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Flashcards in BMS Part II Deck (255):
1

Define locus

site on chromosome where gene is located

2

Define allele

variants of a gene located on same locus

3

Define locus heterogeneity

Different mutations on different genes produce the same phenotype e.g. familial hypercholesterolemia

4

Define allelic heterogeneity

different mutations at same allele cause similar phenotype e.g. Duchenne, CF, B-thalassemia

5

Define phenotypic heterogeneity

different mutations in the same gene cause different phenotypes e.g. Hirschsprung, CF
severity of disease depends on how protein function is affected by mutation

6

Define hemizygous

Male patients only- abnormal gene on X

7

Define compound heterozygote

Mutations are at different locations in the gene e.g. CF

8

Define obligate heterozygote

Clinically unaffected but must carrier mutant allele based on pedigree (e.g. normal parents of affected child)

9

Autosomal recessive diseases:
define
features of pedigree
factors that increase risk
factors that affect clinical severity

1) Two mutant alleles (can be compound heterozygotes), rare
2) skips a generation (parents are asymptomatic carriers), prevalence among siblings (1/4 chance of disease), equal prevalence among M/F
3) Carrier frequency (consanguinity i.e. related, genetic isolates i.e. Tay Sachs, inbreeding i.e. same small pop)
4) Sex influenced phenotype (hemochromatosis- too much iron, disprop affects males)

10

Autosomal dominant:
define
features of pedigree
what is incomplete dominance
factors that affect clinical severity

1) can have 1 or 2 mutant alleles (though 2 is rare), majority of CURRENT known Mendelian disorders
2) disease is in every generation, equal prevalence among M/F, prevalence among siblings (1/2 chance of disease), normal siblings have all normal offspring
3) homozygous BB is more severely affected than heterozygous Bb e.g. familial hypercholesterolemia
4) reduced penetrance, variable expressivity, sex-limited phenotype (e.g. male-limited precocious puberty)

11

X-linked recessive (XLR)
define
features of pedigree
what is unbalanced X inactivation/dosage compensation

1) need two mutated X alleles to exhibit the phenotype
2) No M-M transmission (offspring M of affected M are carriers), prevalence M>>>F, women are carriers, any women with phenotype due to unbalanced X-inactivation
3) if female is Xx but the rate of inactivation of the mutated X is out of prop--> female will exhibit phenotype
females are somatic mosaic

12

X-linked dominant (XLD)
define
features of pedigree

1) only need 1 mutated X allele to exhibit phenotype
2) NO M-M transmission (offspring M of affected M are normal, offspring F are all affected), prevalence F>>>M

13

What is the difference between reduced penetrance and variable expressivity?

1) Penetrance- does disease show up? prop with mutation who express symptoms
Expressivity- how does disease show up? same mutation but severity of phenotype varies
*can have differential expressivity- minor clinical abnormalities vs definite clinical abnormalities

14

What are exceptions to Mendelian inheritance?

1) Mosaicism- somatic or germline (depending on when the mutation occurs)
pedigree clue-- two normal parents who have multiple affected children with AD or XLR
2) New mutations- v rare
pedigree- if you see a new mutation for a genetic lethal disease e.g. Duchenne
3) Misattributed paternity
4) Genomic imprinting - methylation is off in gametes
5) Reduced penetrance/variable expressivity
6) mitochondrial inheritance
7) TNR expansion disorders

15

Difference between chromatin and chromatid?

Chromatin- fibers composed of DNA + histones
Chromatid- DNA after replication (Sister chromatids), made of condensed chromatin fibers

16

Define c-value

Amount of DNA in one gamete
*wide variety between C-value and organism complexity (also with chromosome number and complexity)

17

Compare RNA and DNA in the genome

RNA precedes DNA in evolution
RNA more complex/diverse in actions
DNA more chemically stable --> evolutionary advantages

18

Conclusions from the ENCODE project

1) Chromatin has 7 functional states
2) 60-75%of DNA transcribed into RNA
3) 80% is "functional"-- reproducible biochemical signature
4) non-coding transcripts as abundant as coding ones--> will be important as determinant of health/disease

19

Describe repetitive sequences:
impact
types
example

More recent the repeat- less divergence from predecessor segment --> proteins encoded are more similar
repeats enhance probability of recombination/structural changes
1) Tandem repeats - adjacent repeating sequences, evolutionally recent repeats are more similar --> lead to recombination
2) Short repeats - satellite (100+ bp)/microsatellites (few bp) tandem repeats --> form small satellite when DNA is fractionated by density
3) Retrotransposons - encode reverse transcriptase acting on mRNA creates segmental duplication
LINE-encode reverse transcriptase
SINE- do not, e.g. Alu sequences
*reverse transcriptase comes from viruses

20

Examples of diseases due to repetitive sequences:
R/G color blindness
Continuous gene syndrome

1) R/G color blindness-- recombination bw duplicated genes on the X chromosome;
misalignment in meiosis and improper segregation into sperm cells; one has only red receptor gene (colorblind) and the other has 1 red + 2 green receptors (normal)
2) DiGeorge/Prader Willi/Angelman- deletes large gene sequence repeats
use FISH to tell you region of DNA deleted

21

Define pseudogenes

Non functional, pieces of DNA back in the genome from action of reverse transcriptases; not transcribed bc they lack promoters

22

Describe the three techniques for karyotype staining:
G-banding
FISH
CGH (Comparative genomic hybridization)

1) G-banding- detects large changes
arrest chromosomes in metaphase, stain with Giemsa so bands form (dark G bands)
Chromosomes have unique banding pattern, size, also centromere position (meta, sub-meta, or acrocentric)
can only detect large deletions (one band = 45 genes)
2) FISH- detects smaller changes with known location
Interphase FISH is faster, metaphase FISH is better resolution
need specific probe- test only as good as the probe
green is the internal control, red is the test probe--if red is missing, there is a mutation
but if red is there, there could still be a mutation elsewhere
con: cannot detect single nucleotide changes, resolution decreases as # probes increases
3) CGH- detects changes even if location is not known
oligonucleotides on glass slide/ microarray
PCR test patient DNA (green) with reference DNA (red) and allow both to hybridize on glass slide to detect small changes in the genome
yellow- both bind equally
red- no patient DNA
green- too much patient DNA
can also plot hybridization ratios (L-->R is p-->q arm) to quantify the deletion/duplication
con: only for deletions/duplicates NOT inversions/translocations

23

Describe Robertsonian translocation

D (13-15) and G (21-22) group acrocentric chromosomes only; fusion of chromatids and loss of p arms --> so 3 chromosomes instead of 4
*not necessarily deleterious bc p arms of acrocentric chromosomes have multiple copies of genes for rRNA

24

Three ways to ID chromosomes

1) Size
2) Centromere index = length of p arm / total chromosome length
3) G banding

25

Steps of meiosis

Meiosis I
1) Interphase- chromosomes duplicate
2) Prophase I- chromosomes condense, synapsis- tetrads line up; crossing over (swapping homologous sections), nuclear breaks down + spindle forms
3) Metaphase I- tetrads line up in independent assortment, spindles attach to kinetochore
4) Anaphase I- spindle microtubules contract and pull chromosomes to opposite ends
5) Telophase- nuclear membrane reforms + cytokinesis
Meiosis II
1) Prophase II- spindle reforms, nuclear breaks down again
2) Metaphase II- lining up, spindles attach
3) Anaphase II- spindles contract
4) Telophase II- nuclear membrane reforms + cytokinesis

26

Difference between meiosis in M/F

M- spermatogenesis: meiosis I and II begins at puberty- 4 sperm produced- and continues throughout life
F- meiosis starts in utero- all oocytes formed before birth, arrested in metaphase II until fertilization (one oocyte during ovulation each month)
1 polar body extrudes at ovulation, 2nd at fertilization, they die when egg is fertilized

27

Nondisjunction

Either Meiosis I or II - failure of homologous chromosomes or sister chromatids to separate
leads to abnormal zygotes e.g. Down, Turner

28

Define uniparental disomy

Both chromosomes come from one parent e.g. Prader Willi, Angelman (imprinted genes)

29

How do you use polymorphic markers to determine where meiosis II occurs? Stage of meiosis and parental origin

If the alleles near the centromere are NOT identical --> nondisjunction in meiosis I
If alleles near centromere ARE identical --> nondisjunction in meiosis II
For parental origin - need to analyze polymorphic DNA markers

30

Explain types of rearrangements
Balanced
Unbalanced

Balanced
1) Reciprocal translocation- exchange of segments between non-homologous chromosomes; usually harmless
2) Inversion- single chromosome undergoes breakage and rearrangement
Unbalanced
3) Deletion
4) Duplication

31

Difference between paracentric and pericentric inversions

Paracentric- inversion on one arm of the chromosome (either p or 1)
Pericentric- inversion includes the centromere
Requires two chromosome breaks
Balanced rearrangement in the carrier parent but high instability in gamete formation- miscarriages or affected children

32

Define isochromosome

Chromosome has lost one of its arms and replaced it with a copy of the remaining arm
e.g. Turner's syndrome, Down's

33

Gene density of human DNA

12-15 genes in 1 M bp
22,000 genes over 23 chromosome pairs

34

Difference between ataxia and apraxia

Ataxia- loss of coordination of motor control (e.g. in HD)- muscle/movement disorder
Apraxia- inability to execute movement despite having both desire AND capability - motor nerve disorder

35

Describe the mt inner and outer membrane and mitochondrial matrix

Inner- impermeable to molecules, rich in protein, where electron transport chain/ox phos takes place, contains cristae (convoluted folds which increase surface area)--> # of cristae depend on energy needs of the cell
Outer- permeable to small molecules/ions, contains signaling receptors
Matrix- proteins for TCA, etc. mt RNA and DNA, protein synthesis machinery

36

Differences between mitochondrial and nuclear genome

1) Mt DNA is much smaller--> 2-50 mtDNA in matrix of each mt, ~10-200 mt per cell (20-10000 mtDNA molecules per cell)
2) mt DNA circular
3) no introns

37

T/F:
1) Protein transport is unidirectional
2) Mt have fixed copy number, shape, size

1) True - protein from cytosol imported into mt
2) False- through fission and fusion, mt shape is changing; size depends on energy demand conditions

