (skeletal) vs. involuntary (cardiac, smooth)

    -Many tubular structures (vasculature, GI tract) have contractile tissue

    *Smooth muscle can often look like connective tissue. Try looking at how

    many nuclei there are- more nuclei usually signals smooth muscle

    Nervous tissue

    -Neurons are large cells responsive for conveying electrical stimuli

    -Glia are smaller supporting cells (50:50 90:10)

    -Neuropil is cellular (not extracellular) material between cell bodies- cell

    processes of glia and neurons

    Origins of tissues from germ layers

    Ectodermal Origin Mesodermal Origin Endodermal Origin

    Epidermis of skin Dermis of skin GI (stomach, intestines,

    epithelial lining) but

    NOT including muscle

    layers

    Nervous system Circulatory (heart,

    vascular epithelia)

    Lungs and epithelial

    lining

    Cornea Kidney tubule epithelia LiverLens Skeletal muscle,

    skeleton

    Pancreas

    Connective tissue Bladder/urethral lining

    Lymphatic systems

    Excretory system

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    (kidneys and bladder,

    excluding lining of

    bladder)

    Lining of body cavity

    (mesothelium?)

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    Epithelium 9/29/2011 10:01:00 AM

    1) Explain how epithelia establish barriers and control exchange between

    different environments

    Epithelium lines the free surfaces of the body, so material (ions, proteins,

    oxygen, sugars) needing to pass between tissues of the body and luminalspaces (lungs, gut, ducts) needs to pass through the epithelium. Epithelial

    cells are densely packed and connected to neighboring epithelial cells

    through:

    Tight junctions: forms a ring around the cell that maintains polarity of

    transport proteins between apical and basolateral faces. Not anchored by

    cytoskeletal elements

    Adhering Junctions: forms a ring around the cell, connected to actin

    Desmosomes: Spot welds connected to intermediate filaments

    This most commonly happens in a transcellular pathway, although there

    are examples of paracellular transport through the tight and adhering

    junctions. Because of cellpolarity, different transporters on apical and

    basolateral surfaces can regulate directionality of transport and create

    downhill gradients across the cell.

    The apical side of epithelium in the trachea are coated in cilia that facilitate

    transfer of mucus out of the airway

    Keratinized stratified squamous epitheliumprovides a particularly

    strong barrier with a thick layer of dead, denucleated squamous cells that

    are continuously regenerated as they are sloughed off.

    2) Classify Epithelium based on cell layers and cell morphology

    Simple Squamous Epithelium: fried egg morphology, can have elongated

    cytoplasm. (endothelium, alveoli, mesothelium, portions of kidney tubule)

    Simple Cuboidal epithelium:cuboidal, regular (or just not columnar or

    squamous) morphologyshape. (kidney tubules and ducts)

    Simple Columnar Epithelium:Tall cells, usually with apical surface

    specialization (gut, intestine, respiratory tract, oviduct

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    Stratified Squamous Epithelium: Only top living layer has to be

    squamous. Usually serves to protect underlying tissue. Can be keratinized

    (skin cells) or non-keratinized (mouth, vagina, esophagus)

    Transitional Epithelium: Stratified epithelium thatlines urinary system

    from center of kidney (Renal calyx) to bladder. Allows stretching.

    -rounded (bumpy) surface is identifying characteristic

    Pseudostratified epithelium (Respiratory Epithelium): All cells contact

    the basement membrane, but not all are exposed to the lumen

    3) Explain replacement of epithelium:

    Determined cells can undergo mitosis to form new cells (liver, endothelium)

    or stem cells can undergo mitosis and determination (intestines, skin). In

    stratified epitheliums, the new cells are generated near the basement

    membrane and migrate to the top as cells are sloughed off.

    4) Explain different types of glandular secretion:

    Merocrine:secretory granule fuses with the PM and dumps it contents intothe ECF

    Apocrine:secretory granule leaves cell where its contents are still enclosed

    in plasma membrane (i.e. mammary glands secreting milk)

    Holocrine:Whole cell lyses and dumps contents (ex/ sebaceous gland)

    Paracrine: Cell releases contents that then signals nearby cells. Commonly,

    this occurs with signaling molecules that degrade quickly and can thus only

    act on nearby cells.

    Autocrine: Cell releases contents that then binds to same cell (B-cells)

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    Cell-cell/Cell-matrix interactions 9/29/2011 10:01:00 AM

    Multicellularity allows cells to specialize and carry out more tasks

    simultaneously

    To become multicellular:

    - Contact and communicate with other cells

    - Regulate passage of environmental substances into and out oforganism

    - Recognize self/non-self

    Occluding/tight junctions: do not involve cytoskeletal elements

    Formed by claudin and occludin (transmembrane proteins)

    associations

    Form tight seal that that not allow most molecules to pass through

    Found usually on apicolateral surface of cells (epithelial usually)

    Anchoring junctions: do involve cytoskeletal elements

    Actin Microfilament

    associated

    Intermediate

    Filament associated

    Cell-cell junction

    (mediated by

    cadherins)