38

Explain mt DNA replication

Can occur independently of the cell cycle
Replication and transcription take place in the D-loop
DNA polymerase gamma- 3 activities (polymerase, exonuclease/proofreading, lyase for DNA repair), nuclearly coded protein
Twinkle- helicase
afterwards, mtDNA packaged 1 or 2 at a time into nucleoids
mtDNA repair less efficient bc there is high copy number buffer

39

Explain mt DNA transcription

Takes place in the D-loop, no cap added
polycistronic, encodes 13 mRNA, 2 rRNA, and 22 tRNA
requires mt RNA pol, regulated by TFAM (transcription factor activator), and mt transcription factors B1 and B2

40

Explain mt translation

-Encodes 13 proteins
-1 tRNA can pair with any of 4 codons- need only 22 tRNA (all are mt encoded)
-Proteins nuclearly coded and imported:
Initiation- IF2 and IF3
Elongation- mtEFTu, mtEFTs, mtEFG1
Termination- only 2 stop codons
*most mitochondrial proteins come from cytosol (not RER and Golgi)

41

Explain the 6 distinguishing features of mtDNA genetics

1) High mtDNA mutation rate
-close to source of ROS, DNA pol gamma is not great at proofreading, no introns so mutations happen in coding exons
2) Maternal inheritance - paternal mitochondria destroyed when sperm fertilizes the egg
3) Genetic bottleneck - during oogenesis, reduction in primary oocyte then amplification --> redistributes mt DNA among daughter cells--> can have different levels of mutated mtDNA
4) Random segregation of mt and mtDNA between daughter cells
-heteroplasmy- mixture of mutant and normal mt in the same cell
5) this is associated with the threshold effect--> cross threshold of mutant mtDNA for phenotypic expression
-daughter cells have wide variation in prop of mutant/normal mt
-threshold level varies among mutation and tissue
6) Changes with age
-number of mtDNA mutations increases with age due to ROS and defective DNA pol gamma, also levels of ox phos decline

42

Common features of mt diseases

1) Lactic acidosis - ox phos impaired --> systems that need the most energy are most impaired (heart, brain, muscles, lungs)
2) Ragged red fibers- Appear due to proliferation of large, diseased mt in myofibers/muscle fibers (in 1/3 diseases)
-in diseases with RRFs- ataxia, retinopathy, and dementia
3) Absence of cytochrome c oxidase (partly encoded by mtDNA) - needed for electron transport chain
4) Affect multiple systems (ones that need a lot of oxygen) e.g. heart, lungs, muscles, brain

43

What are the 4 assumptions for a population in equilibrium?

1) Random mating
2) No changes in population due to migration
3) No random fluctuations in allele frequency due to natural selection, genetic drift, etc.
4) No positive or negative selection- all genotypes reproduce equally well

44

What are the 5 factors that influence genetic variation (/shift allele frequency)?

1) Natural selection- heterozygotes have the advantage *
2) Genetic drift- allele frequencies change by chance alone, esp in small pop, due to sampling error - increases homozygosity since some alleles are wiped *
3) Founder effect- one rare allele at high frequency in a particular pop derived from 1 ancestor *
4) Pop bottleneck- some alleles at high frequency because of population constrictions *
*changes allele frequencies
5) Inbreeding- increases homozygosity frequency, can also happen bc of arranged marriage

45

What is the Hardy-Weinberg equation

For 2 alleles, A and B
p+q=1
Total number of A= p^2 + 2pq
Total number of B=q^2 + 2pq

46

What is the coefficient of inbreeding f

probability that a person with 2 identical genes received both from the same ancestor (e.g. grandmother with CF mutation passing on the disease to great-grandchild)
for first cousins- f=1/8, for their offspring, f=1/16

47

Define Bayesian terminology
prior prob
conditional prop
joint prob
posterior prob

Prior prob= Mendelian prob of carrier
Conditional prob=other information in family
Joint prob= prior x conditional
Posterior prob=ratio of joint prob of one outcome/sum of all joint probs

48

Define polymorphism and types of polymorphisms.
Define population stratification
Define heterozygote advantage

1) Differences in DNA sequence between individual
- mutation (e.g. end paragraph early), silent (eg spelling change), functional (eg replace . with ! punctuation)

Also defined as multiple phenotypes in same population (pop genetics)
2) nonrandom mating between groups causes different allele frequencies between subpopulations
eg. different HW probabilities of CF based on race/ethnicity
3) heterozygote has higher fitness than homozygotes e.g. sickle cell anemia
*maintain polymorphism when selecting for heterozygotes

49

Define heritability

=variance in dizygotic twins - variance in monozygotic twins / variance in dizygotic twins
1= trait due completely to heritability

50

Describe genetic risks for common multifactorial diseases and how you ID

Common variant- less individual, more population risk (higher RR)
ID through GWAS --> isolate DNA from thousands of individuals with disease---> ID SNP alleles co-inherited with disease
rare variant= mutation, less population, more individual
ID through deep genome sequencing

51

What was the ENCODE study and major findings?

1) Looking to see if the 97% of the genome that doesnt encode for protein had functionality
found that 80% of genome is involved in at least 1 biochemical event --> can be associated with disease

52

What is the epigenetic hypothesis?
What is the importance of proteomics/metabolomics in looking at disease?

1) Risk of common disease due to epimutations- changes in epigenetic signature that affects gene regulation and can lead to disease
-provide basis for variance in phenotypes among common diseases
-interface between environment and gene expression
-can look at methylation status of DNA through hybridization
2) Proteomics- looking at proteins
Metabolomics- different disease cells have different metabolic profiles (e.g. Warburg effect- cancer cells use high rate of glycolysis instead of ox phos)
can use these -omics analyses to target drugs

53

Causes of pathological hypoxia

1) Reduced 02 supply- high altitude or lung disease
2) localized ischemia (reduction in blood flow)
3) Abnormality of tumor microvessels

54

How does high altitude lead to hypoxia? What are symptoms of hypoxia at high altitude?

1) Air pressure drops as altitude increases
Air holds less molecules per area- lowers gas pressure
Lower number of 02 molecules per area--> lower 02 pressure
2) slower rxn time
learning/spacial memory impaired
MRI changes, hallucinations

55

What are the types of hypoxia inducible factors and where are they located?

Hif1, Hif2, Hif3
Hif1 and Hif2 are active as heterodimer- alpha is unstable cytosolic protein, beta is stable nuclear protein
need to go to nucleus and bind to beta subunit and then can activate transcription of hypoxia-inducible genes

56

Explain prolyl hydroxylation of Hif-1

Post-translational regulation
Prolyl hydroxylases (PDH 1-3) attach OH to Hif-1 --> need 02 and alpha-ketoglutarate
PDH active with Fe2+ cofactor, inactivated when oxidized to FE3+ (e.g. mt damage due to ROS)

57

How is Hif-1 degraded?

After hydroxylation, recognized by VHL (component of complex E3 ligase) --> ubiquinated and degraded

58

3 ways that Hif-1 can escape degradation?

1) Siah1/2 adds multi-UB chain to PDHs 1-3 and causes them to be degraded
2) VHL can also be ubiquinated and degraded
*also have mutations in VHL
3) VDU deconjugating enzyme removes the Ub chain from Hif-1
*VHL can ubiquinate Hif-1 OR VDU
Fastest way to activate hypoxia response is to prevent Hif-1 from degradation (rather than waiting for it to be transcribed)

59

How does hypoxia response intersect with inflammatory response pathways?

Hif-1 can activate the IkB kinase- which then phosphorylates the IkBalpha (which is then degraded)--> NFk can go into nucleus and activate transcription of inflammatory response (including making Hif-1)
*IkB kinase is needed for both inflammatory and hypoxia response --> need to coordinate mechanisms to achieve high levels of Hif-1 protein

60

What is the purpose of mir-210 in hypoxia response?

Transcriptional regulation
microRNAs that inhibit translation of normoxia genes during the hypoxia response --inhibits global protein synthesis

61

How is more Hif-1 protein synthesized when it induces the shutdown of global protein synthesis during hypoxia?

mTOR and S6-kinase promote preferential translation of mRNA encoding Hif1alpha
through PI3K MAPK pathway

62

What are the major effects mediated by Hif-1 during hypoxia?

1) Increased glycogen synthesis
2) Switch from ox phos--> glycolysis
3) Activates angiogenesis (new blood vessel devlpt)
4) Activates inflammatory response (by activating kinase)
5) alpha ketoglutarate produced, converted to citrate
6) mir-210 activated- inhibits normoxia expression

63

How does hypoxia induce increase in Hif-1 protein?

Reduced degradation (1) + increased gene expression (2)
1) Reduces function of prolyl hydroxylases (PHDs) which add OHs to proline of Hif-1alpha to target for degradation
2) prevents PHD inhibition of IkB kinase --> NFk factor activates transcription of HIf-1

64

What are the major post translational modifications of Hif1alpha?

1) proline hydroxylation --> required to be recognized by VHL for degradation
2) lysine acetylation--> promotes interaction with VHL--> degradation
3) Asparagine hydroxylation--> inhibits HIf1 interaction with transcription coactivator

65

What are the key features of trinucleotide repeat disorders?

1) Characteristics/consequences of expansion differ
2) Transmitting parent influences tendency for repeat expansion
3) if you have abnormal number of TNRs but no symptoms= pre-mutation
4) Display genetic anticipation-- phenotype occurs earlier and/or phenotype is more severe from generation to generation

66

Huntington's Disease
-mode of inheritance- parental preference for TNR?
-location of TNR expansion - how it correlates with phenotype
-clinical features of disease

1) Autosomal dominant
high tendency for repeat expansion during spermatogenesis-- high likelihood a man in premutation stage will pass on HD to his children (paternal expansion)
2) Expansion in first exon of Huntington protein --> Causes aggregation of glutamines --> misfolded protein --> neuron degeneration and toxicity
>39 CAG
3) chorea, dystonia, ataxia, bipolar disorders, cognitive impairment/dementia
(Alzheimers + Parkinsons + Bipolar)

67

How do you detect TNR disorders?

PCR- use primer in affected part of gene sequence
larger products implies more repeats- can estimate # repeats based on size

68

Fragile X syndrome
-mode of inheritance- parental preference for TNR?
-location of TNR expansion - how it correlates with phenotype
-clinical features of disease

1) X-linked
maternal expansions in oogenesis--> high likelihood a female in premutation stage will pass on to children (females more likely to be carriers, males more likely to be affected)
*most common inherited cause of intellectual disability
2) Expansion in promoter (5' UTR non coding region) of FMR1 gene
FMR1 protein is found in neurons- chaperones mRNA down axons where protein is translated- associated with maturation of neurons
3)
Premutation phenotype- tremor-ataxia syndrome (neurodegenerative), primary ovarian insufficiency (menopause before 40) *premutation phenotype bc transcription still happens--> insertions--> Excess RNA which sequesters normal RNA binding proteins needed for OTHER mRNA transcripts
Phenotype in affected males: low IQ, large testes, long narrow face + large ears
Phenotype in affected females: 50% of females with full mutation in ONE allele have intellectual impairment (phenotype associated with unbalanced X inactivation)
*in full mutation range- gene becomes methylated

69

Friedreich's Ataxia
-mode of inheritance- parental preference for TNR?
-location of TNR expansion - how it correlates with phenotype
-clinical features of disease

1) Autosomal recessive
2) Expansion in first intron (non-coding region) of frataxin/FRDA gene which encodes mitochondrial protein (degeneration of cells in myelinated neurons --> demyelination)
excess repeats --> long intron--> cant sit on exon and create spliceosome --> unspliced protein degraded
3) Frataxin high in heart/spinal cord--> Cardiomyopathy, ataxia (progressive limb weakenss/gait disturbance), loss of vibration senses and leg tendon reflexes

70

Myotonic dystrophy
-mode of inheritance- parental preference for TNR?
-location of TNR expansion - how it correlates with phenotype
-clinical features of disease

1) Autosomal dominant
both parents can transmit TNR expansion up to 1000 repeats
>1000 repeats- from mom ONLY (e.g. congenital MD)
2) expansion in 3' UTR of DMPK gene
RNA-mediated toxicity- Expansions cause hairpin loop in 3' UTR --> sequesters binding/splicing factors --> messes up transcription/translation for other nearby genes (this also happens in Fragile X syndrome)
3) Myotonia (inability to relax voluntary muscle after rigorous effort), muscular dystrophy (most common cause of adult onset), Type II diabetes, cardiomyopathy

71

Difference between pharmacogenetics and pharmacogenomics

Pharmacogenetics- how one particular drug interacts with the body e.g. inherited genetic differences that influence response to a drug
Pharmacogenomics- how genetic differences within populations explain different responses to a drug

72

Difference between pharmacokinetics and pharmacodynamics

Pharmacokinetics- the way your body processes a drug (clearance, metabolism, transport) --> what body does to drug
Pharmacodynamics- site of activity of drug and downstream effect --> what drug does to body

73

What are CYPs and role in drug metabolism?

CYP= cytochrome p450 oxidase, found in the liver and contain heme
have a flap which opens up- drug comes in and heme group chemically modifies it
break down metabolites/xenobiotics, activate drugs (e.g. codeine--> morphine), catalyze reaction (e.g. by accepting e-), increase solubility of drugs (e.g. adding hydroxyls)
substrates include xenobiotics, vitamins, fatty acids, sterols, signaling molecules (multiple possible drug/CYP interactions)

74

What is warfarin, how does it work, how does it interact with CYPs?

-Warfarin- blood thinner
-Inhibits Vitamin K reductase- enzyme in Vit K recycling that reduces oxidized Vitamin K --> reduces levels of active Vit K--> impairs synthesis of vit K dependent clotting factors
-CYPs hydroxylate warfarin and make it more soluble
*with warfarin overdose- administer Vit K to outcompete warfarin effect

75

What is the pharmacodynamic effect of warfarin? What two variants have highest effect on warfarin sensitivity

-Warfarin inhibits VKORC1 (Vit K reductase) and prevents it from reducing Vitamin K into active form
-variations in VKORC1 and CYP2C9 affect response- mutation can increase affinity of drug for VKORC1 target and make patient more sensitive (need lower doses)
OR mutations to CYP2C9 can make patient intermediate/poor metabolizer (need lower doses because drug is in the system longer before being broken down)

76

Define linkage disequilibrium

combinations of alleles/genetic markers that occur more or less frequently than expected at random

77

Plasma membrane
-purpose in the cell
-what is it composed of
-viewed with which microscope

-interface between cell and environment, permeability barrier
-PM= lipid bilayer
lipids are amphipathic, also have membrane proteins
-viewed on EM (LM shows both plasma membranes + extracellular matrix)

78

What is spectrin and what is its role/significance?

Spectrin- cytoskeletal protein that is attaches to membrane proteins, stabilizes biconcave shape of normal RBCs
with mutation--> RBCs more spherical, more sensitive to hypertonic--> degraded in spleen

79

What are the major functions of membrane proteins?

1) Linker/anchor molecules e.g. binding with spectrin
2) Transporter molecules
3) Receptors
4) Enzymes

80

Microvilli
-purpose in the cell
-what is it composed of
-viewed with which microscope

-evaginations of apical cell membrane- increase absorptive surface for absorption, secretion, adhesion
-composed of a core actin filaments as well as myosin Ia motor protein and calmodulin --> ends in a terminal web of actin minus ends + spectrin + myosin II
-have a glyocalyx at the surface
-viewed on EM, seen as a brush border under LM

81

What is the glycocalyx?

-glycocalyx- fuzzy coat on tops of microvilli
-consists of sugar side chains attached to glycoproteins, glycolipids, and proteoglycans (sugar added to membrane proteins/lipids during biosynthesis in the RER) --> can be used for binding, adhesion, protection

82

Junctional complexes
-purpose in the cell
-viewed with which microscope

Provide contact between neighboring cells- promote adhesion and prevent passage of molecules
IDed on LM by small reddish dots - terminal bars; also IDed on EM

83

What is the nuclear lamina?

network of intermediate filament proteins that provide support to the nuclear envelope

84

What is the nucleolus? What can you see under EM?

site of synthesis of rRNA
can see lighter fibrillar centers (DNA between rRNA genes, has RNA pol I for transcription)
granular region- ribosome assembly

85

What are the main components of cyotosol?

1) Cytoplasm
2) free ribosomes/polysomes (in active translation)
3) Inclusions e.g. glycogen, lipid (dont have membranes, nonliving components), secretory vesicles (do have membranes)
4) Cytoskeleton i.e. actin, intermediate, microtubules (cannot see under LM)

86

Smooth ER
-function in the cell
-viewed with which microscope

-lipid and steroid biosynthesis, detoxification, metabolism, calcium storage
-view with EM
*lipids have no staining- washed out

87

Rough ER
-function in the cell
-viewed with which microscope
-why is there basophilic staining

-studded with ribosomes for protein synthesis (membrane, lyosomal, secretory)
-view with EM, continuous with nuclear envelope
*lots of RER in secretory cells
-basophilic dark staining bc (-) rRNA on ribosomes binds (+) dyes

88

Golgi apparatus
-function in the cell
-viewed with which microscope
-why are vesicles needed

-flattened sacs- polarity because proteins enter at cis face (more RER there) and leave at trans face (more vesicles there)
-view on EM, can see on LM only through silver stain
-need vesicles to transport material b/w golgi cisternae

89

Difference between constitutive and regulated secretion?

Constitutive- continual secretion of proteins e.g. plasma cells
regulated secretion- initiated by external stimulus, lots of storage and secretory granules

90

What is receptor mediated endocytosis?

How ligand-receptor complex can enter the cell:
associate with coat proteins to form coated pit--> coated vesicle--> fuse with early endosome --> lysosome

91

Lysosome
-function in the cell
-viewed with which microscope

-site of intracellular digestion
-hard to ID even at EM (cant see on LM) but if you use trypan blue can see places where dye accumulated in lysosomes
ONE exception is eosinophil (white blood cell) - lysosomes have crystalline inclusions which can be seen on EM

92

Peroxisomes
-function in the cell
-viewed with which microscope

-hydrogen peroxide metabolism, high enzyme concentrations- also have crystalline inclusions (like lysosomes)
-hard to ID even at EM (like lysosomes)

93

How do cells interact with their environment?

Secrete and respond to signals
reversible (motile/change shape)
irreversible (divide/differentiate/die)

94

What are ligands? What are key features?

ligand- triggers signal by binding to receptor
-agonist or antagonist to receptor
-lock/key fit
-receptor not active forever
-receptors typically inactive until bound

95

What are the 4 major classes of receptors?

1) Ion channels
2) Steroid hormone receptors
3) Protein kinase receptors
4) 7-alpha-helix receptors

96

Ion channels
-what are they
-where are they
-example: ligand-gated
-related diseases and example

-pore-forming proteins that allow flow of ions down electrochemical gradient (do not need energy)
-can be anywhere there is a lipid bilayer- cell surface, organelles
-ligand-gated: binding of ligand triggers flow of ions across membrane - ligands are neurotransmitters (for nerve cells and muscle contraction)
-mutations can cause loss or gain of function e.g. cystic fibrosis caused by loss of function mutation in CFTR chloride channel, tetrodotoxin (pufferfish) blocks Na+ channel

97

Nuclear steroid hormone receptors
-what are they
-where are they
-example: estrogen
-related diseases and example

-steroid hormones control gene expression by binding to and activating hormone receptors in nucleus or cytoplasm --> receptor binds to response element on DNA--> activation domain triggers transcription factors
-anywhere in cell- hydrophobic so can cross cell membrane
-estrogen: estrogen binds to receptor, it dissociates from chaperone and dimerizes, enters nucleus to bind response element to activate transcription
-estrogen receptors overactive in majority of breast cancer; tamoxifen is competitive antagonist to estrogen and represses transcription

98

Protein kinase receptors
-what are they
-where are they
-example: ras
-related diseases and example

-receptors that induce signal transduction cascade; dimerize when ligand binds- the cytosplasmic kinase domains phosphorylate and activate
-3 domains: 1) extracellular (binds ligand); 2) transmembrane; 3) cytoplasmic (kinase activity or binds PK protein)
-Ras is a small G protein that targets gene transcription in the nucleus (proteins in cell division) by stimulating a MAPK phosphorylation cascade
-ras one of most mutated proteins in cancers- we know the 2 mutations, they destroy intrinsic GAP function of ras so it is always active

99

Define:
Grb
GEF
SoS
G-protein
GAP

Grb- G protein receptor binding--> binds to phosphorylated protein kinase receptors
GEF- activates G proteins through GDP--> GTP
SoS- GEF that binds to Grb and activates ras
G-protein- binds guanine nucleotides (e.g. GTP,GDP) and acts as molecular switch during signaling
GAP- inactivates G protein through GTP--> GDP

100

7 alpha helix receptors
-what are they
-where are they
-example: beta adrenergic receptor
-related diseases and example

-most abundant- detect odor, light, taste, hormones, neurotransmitters
-coupled to trimeric large G proteins; when ligand binds, receptor is a GEF and activates G protein --> alpha and beta/gamma subunits dissociate
-3 classes of Galpha: Gsalpha (activates adenylate cyclase--> PKA --> Ca2+), Gialpha (inhibits adenylate cyclase--> PKA), Gqalpha (activates PLC--> PKC--> Ca2+ influx from SER)
-beta adrenergic receptor desensitization- bound by epinephrine and norephinephrine --> this complex is phosphorylated by BARK--> bound by Beta-arrestin--> blocks interaction with trimeric G protein

101

What is calmodulin?