    Adherens junctions Desmosomes

    Cell-matrix junction

    (mediated by

    integrins)

    Focal adhesions Hemidesmosomes

    Homophilicvs. heterophilicbinding

    Cadherin family:

    Calcium-dependent adhesion molecules

    Low Ca++: cadherins are largely disorganized

    ~1mM Ca++: Dimers form extracellularly, nucleates other dimers molecular zipper

    Adherens Junctions: Desmosomes:

    E-, N-, VE-cadherins (classical) Desmocollin, desmoglein

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    Linked to actin cytoskeleton Linked to IF via

    plakoglobin/desmoplakin

    via / catenin

    *Cadherins mediate cell-cell sorting where cells expressing the samecadherins (hemophilic binding) associate with each other and organize into

    structures

    Other adhesion molecules:

    Ig superfamily:

    Ca-independent family of proteins that are weaker than cadherins and can

    be both homophilic and heterophilic

    Selectins

    Ca-dependent group of adhesion molecules that bind oligosaccharide lectins

    (heterophilic)

    Low affinity binding. Responsible for leukocyte rolling

    ECM

    Hydrated, polysaccharide gel substance with fibrous proteins embedded,

    important for physical stability and diffusion of important substances.

    Usually formed via fibroblasts. The basal lamina is a specialized form of ECMlaid down by epithelial cells.

    ECM components

    Glycosaminoglycans(GAGs):

    Unbranched, repeating disaccharide units.

    Hydrophilic, highly negatively charged- responsible for resisting

    compressive forces

    Ex: hyaluronan

    Proteoglycans:

    Protein + GAG = proteoglycan

    Formed by a tetrasaccharide link

    Structral and cell-cell signaling roles

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    Fibrous proteins:

    Collagen long inflexible fibers providing strength

    Elastin - mesh-like fibers that provide flexibility

    Fibronectin recognized by attachment proteins

    Laminin - recognized by attachment proteins

    Integrins

    Transmembrane receptors (heterodimeric) with affinity for RGD motif in

    fibronectin.

    Linked to actin/IF cytoskeleton via talin.

    Can communicate bi-directionally.

    ECM cell: Leukocyte recruitment

    Cell ECM: ECM remodeling for pathfinders

    Communicating junctions: mediate chemical or electrical signals from one

    cell to another

    Gap junctions

    Mediated by membrane channels called connexons, which are formed by 6

    connexin proteins

    Permeability affected by pH and Ca++ (open with high pH, low Ca++)

    Chemical synapsesFast type of paracrine signaling

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    Blood 9/29/2011 10:01:00 AM

    Element Function

    Identifying

    characteristic

    Abund

    ance Lineage

    Erythrocyte

    transport O2,

    transport a little

    CO2

    no nucleus or other

    organelles, biconcave

    shape, appear

    electron dense

    most

    abundant

    proerythroblasts ->

    erythroblasts ->

    reticulocytes (no

    nucleus) -> RBCs (left

    bone marrow, no more

    RNA)

    Thrombocyte

    (platelets)

    trigger thrombi

    to limit blood

    loss at injury

    site; aggregate

    w/ platelets,

    alter blood flow,

    initiate

    coagulation

    cascade

    discoid, small

    fragments (small

    specks interpsersed

    w RBCs) many

    megakaryoblast -

    >megakaryocyte (in

    bone marrow) -->

    1000 platelet clumps

    (made of cytoplasm)

    Neutrophil

    phagocytosis &

    destruction of

    bacteria

    multilobed; clear or

    light pink granules 60-70% myeloid cell

    Eosinophil

    destroy larger

    parasites /

    modulated

    allergic

    response

    multi-lobed (2);

    pink/orange granules 1-3%

    myeloid cell

    Basophil

    release

    histamine &

    heparin

    (anticoagulation

    )

    S-shaped nucleus,

    blue/black granules phagocytosis

    irregular, kidney-

    shaped nucleus; no

    granules 4-10%

    lymphoid cell: enter

    lymphoid organs -->

    macrophages (Myeloid

    lineage)

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    Lymphocyte

    B cells -->

    plasma cells; T

    cells -->

    immune rxns

    large, round, dark

    nucleus 20-30%

    myeloid cell: B cells --

    > plasma cells

    Megakaryocyte

    produce

    platelets;

    located in bone

    marrow

    largest cell; has

    clusters of

    proplatelets

    Reticulocytes

    produce RBCs;

    located in bone

    marrow

    look like RBCs but

    still have RNA (blue

    stain in cytoplasm)

    Plasma cells

    found in

    connective

    tissue; secrete

    targeted

    antibodies

    lots of rough ER that

    is dilated; clumps of

    heterochromatin; no

    storage vessicles

    Macrophages

    found in

    connective

    tissue;

    phagocytic; act

    as antigen-

    presenters

    many cytoplasmic

    processes, numerous

    lysosomes

    Remember: Never Let Monkeys Eat Banas

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    9/29/2011 10:01:00 AM