Ca2+ binding protein
Active when 4 Ca2+ bound, in turn activates CAMK by binding to inhibitory domain and allowing active site to be accessible for kinase activity
CAMK is important -- linked to learning, memory, and Alzheimer's

102

How does tamoxifen work?

competitive antagonist for estrogen- binds to estrogen receptor to prevent estrogen from binding
goes into nucleus and activates genes that inhibit target genes (estrogen activates them)

103

What are the 4 classes of membrane lipids and major features

1) phosphoglycerides
-kink in 1 of 2 fatty acid chains - increases fluidity
-polar head group
-phosphotidyl serine normally on inner leaflets
2) sphingolipids
-derived from sphingosine (amino alcohol)
-sphingolipids normally on outer leaflets
3) glycolipids
-also derived from sphingosine- but sugars added (sugars on external face only)
-primarily in myelin + PM outer leaflet (not internal membranes)
*cholera binds to glycolipid
4) sterols
-rigid ring structures to provide shape, in both leaflets (cholesterol has polar OH near surface)
plasma >> internal membrane
*lipids spontaneously assemble into lipid bilayer--> liposome

104

Key features of plasma membrane lipids

1) asymmetric distribution between membrane leaflets (though cholesterol roughly equal)
2) lipids can flip within leaflet but not between
3) lipid raft- non random lipid distribution, mostly cholesterol and sphingomyelin that sequesters membrane proteins

105

Key features of membrane proteins:
1) Types
2) What happens if lipid bilayer is separated
3) How to molecules pass through

1) Types:
integral membrane- incorporated into membrane with hydrophobic AA and traverse whole width, OR lipid tail covalently attached
peripheral membrane- on surface through non-covalent bonds, usually electrostatic interactions
2) if lipid bilayer is separated-- integral protein will associate with one leaflet or the other (more on cytosplasmic leaflet --> add'l interactions w/ cytoplasmic proteins, more on internal face--> attached to cytoskeleton)
3) hydrophobic molecules can pass through the PM, others have to use transporters

106

Describe the three types of membrane transporters and give examples

1) Ion channel - ion specific pores that allow ions to flow down the concentration gradient - fastest but least specific, passive (doesnt require energy) *neurotransmission
2) Carriers- enzyme-like that mediate transport down the concentration gradient- intermediate specificity and speed, passive (no energy)
-uniport- one transported molecule at a time (random switches whether or not solute is bound)
-symport- one molecule co-transported against gradient into cell e.g. Na-Glucose symporter brings glucose into cell- cooperative binding, need both bound for conformational change (glucose leaves cell through uniporter)
-antiport- one molecule into cell, one co-transported against gradient out of cell --> free energy drives 2nd transport
3) Pump - three types - need energy to move molecules against concentration gradient
-P-type- kinase pumps that transport ions
e.g. Ca2+ in sarcoplasmic reticulum of muscles- Ca2+ binds when nonphosphorylated--> ATP binds and ATP hydrolysis phosphorylates AA--> conformational change --> Ca2+ enters cell
e.g. Na+/K+ pump in intestinal epithelial cells (lot of energy bc both Na and K against concentration gradients) Na+ out, K+ in -- need to keep intracellular Na+ low because glucose gets into the cell through the Na+ symporter
-ABC type- transports small molecules e.g. anti-cancer drugs through ATP hydrolysis
bacteria- import
eukaryotes- export
molecule binds non-ATP state, ATP binds and dimerizes--> conformational change--> ATP hydrolysis releases substrate

107

How does multiple drug resistance work? (esp in cancer therapy)

Cancer cells induce expression of ABC transporter to high levels- they bind the cancer drug and released from the cell --> drug effect reduced
40% of patients in chemo

108

Cytoskeleton: Intermediate filaments
1) what are they
2) purpose in cell
3) where are they in cell
4) structure

1) component of cytoskeleton, nuclear boundary, organizes cell architecture e.g. keratin, neurofilaments (axons)
2) stress absorber (protection from stress), cell migration and movement, mechanical support/structure, also signaling
3) surround nucleus, extend to cell periphery, also junctions (cell-cell, cell-connective tissue)
4) no motor (structure is NOT polar), formed of coiled coil dimers that form filaments

109

Cytoskeleton: Actin
1) what are they
2) purpose in cell
3) where are they in cell
4) structure
5) binding proteins

1) globular protein- most abundant protein in cells, thinnest ones
2) movement
3) in all cells, present throughout cell, most highly concentrated near plasma membrane, relocates to contractile ring in middle of cell when division takes place
4) polar, polymers of actin protein (bound to ADP/ATP) that form helical filaments
faster growth at + end, slower at - end (stabilized by ADP-actin)
regulation does not happen at actin filament itself
5) myosin and tropomyosin

110

Similarities between actin and microtubules

1) formed from globular proteins - nucleation and polymerization
2) + end is preferred addition end --> confers polarity
3) ATP/GTP cap at + end
4) nucleotide at + end determines stability
5) can regulate through binding proteins
6) polar

111

Cytoskeleton: Microtubules
1) what are they
2) purpose in cell
3) where are they in cell
4) structure
5) binding proteins
6) toxins

1) filamentous intracellular cytoskeleton component, thickest ones, made of tubulin
2) vesicular/organelle transport, centrosome forms mitotic spindle during division, cilia/flagella + basal bodies
3) throughout cytoplasm
4) polar, polymers or alpha/beta tubulin arranged in tubes to form 13 protofilaments--> microtubules
beta tubulin binds GTP on + end
5) bind to side or sever and can stabilize/destabilize +/- end e.g tao (role in Alzheimer's), +tip protein (stays with + end to track its progress, can also carry cargo to actin)
6) colchicine- depolymerizes microtubule
taxol- stabilizes microtubule

112

Cilia
1) types
2) purpose
3) where are they
4) structure

1) primary- one per cell, non-motile
motile- dynein motor - rhythmic beating motion
2) primary- sensory, developmental signaling
motile- keep airways free of dirt, propel sperm
3) primary- apical surface of cells
motile- apical surface in lungs, respiratory tract, middle ear
4) made of microtubules
primary- 9 doublets in ring
motile- add'l doublet in the middle (9+2)

113

What is the centrosome?

microtubule organizing center, only in animal cells
gamma tubulin ring complex nucleates microtubules and caps the - ends
centriole + ends oriented away from the nucleus
regulate cell cycle progression

114

What is dynamic instability?

+ ends of microtubules transition between growth and shrinking
shifts between catastrophe when GTP cap comes off + end to rescue where it is put back on

115

How does a cell migrate?

Polymerization or motor driven
1) leading edge through actin polymerization (protruding)
*this alone can drive cell movement e.g. Arp2/3 that nucleates actin filaments from the side (Listeria bacteria hijacks Arp2/3 and polymerizes actin to create actin tails and create infection)
2) Motor- tail follows through actin-myosin interaction (contracting)
e.g. how neutrophils migrate to site of infection

116

What do all cytoskeletal motors have in common?

1) All ATPases (mechanochemical enzymes)
2) have different isoforms- move in a particular direction
3) vesicles can move on microtubule/actin - have many different types of motors
4) motors are downstream targets of signaling cascades

117

Cytoskeletal motor: Myosin

-actin motor, most are + end
-big gene family- lots of classes
-6 chains: 2 head chains, 2 light chains bound to head, 2 heavy chains e.g. alpha helices in a coiled coil
-classified based on the head (where ATPase is)
-tail domain is variable- can bind cargo, form filaments, etc.

118

Cytoskeletal motor: Kinesin

-microtubule motor; head domain related to myosin and G proteins
-moves - to + end
-large gene family
-structure: head domain (ATPase), light chains bound to tail, stalk region (can dimerize), tail binds to targets

119

Cytoskeletal motor: Dynein

-very large microtubule motor
-moves + to - end
-unrelated structurally to myosin/kinesin
-only motor in cilia/flagella
-small family of proteins
-structure: 6 AAA domains contain ATPase, connected to head domain through a stalk, tail binds cargo
dynactin protein helps it bind to microtubules

120

Explain myosin movement mechanism and how it is regulated

1) Rigor mortis- no ATP bound to actin + myosin
2) when ATP binds- actin + myosin break part --> conformational change
3) ATP hydrolysis leads to weak rebinding
4) P released leads to strong rebinding --> power stroke
*this is the rate limiting step
*movement speed determined by myosin ATPase
Regulated at RLS of P release

121

Explain kinesin movement mechanism and similarities/differences with myosin

1) Trailing head has ADP, weakly on microtubule; nothing on leading head
2) ATP binds leading head, trailing head rotates forward
3) ATP hydrolysis on the new trailing head, new leading head binds microtubule and ADP leaves
4) P dissociates trailing head and weakens binding to microtubule
2) Same concept as myosin movement- ATP binding weakens, release of ATP hydrolysis P is the power stroke
-Difference: kinesin has 2 heads and is processive (doesn't fall off)

122

What is PCD or Kartagener syndrome?

Immotile cilia because dynein stays in nucleus and doesn't go out to cilia -- respiratory tract infection + males sterile

123

How are nuclear proteins trafficked to nucleus?

1) specific AA signal sequence recognized by importin
2) complex goes through nuclear pore
3) RanGTP binds to importin, nuclear protein disassociates
4) RanGTP+importin goes back to cytosoplasm, GAP hydrolyzes to RanGDP and importin is released
*small proteins can diffuse through membrane
*GEF for Ran only in the nucleus

124

How is protein trafficked to peroxisome? What is the associated disease?

1) Protein translated in cytosol
2) peroxisome membrane receptor recognizes specific AA signal sequences
3) Catalase in peroxisome H202--> H20; heme is added to prevent protein moving back across membrane
4) Zellweger syndrome- lethal! enzymes proteins are not recognize and remain in cytoplasm --> degraded--> nonfunctional peroxisomes

125

How is secretory protein trafficked to ER?

*Co-translational - for secretory, PM, golgi, lysosomal proteins
1) Translation begins at free cytoplasmic ribosome
2) AA signal on N terminus of protein - SRP binds at halts translation
3) complex attaches to SRP receptor on rough ER membrane
4) GTP hydrolysis on SRP / receptor --> conformational change--> SRP and SRP receptor leave
5) translation continues - nascent protein translocated into ER lumen

126

How are proteins modified for secretory pathway? How does glycosylation work?

1) signal peptide removed
2) hyroxylation of lys, pro
3) disulfide bonds
4) chaperones for folding
5) glycosylation - e.g. 9 mannose oligosaccharide added to Asn - modified throughout the pathway

127

What components are required for vesicular transport?

1) Coat protein to form invagination
-Clathrin- from PM--> early endosome, from TGN--> late endosome and vice versa
-COPI- backwards within Golgi via KDEL sequence (need Arf-GTP for COPI to bind to membrane)
-COP II- from ER--> cis-Golgi (need Sar1-GTP for COPII to bind to membrane)
*proteins dont go back to the ER once they have been post-translationally modified
2) v/t SNAREs - lock/key combo for vesicle/target recognition/fusion (mediated by Rab-GTP)
*Botulinum toxin cleaves SNAREs in neurotransmitters

128

What is the TGN? How are lysosomal proteins marked and what are related diseases?

1) TGN- Trans-golgi network- sorting station at trans face of Golgi --> lysosome, constitutive secretion, OR regulatory vesicle
2) phosphate is added to 6-mannose on oligosaccharide on glycosylated protein in cis-face
recognized by MPR on trans face
sent in clathrin vesicle to late endosome
in endosome- low pH so protein and MPR disassociate, MSP removed
3) Human I-cell disease- enzyme mutation so there is no M6P added to lysosomal protein- not recognized to be sent to lysosome so they are ALL secreted

129

How does Legionnaire's disease work?

bacteria evades delivery to lysosome for destruction by finding a way to bind phagosome to normal transport vesicle

130

Endocytosis:
purpose
mechanism e.g. cholesterol
related disease

1) receptor recycled back to PM, receptor sent to lysosome ("receptor down-regulation"), OR antibodies sent to basolateral membrane for transcytosis
2) free LDL binds receptor on PM --> LDL + receptor endocytosed into clathrin vesicle --> uncoated--> sent to early endosome for sorting and disassociated in the low pH--> LDL sent to lysosome --> receptor recycled or sent to lysosome
3) Hypercholesterolemia- LDL receptor is mutated and missing the tail which binds to adaptin protein that helps form clathrin-coated pit --> LDL not internalized --> accumulates in blood and causes artherosclerosis and myocardial infarction

131

What are GTP binding proteins? examples for vesicular transport?

1) GDP bound- inactive
GEF- promotes release of GDP
GTP bound- active, conformational change
GAP- increases inherent GTPase activity for GTP--> GDP
2) Ran (nuclear trafficking)
Arf (for COP1) and Sar1 (for COPII) for transport vesicle formation
Rab (for transport vesicle recognition)

132

Epithelium:
1) what is it
2) purpose
3) key characteristics

1) Epithelium- line internal and external surfaces of blood vessels and organs
2) Protection (skin)
selective permeability barrier (blood vessels)
absorption (small intestines)
secretion (glands)
excretion (kidney tubules)
sensory reception (eye, ear, tongue, nose)
reproduction (germ cells)
transport via cilia modification
3) Characteristics:
-structural polarity
-avascular (need diffusion)
-lots of cells, little intercellular space
-regenerative capacity

133

Simple squamous epithelium:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) Endothelium (blood vessels), endocardium (lining of ventricles/atria), mesothelium (lining of walls/closed cavities e.g. spleen)
2) blood/lymphatic vessels (vein/capillary/artery), alveoli, kidney (loop of henle/bowman's capsule), inner/middle ear, pleural cavities in lungs
3) gas exchange, fluid transport, lubrication
4) One layer of flattened cells and flattened nuclei

134

Simple cuboidal epithelium:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) N/A
2) gland ducts, ovary coverings, kidney tubules
3) secretion, absorption, protection
4) one layer of cubed cells with rounded nuclei in the middle of cell

135

Simple columnar epithelium:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) N/A
2) nasal sinuses, most of digestive tract, large glandular ducts, uterus, testis, etc
3) absorption, secretion, protection, transportation (if it has modification)
3) long rectangular shaped cell with oval nuclei towards basal side

136

Goblet cell:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) N/A
2) digestive and respiratory epithelial lining
3) lubricate luminal surface- filled with mucus droplets/glycoproteins that are secreted
4) wine glass shaped, near apical surface, washed out

137

Stratified squamous epithelium:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) N/A
2) mouth, esophagus, vocal chords, vagina, epidermis, anal canal
3) protection, prevent dehydration
4) cell layer closest to lumen is nucleated (flat nuclei), many cell layers
no cilia/stereocilia
*keratinized (no nuclei) top layer in skin - white is sloughing of epithelial cells, fingers of connective tissue jutting in to provide nourishment

138

Stratified cuboidal epithelium:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) N/A
2) ducts of sweat glands
3) protection, secretion, absorption
4) two layers- surface layer cuboidal
no cilia/stereocilia

139

Stratified columnar epithelium:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) N.A
2) eye conjunctiva, large excretory ducts, salivary glands, mammary glands
3) secretion, protection
4) two layers- surface layer columnar
no cilia/stereocilia

140

Pseudostratified epithelium:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) N/A
2) urethra, nasal cavity, epididymus, bronchi, trachea
3) secretion, absorption, lubrication, protection, transportation via cilia
4) all cells touch basement membrane but not all touch luminal, nuclei not in a straight line but none near apical area
has cilia or stereocilia

141

Transitional epithelium:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it

1) N/A
2) lines urinary tract, renal calyx
3) protection, can be stretched (invaginations that fold out to increase surface area when bladder is full)
4) stratified, top layer is domed/flat but top layer of nuclei is always rounded

142

Difference between microvilli, cilia, stereocilia

Microvilli- made of actin, dense brush brother, base is terminal web
Cilia- made of microtubules, more airy, base is basal bodies
Stereocilia- made of actin, spaced out finger-like projections *not involved in movement

143

Difference between exocrine and endocrine glands?

*all glands = epithelial
Exocrine- secrete via duct
Endocrine- secret directly into blood stream via fenestrated capillaries (with pores) /lymphatic vessels

144

Tight junctions:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it
5) structure
6) cytoskeleton

1) Zona occludens
2) top of lateral wall towards apical domain- circle around entire cell *only in epithelium
3) prevent things from environment getting into underlying tissue- want to stop leakage early
4) looks like stitches - kissing points
5) occludin and claudin loops on both sides bring the two membranes together
6) Actin

145

Belt desmosomes:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it
5) structure
6) cytoskeleton

1) Zonula adherens, Adherens junctions
2) close to apical side- circle around entire cell
3) initial adhesion between two cells
4) beneath tight junctions
5) E-cadherins are the main adhesion points- dimers from trans interactions
Need Ca2+ (linked to actin cytoskeleton through catenins)
Loss of E-cadherin during cancer--> cells dont adhere, instead become mesenchymal and migrate/invade
6) Actin

146

Spot desmosomes:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it
5) structure
6) cytoskeleton

1) Macula adherens
2) beneath belt desmosomes- in spots around cell
3) provide mechanical strength to hold the tissue together
4) looks like rivets- very dense regions due to plakin
5) Cadherins are main adhesion points (desmocollins + desmogleins), connected to intermediate filaments through plakins
6) Intermediate filaments

147

Focal adhesions:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it
5) structure
6) cytoskeleton

1) N/A
2) basal domain
3) important for stability of hemidesmosomes, growth, survival, cell migration
4) can't see under EM- need special immuno stain
5) integrins (alpha/beta subunits) linked to actin cytoskeleton through vinculin/talin, bind to extracellular matrix proteins through RGD peptide sequence
6) Actin

148

Hemidesmosomes:
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it
5) structure
6) cytoskeleton

1) N/A
2) basal domain
3) mediate interaction between cells and basement membrane
4) alpha6beta4 integrin is the adhesion protein, connected to intermediate filaments through plaque proteins (plectin, BPAG1)
5) rivets between cell and matrix
6) Intermediate filaments

149

Gap junctions
1) what else is it called
2) where is it found
3) what is its purpose
4) how do you recognize it
5) structure
6) cytoskeleton

1) communicating junctions
2) cardiac/smooth muscle cells, epithelial cells
3) allow small molecules e.g. cAMP, Ca2+ to pass through cells quickly so they can coordinate a response e.g. contraction
4) two cell membranes are close together- extensive structure
5) hydrophilic intracellular connexon channels formed through 6 connexin proteins- can be open or closed regulated by pH, voltage, [Ca2+]
6) NONE

150

Diseases associated with junctional defects:
1) Tight junction- Claudin 16 mutation
2) Belt desmosomes- P-cadherin mutation
3) Gap junctions- connexin mutation
4) Spot desmosome- Desmoglein autoimmune
5) Hemidesmosome- BPAG1 autoimmune

1) Tight junction/ Claudin 16 - low Mg in blood and high Ca2+ in urine, calcified kidneys
If the tight junctions are leaky, then Mg/Ca2+ can leak out
2) Belt desmosomes/ P cadherin mutation- P cadherins in hair follicles and retina --> hair loss and retinal degeneration
3) Gap junctions/connexin mutation- associated with hearing loss
4) Spot/desmoglein- pemphigus, causes splitting/vesicles within epidermal cell layers
5) Hemi/BPAG1- pemphigoid, causes splitting/vesicles between epidermis and basement membrane

151

Leukocyte extravasation:
1)what are selectins
2) what are IG-CAMS
3) describe mechanism

*Cells need dynamic adhesion during locomotion (non-junctional adhesion)
1) Selectin- Ca2+ dependent transmembrane protein that binds oligosaccharides, found on lymphocytes (T/B cells), epithelial cells, platelets
2) IG-CAMs- cell adhesion molecule (CAM) with immunoglobin-like domain; ICAM and VCAM
3) P-selectins on endothelium form loose adhesion with glyocproteins on leukocyte --> leukocytes start rolling
-beta2 integrins on leukocyte surface are activated, bind strongly with ICAM and VCAM on endothelium --> leukocytes stop
-integrins mediate leukocyte migration through the junction of two endothelial cells into the tissue

152

Define for ECM:
1) ground substance
2) connective tissue
3) parenchyma
4) stroma

1) Ground substance: gel like substance in ECM
-water and ions
-structural proteins e.g. collagen, elastin
-gylcosaminogylcans
-proteoglycans
-glycoproteins e.g. fibronectin, lamins
2) Ground substance + other cells (e.g. macrophages, fibroblasts)
3) Parenchyma- specialized epithelium with particular function
4) Stroma= connective tissue, supportive framework beneath basement membrane composed of dense, irregular connective tissue (few cells, lots of extracellular matrix)
*size varies between tissues (lot in bone, little in liver)

153

Basement membrane:
1) difference between BM and basal lamina
2) structure

1) Termed basement membrane when viewed on LM, termed basal lamina when viewed on EM
2) first lamina lucida- laminin 5 and collagen type XVII
then lamina densa- laminin I (glycoprotein), type IV collagen, perlecan (proteoglycan)
then lamina reticularis- fibroblasts, type III collagen

154

Collagen:
1) purpose
2) structure
3) Biosynthesis and assembly

1) Purpose- impart strength
2) Structure:
-triple stranded alpha helix
-sequence: Gly-X-Y *single Gly mutation can alter structure
3) Synthesis starts in cytoplasm- SRP binds N-terminus AA signal and brings ribosome to RER
- signal peptide is removed- alpha chain translated
- Hydroxylase + Vitamin C add OHs to proline, lysine (*Scurvy)
- Lys can be glycoslyated
- alpha chains form triple helix through H bonds
-secreted out of the cell through vesicle
-extension propeptides at N and C termini cleaved--> tropocollagen self-assembles into fibrils and cross-links at the hydroxylated Pro, Lys--> forms collagen fiber
*overlap of chains leads to banding pattern in the EM

155

Describe the types of collagen and where they are found

1) Fibril-forming
Type I- 90% of all collagen; in bone, lung, tendons, cornea, internal organs, and skin (osteogenesis imperfecta)
Type II- cartilage
Type III- in skin, blood vessels, and internal organs (liver, kidney, spleen); can be stained silver bc they have more sugar, thinner, branched - aka reticular fibers (Ehlers-Danlos)
2) Fibril-associated
Type IX- cartilage (outside of Type II)- important for joint integrity (leads to arthritis)
3) Non-fibrillar/network forming
Type IV- basal lamina collagen, no N/C termini cleavage so they interact and form "chicken wire" array
Type VII- anchoring at N-terminus at basement membrane below lamina densa (otherwise you have blistering)
4) Transmembrane
Type XVII- connects epithelium to BM (otherwise you have blistering), above lamina densa

156

Elastin:
1) purpose
2) where is it found
3) structure
4) Related disease

1) resilience- stretching and recoil of connective tissue
2) in aorta, skin, lung
3) elastic fibers surrounded by microfibrils composed of fibrillin glycoprotein
4) Marfan syndrome- long limbs and aorta can rupture (mutation in fibrillin--> increased TGFb --> too much growth)

157

GAG:
1) purpose
2) where is it found
3) structure
4) types

1) lubricant, shock absorber
2) viterous humour in eye, synovial fluid in the joints
3) long chains of repeating disaccharides, negatively charged and polar so binds water
4) hyaluronic acid- unique bc not sulfated or attached to protein (found in joints)

158

Proteoglycan:
1) what is it
2) types
3) where is it found
4) purpose

1) core protein with covalently attached GAG
2,3,4) Aggrecan- in the cartilage with collagens type II and IX- forms large aggregates with hyaluronic acid (non covalently attached)- mechanical support
Perlecan- in the basal lamina- structural and filtering
Syndecan- fibroblasts and epithelial surface - core protein has transmembrane domain + heparan sulfate GAG- coreceptors for signaling molecules e.g. growth factors

159

Glycoproteins:
1) types
2) structure
3) purpose

1) Laminin - component of basal lamina - multiple different binding sites for collagen, RGD site for integrins, perlecan - three polypeptide chains, looks like a cross
laminin 1- lamina densa with type IV collagen and perlecan
laminin 5- with collagen type XVII in anchoring filaments
2) Fibronectin - binds extracellular molecules including integrin, collagen, syndecan, heparin, etc. - dimer with disulfide bonds
-can be soluble form for wound healing, can be attached to cell surface, or can form insoluble matrix in stroma
*low levels in tumor cells- uncontrolled growth

160

Integrin:
1) structure
2) what do they do
3) types
4) how are they regulated

1) Dimer of alpha/beta subunits
2) involved in leukocyte extrasavation, focal adhesion formation, platelet binding to fibrinogen, bind RGD sequence in ECM
integrate intra/extracellular signals and link cytoskeleton with ECM--> beta subunit binds to actin cytoskeleton through actin-binding proteins and initiate signaling pathways through focal adhesion kinase
3) B1- extracellular matrix
B2- endothelial surface of WBCs (leukocyte extravasation)
B3- platelets, bind fibronigen for clots
B4- hemidesmosomes
4) independent signaling pathway - intracellular signaling cascade interacts with integrin to activate e.g. in leukocyte extravasation when selectins bind to leukocyte

161

Cell cycle:
1) what is interphase?
2) What happens during G1 and G2?
3) What/when is the restriction point?
4) What is G0?

1) Interphase = G1 + S + G2
2) During G1 and G2, cell accumulates mass and monitors internal/external conditions
3) At the end of G1, cell examines conditions to see if its favorable to continue; committed step
4) If conditions are not favorable, cell goes into G0 and hangs out until they are e.g. oocyte in ovarian devlpt

162

Important features of cell cycle control:

Events happen at a specific time, in a specific order, only 1x, through an irreversible molecular on/off switch

163

What are cyclins and how do they function?
What are Cdks and how do they function?
What are the 4 types of Cdks and what is their purpose?

1) Proteins that control progression of cells through the cell cycle by activating Cdk kinase activity
different types of cyclins for the different phases- degraded after their role is done
2) Kinases that phosphorylate downstream proteins to control cell cycle; need cyclin to be active
3) G1 Cdk- passage through restriction point
G1/S Cdk- commits cells to replication
S Cdk- initiates replication
M Cdk- promotes mitosis

164

What is the origin of replication? What is the origin replication complex?

AA sequence where replication is initiated
ORC- large multi-protein complex that binds (melts the DNA strands open) -- acts as a landing pad for other regulatory proteins

165

S-phase cell cycle control:
1) How is entry into S phase regulated?
2) How is re-replication prevented?
3) How does control system reset itself?

1) Cdc6 protein increases concentration at start of G1
-binds to ORC and recruits mcm proteins to the complex--> forming the pre-replicative complex (pre-RC) by end of G1
-active S-Cdk (Cdk + S-cyclin) triggers S-phase by phosphorylating pre-RC, recruiting DNA pol and other proteins to the ORC, and activating mcm proteins to act as helicases
2) Re-replication prevention: both S-Cdk and M-Cdk phosphorylate Cdc6 --> dissociates from pre-RC which is disassembled --> Cdc6 degraded
S-Cdk and M-Cdk also phosphorylate Mcm proteins--> they are exported from nucleus
3) S-Cdk activity high during G2 and mitosis
BUT resets at end of mitosis--> Cdc6 and Mcm proteins are dephosphorylated and active again

166

M-phase cell cycle control:
1) How is entry into mitosis regulated?
2) What are the feedback loops
3) How does control system reset itself?

1) M-cyclin partially activates M-Cdk through phosphorylation
-CAK adds an activating phosphate
-Wee1 adds an inhibitory phosphate that is removed by cdc25 in order activate M-Cdk
-active M-Cdk phosphorylates downstream proteins for spindle assembly, chromosome concentration, nuclear envelope breakdown, etc.
2) 2 positive feedback loops:
Active M-Cdk phosphorylates and activates more Cdc25
Active M-Cdk inhibits Wee1
3) Ub ligase is activated by M-Cdk, it attaches a Ub chain to the M-cyclin which is degraded; now M-Cdk no longer exists (is just CDK)

167

G1 phase cell cycle control:
1) What is in place to ensure absence of Cdk activity?
2) How does the cell pass through restriction point?
3) What are the feedback loops in place?

1) CKI production increased during G1; CKI bind and inactive cyclin-Cdk complex
Rb binds and inactivates E2F transcription factor
Ub mediated degradation of Cdks
2) With extracellular signal, G1-Cdk becomes active and phosphorylates Rb --> reduces affinity of Rb for E2F--> E2F induces transcription of G1/S Cdk and S cyclins
3) E2F autoregulates and increases its own expression
E2F leads to production of G1/S Cdk and S Cdk --> phosphorylates more Rb--> releases more E2F
G1/S Cdk and S Cdk also inactivate ubiquitin ligases and CKIs--> accumulation of G1/S Cdk and S Cdk

168

What are the 2 DNA checkpoints during the cell cycle?

1) late G2: damaged DNA sends a signal that blocks cdc25 activity - so M-Cdk inhibitor phosphate is not removed and it is not activated --> mitosis doesn't happen
2) late G1: DNA damage activates p53--> activates CKIs --> inhibit cyclin/Cdk complex --> inhibits G1/S and S Cdks --> cell doesnt leave G1 and enter S phase

169

Apoptosis:
1) define
2) functions
3) morphological markers
4) biochemical markers

1) programmed cell death (suicide) *high Ca2+ signals apoptosis
2) tissue sculpting, eliminating used B/T cells, tissue homeostasis, removing damaged cells
3) intact cell membrane, chromatin condensation, large clear vacuoles, apoptotic bodies (filled with DNA, show up bright on fluorescence), cell shrinkage, and blebs (shriveled cells since actin cytoskeleton degrades)
4) Phosphatidyl serine flipping (flips to outer leaflet under apoptosis)-- will see green markers on outside of cell
Flow cytometry- quantitive method, can track % affected cells
TUNEL assay- fluorescent dUTP binds with nicked ends of apoptotic cells (fast sensitive and easy, but has false positives since necrotic cells have nicked DNA too)
DNA laddering (gel electrophoresis)- DNA cleaved at linker regions by DFF40 DNAase (normally DFF40 is dimerized to DFF45 and inert, but Caspases 3 and 7 cleave them)
Caspase activation (western blot)- cleaved active caspase has lower bands in the lane

170

What are caspases? What are the types of caspases?

Caspase- enzymes that are involved in apoptosis, must be cleaved to be active
initiator caspases- give the order (2, 8, 9, 10)
they cleave
effector caspases- which carry out the order (3, 6, 7)
by cleaving and activating DFF45 --> activates DFF40--> oligomerizes and becomes DNAase --> cuts up DNA

171

p53:
1) What is p53?
2) How is it regulated?
3) Why is it activated?
4) How is it activated?
5) What does it do?

1) p53- tumor suppressor transcription factor- has DNA repair proteins, can arrest growth, initiate apoptosis
2) Kept at low levels in normal cells through mdm2- tags it with Ub chain for degradation, also exports it from nucleus to cytosol
3) Activated with DNA damage/cell stress- UV radiation, hypoxia, telomere attrition
phosphorylation of p53/mdm2 inhibits their association
4) Activated by MAPK or checkpoint kinase families
5) Activates CKIs if cell does not pass G1 checkpoint to inhibit G1/S and S Cdks

172

Autophagy:
1) define
2) when is it triggered
3) what are the four stages

1) cell self-cannibalizes/eats itself; can be a survival mechanism-
sequestration of cellular organelles into vacuoles that fuse with lysosome that digest the enclosed material
2) Triggered when nutrient levels are low- breaks down proteins/organelles and recycles them for metabolism; also for housekeeping process
3)
I- Induction- mTor inactivated (eIF4BP not bound to eIF4E which can trigger translation of autophagy-related genes)
II- Autophagosome formation- form membrane around targeted portion
III- Autophagosome-Lysosome fusion- docks and fuses to lysosome, contents released and degraded
IV- Autophagosome breakdown- autophagosome body is also broken down

173

Explain the intrinsic and extrinsic apoptotic pathways

Intrinsic:
apoptotic signal --> activates p53--> PUMA/NOXA sensors --> BAX/BAK proteins go onto mt and create channels (inhibited by BCL2, which is inhibited by BID/BAD) --> cytochrome c leaks out --> with APAF1, creates apoptosome --> activates caspase 9--> activates caspases 3, 6, 7--> apoptosis
Extrinsic:
ligands Apo2L, TRAIL (tumor necrosis factors) activate pro-apoptotic receptors DR4 and DR5--> recruit FADD and create DISC --> FADD recruits caspases 8 and 10 and activates--> 8 and 10 go into cytoplasm--> activate 3, 6, 7 --> they cleave and activate DFF45 --> activate DFF40--> oligomerizes and becomes DNAase --> cuts up DNA

174

Describe the process of fertilization

Takes place in fallopian tube closer to ovary
1) Oocyte stalled in 2nd meiotic division
2) sperm move past corona radiata cells to zona pellucida- are capacitated by chemicals in uterus - allow them to release acrosomal enzymes to dissolve part of zona pellucida shell (need lots of sperm for this)
3) finally, one sperm fuses with oocyte cell membrane and releases its contents (just pronuclei, no organelles) -- union of sperm and oocyte
4) Causes intracellular calcium tsunami
5) This triggers cortical reaction- cortical granules fuse with membranes and extrude contents into extracellular space to prevent polyspermy
6) oocyte completes 2nd meiosis cycle
7) male/female pronuclei fuse together- 2n diploid (now know sex of embryo)
8) initiation of cleavage- zygote begins to undergo mitosis

175

Describe what happens in week 1 of embryonic development (period of devlpt from zygote--> implantation of blastocyst)

1) For ~ 4 days, zygote keeps cleaving within the zona pellucida--> forms a morula of 32 cells
2) morula undergoes compaction - inner cell mass/embryoblast and outer cell mass/trophoblast (becomes placenta)
3) embryo develops fluid filled cavity--> now called blastocyst; inner cell mass protrudes on one side- called embryonic stem cells
4) Day ~5- blastocyst hatches from zona pellucida
5) Day ~6, blastocyst implants on uterine epithelium/lining (upper posterior wall of uterus)
6) Trophoblast invades uterine lining, induces vascular changes, produces HCG (can detect on pregnancy test), corpus luteum makes etrogen/progesterone
7) peripheral cells of trophoblast become syncytial tissue --> invade endometrium and pull in the blastocyst
(cytotrophoblast are the source of progenitor cells and contribute to the syncytiotrophoblast)

176

What is ectopic pregnancy? What are common sites?

embryo implants somewhere other than uterus- can happen because the embryonic membranes induce the same changes in any epithelium no matter where
most common site - ampulla of the fallopian tube
most common site in the peritoneal cavity- rectouterine pouch

177

Describe the formation of amniotic cavity, bilaminar germ disc, and definitive yolk sac)

Day ~8
1) Embryoblast differentiates into bilaminar germ disc--> hypoblast (ventral- endodoerm) and epiblast (dorsal- ectoderm)
2) Amniotic cavity forms within the epiblast
3) Hypoblast gives rise to primitive yolk sac (what used to be the blastocyst cavity)
Day ~12
4) new wave of cells creates another yolk sac lining--> Definitive yolk sac (where blood cell production occurs)

178

Define buccopharyngeal and coacal membrane

Only two locations where the endoderm (hypoblast) and ectoderm (epiblast) adhere-
buccopharyngeal- cranial end of GI tract (mouth)
coacal- becomes anorectal tract

179

Describe primitive streak
Describe asymmetry
Describe gastrulation

1) Primitive streak forms on caudal region of dorsal surface - defines the orientation of the embryo
-formed by nodal
-this is how you know zygotic transcription is starting
2) motile primary cilia in primitive pit sweep fluid on dorsal surface from R--> L, more vesicles on L side with sonic hedgehog--> initiating molecular cascade on L side with nodal/lefty--> causes asymmetry
*defective cilia results in situs inversus ciliopathy
3) Gastrulation- epiblast cells become mesenchymal and deep dive through primitive streak --> migrate between epiblast (ectoderm) and hypoblast (endoderm) to create mesoderm * all 3 germ layers are formed from epiblast cells

180

Describe the development of the notochord:
-which cells give rise to it
-where is it located in the embryo
-what is its function

-1) After gastrulation, cells from the mesoderm give rise to the notochordal process at the primitive node, it grows caudally as the primitive streak gets smaller
2) migrates ventrally to endoderm
3) hollow tube --> solid rod
-tissue derivative: nucleus pulposis of intertebrate discs in adults
-induces formation of neural tube, development of nervous system (also acts as rigid axis around which embryo develops, foundation for vertebrae, secretes sonic hedgehog)

181

Anatomical terms to describe embryo:
1) dorsal vs ventral
2) anterior vs posterior
3) rostral vs caudal

1) Dorsal- back
ventral- belly
2) Anterior-ventral
(there is some confusion because its towards the head to embryologist)
posterior- dorsal
3) Rostral- anterior/head end
caudal- posterior/rear end
*need to know 2 of the 3: dorsal/ventral, L/R, cranial/caudal

182

Explain the etiology behind Hutchinson-Guilford progeria. What other disorder is linked to HGPS?
How does this relate to certain types of muscular dystrophy?

1) There is a mutation in the lamin A gene where exon 11 is missing--> therefore a cleavage site is missing --> lipid tail that is normally cleaved is not--> hydrophobic so it sticks to the PM --> disrupts lamina architecture --> causes progeria (autosominal dominant)
2) Linked to atypical Werner's syndrome because WRN mediates interaction between lamin and helicase
3) Mutations in the rod of the lamin A affect its structure and can cause muscular dystrophy

183

Explain the etiology behind Factor V and VIII deficiency bleeding disorder.

The two proteins LMAN and MCDF2 recruit clotting factors V and VIII into COPII vesicles from ER to Golgi
With mutation in either protein--> coagulation factors dont leave the cell --> blood clots cant form
Autosomal recessive
Used immunofluorescence or Co-IP to confirm interaction of the two

184

Explain the etiology behind Griscelli disease. What are the differences between Type I and Type II?

1) In order to bring melanin out of the cell, the melanosome is bound by Rab27aGTP (the GTP opens Rab lipid tail to bind), the effector melanophilin, and the tail of myosinVa (Exon 6 and GTD) which moves along actin
Type I- mutation in myosinVa (e.g. exon 6), melanin remains in cell--> pigmentation abnormality (silver hair, bronze skin) and neurological defects (because there's no replacement for myosinVa in neurons)
Type II- mutation in Rab27a, which also mediates fusing of lytic granules on target cells in immune response--> immune deficiency in addition to neurological defects

185

What are stem cells?

Cells that are:
1) undifferentiated
2) can self-renew
3) Can be induced to differentiate

186

What are the two types of stem cell self-renewal? What is the stem cell niche?

1) Symmetric- both daughter cells are stem cells (predominates during devlpt, injury)
Asymmetric- one daughter cell is stem cell, the other is differentiated (either because of segregation of factors, or because one cell leaves the niche)
2) mechanical and chemical microenvironment where stem cells reside, cancer or tissue injury can change the niche and cause cells to proliferate
e.g. Paneth cells in intestinal crypt

187

Describe the two pathways of self-renewal

1) Intrinsic- transcription factors that repress transcription of genes that would differentiate cells are Sox2, Nanog, Oct4, and Ronin
3) Extrinsic- LIF (JAK/STAT3), BMP (TGF/SMAD) work to inhibit MAPK cascade

188

What are the 3 different kinds of stem cells?

1) adult stem cells- undifferentiated cell in differentiated tissue (V RARE)
induced pluripotent stem cells- artificially derived from adult somatic cell through forced gene expression (used in stem cell research)
2) embryonic stem cells- from preimplantation embryo
3) cancer stem cells- drive tumorigenesis and have differentiated daughter cells

189

Define:
differentiated
undifferentiated
dedifferentiated
totipotent
pluripotent
multipotent
unipotent

Differentiated- cell with specialized structure and function (cancer- cells look like tissue where they came from)
Undifferentiated- cells aren't yet specialized (cancer- cells are immature)
Dedifferentiated- differentiated cell reverts to earlier developmental stage e.g. iPSCs
Totipotent- cells from first few division after fertilization, can form placenta or embryo
Pluripotent- cells that can differentiate into any of the 3 germ layers of the embryo e.g. embryonic stem cells
Multipotent- can differentiate into many cells in a related family e.g. hematopoetic stem cells
Unipotent- cells produce only one cell type but can self-renew e.g. muscle stem cells

190

Describe differences between adult and embryonic stem cells

Adult stem cells- maintain/repair tissue e.g. brain, bone marrow, blood vessels, muscle, skin, liver--> restricted use
Embryonic stem cells- from inner cell mass of pre-implantation blastocysts, that are isolated and grown; cultured to prevent differentiation, can induce certain type of differentiation

191

Describe how stem cells are IDed, isolated, and maintained

IDed: fingerprints (cell surface markers) in particular cell types
Isolated: flow cytometer that looks for fluorescent fingerprints on cells and isolates them
Maintained/cultured: embryonic stem cells plated onto layer of inactivated mouse fibroblast feeder cells that provide nutrients, LIF added because it inhibits differentiation

192

How are adult/embryonic stem cells stimulated to differentiate? How is differentiation maintained?

1) Stimulation:
-Change chemical composition of the culture medium
-Alter the surface of the culture dish
-Modify cells by introducing new genes
2) Maintenance:
-TF enhances its own promoter
-proteins act on chromatin to keep gene accessible
-cell makes signal molecule and receptor (if differentiation is dependent on signaling molecule)
-cell interacts with neighboring cell so they all remain differentiated

193

Describe the role of stem cells in regenerative medicine:
sources of human pluripotent stem cells
challenges to reprogramming
how to overcome challenges

1) best is adult stem cell, also germ cells of fetus, ICM of blastocyst, morula stage embryo
can make induced pluripotent stem cells (need to transfect 4 candidate genes to reprogram
2) low efficiency, genomic insertion, tumors, incomplete reprogramming
3) select for certain cells, use alternate vectors,

194

Describe the new cancer cell theory and cancer stem cells (CSCs). Why is cancer so hard to treat?

-New theory- tumors arise from cancer stem cells and growth is due to disrupted regulatory mechanism of renewal
-CSCs can self renew and make daughter cells that form the tumor mass --> we treat that but NOT the CSC itself --> leading to relapse--> why so many cancers are hard to treat
-CSCs protect against treatment bc they divide slowly, have ABC transporters for multiple drug resistance, good DNA repair mechanisms

195

Why was the Drosophila fruit fly used as model organism for studying development? Why the mouse?

1) Drosophila: genetic/cell bio pathways are evolutionarily conserved in fly, mice, men; fast, cheap; can perform genetic screens; complex morphologically
2) Mouse: most genetically malleable vertebrae species (e.g. knockout, knockin, transgenics)--> Stepping stone from drosophila to humans

196

Define:
knockout
knockin
transgenic

*ONLY IN MICE (for vertebrates)*
knockout- can select for and delete one gene using homologous recombination
knockin- replace one version of a gene with another using homologous recombination
transgenic- adding more copies of a disease

197

Define homeotic mutations and genes.

Homeotic mutations- where one structure is replaced with another, or is duplicated
Homeotic genes- regulate body plan an d devlpt of anatomical structures

198

What is homeobox? What is its structure? What are the classes?

1) Homeobox- conserved, homologous 180bp DNA sequence that encodes homeodomain protein- it is a transcription factor that binds to AT-rich DNA to initiate cascade of gene expression
2) 3 alpha helices, only 3rd binds DNA; other proteins mediate hemeodomain's binding specificity in vivo
3)
I. Hox genes- cause homeotic mutations, 4 clusters in mice/humans, 1 cluster in drosophila
II. not clustered genes- mediate devlp of different cell types/body parts (e.g. Otd, Otx1/2)

199

How do Hox genes function in patterning? Distinguish between spacial and temporal colinearity. What is the Hox code?

1) 3'--> 5' orientation of Hox genes corresponds to anterior--> posterior axis e.g. gene on 3' end would be mouth, or eyes
-each Hox gene expressed in specific embryonic domain --> generates particular body part
-genes are physically linked within each cluster
2) Spatial colinearity- 3' genes are more anterior
-Temporal colinearity- 3' genes expressed earlier
3) Hox code- distinct patterns of Hox genes depending on position along axis - dictates devlpt of different structures

200

Define orthologs and paralogs

Orthologs- homologous genes between species
Paralogs- homologous genes within species

201

How do Hox genes affect phenotype?

1) Loss of function mutation (knockout) --> anterior transformation
2) Gain of function --> posterior transformation
3) single mutants--> less severe phenotype

202

Define the function of other homeobox genes and how they are important in the generation of specific cell types

Otd- formation of head structures (Drosophila)
Otx1 and Otx2- (Mice, humans)
Otx2 mutation lethal (2 die), Otx1 mutation is less severe (l1ve)
Otd and Otx1/2 generate head/forebrain by triggering transcriptional cascade --> thus the transcription factor necessary and sufficient to generate the body part
Orthologs--> Drosophila Otd gene is functionally redundant to the Otx1 gene (discovered this by knocking in Otx--> Otd in mice) -- they regulate same downstream genes

203

Describe how mutations in homeobox genes contribute to human disease

Lots of homeobox genes linked to Mendelian disorders, have been mapped e.g. HOXD13 -- synpolydactyly
also contribute to common disorders

204

Describe how homeobox genes are important for stem cell therapy

Since clusters of Hox genes code for particular body parts--> if you know the Hox code (cascade of homeobox transcription factors)--> can induce embryonic stem cells to mature into different cell types that are damaged or lost to injury or disease e.g. replacing motor neurons for spinal chord injury --> transplant them into animal models

205

What are the contributions of genetic and environmental causes to birth defects?

Genetics (25%)
-chromosomal abnormalities
-single gene defects --> interrupt specific organ development
Environmental (10%)
-chemical toxins, infections, maternal deficiency
-alcohol, retinoic acid
Multifactorial (65%) - combo of the above

206

When during development is the embryo most vulnerable to teratogens?

-Most sensitive when organs begin to form - in first two months of gestation
-not that sensitive before implantation because embryo either dies or is able to regenerate cells that are affected without developing defects
-timing is important e.g. thalidomide exposure in week 4 --> arms don't form (but fingers are fine), fetus most sensitive to rubella exposure in first trimester

207

Explain the TGF signaling cascade (developmental pathway). How can it be inhibited?

1) TGFb ligand binds as dimer to serine-threonine receptor kinase
2) receptor phosphorylates and dimerizes and recruits and activates Smad2/3 by phosphorylating
3) Smad 2/3 dissociates from receptor and joins Smad 4
4) Complex goes to nucleus and activates gene transcription esp CKIs
e.g. BMP4, Nodal
*The BMP4 ligand in particular is inhibited by noggin, chordin, and nodal-- prevent ligand from dimerizing
TGFb is an antigrowth factor

208

What does FGF do?
Explain the FGF signaling cascade (developmental pathway).
What is FGF regulated by?

1) FGF necessary to activate 5' Hox genes for posterior development
FGF promotes chordin/noggin expression --> inhibits BMP4 --> promotes posterior development
2) 4 FGF receptors that are bound to syndecan proteoglycan that is attached to heparan sulfate proteoglycan
- Heparan sulfate presents FGF to tyrosine kinase receptors
3) Regulated by retinoic acid

209

From where does the mesoderm arise?
What is the purpose of the mesoderm?
What protein is responsible for mesoderm formation?
What is the changing role of this peptide at different stages in development?

1) From epiblast cells at the posterior end that move through primitive streak
2) Mesoderm --> connective tissue
mesoderm induces formation of nervous system from overlying ectoderm
3) Nodal (in the primitive node)--> also formation of primitive streak which defines dorsal/ventral, L/R axes
4) Early nodal expression induces anterior visceral endoderm --> signaling molecules establish anterior/posterior axis (anterior=head)
induced AVE produces inhibitors --> nodal expression repressed except for posterior end --> primitive streak forms
Once nodal expression begins in primitive node--> noggin and chordin bind to BMP4 and inhibit--> allow dorsal mesoderm and neural tissue development

210

What are different mechanisms that could lead to deficient posterior mesoderm formation? What does it lead to?

1) too little brachyury, chordin, or noggin
too much retinoic acid
too little FGF
2) Leads to sironomelia- the feet fuse together
*too much goosecoid leads to two heads

211

Explain the retinoic acid signaling cascade (developmental pathway). What are sources of RA?

1) Too much retinoic acid --> FGF turned off prematurely --> no 5' Hox gene activation --> deficient posterior mesoderm formation --> deficient posterior structure
2) RA is a teratogen, can be found in Accutane acne med

212

What is the sonic hedgehog signaling pathway? What is the function of sonic hedgehog?
Where is Shh localized?
What systems are affected by Shh signaling?
From where is Shh secreted?

1) takes place in primary cilium
Shh binds the Patched receptor which prevents it from inhibiting Smoothed protein --> now Smoothed can prevent the Gli transcription factor from being proteased into smaller factors --> Gli is brought to nucleus by IFT where it acts as an activator
2) provides positional information to direct polarity for tissues as they develop in limb, CNS axes
3) Localized in 2 areas with critical signaling functions: zona polarizing activity (ZPA) in limbs and notochord/floor plate in CNS
4) CNS and limbs
5) in embryology, from the notochord

213

What are causes/diseases that are linked to decreased hedgehog signaling?

1) holoprosencephaly- incomplete midline formation due to increased Gli3REP (need Gli3ACT in CNS in order to have midline structures develop)
2) cyclopia- caused by cyclopamine (plant alkaloid)- binds smoothen and prevents it from activating the Hh pathway
3) Holo also caused by low cholesterol -- need 3' cholesterol for mature Shh

214

What are causes/diseases that are linked to increased hedgehog signaling?

1) Polydactyly- due to decreased Gli3REP activity (doesnt matter if there is Gli3 ACT- what you need is gli3REP gradient in order to have right #)
2) Dorsal CNS hypertrophy- decreased Gli3REP (doesn't matter if there is Gli3ACT)
3) medullablastoma- mutations in Patched so it can no longer inhibit Smoothened (loss of heterozygosity)
4) basal cell carcinoma - mutations in Patched or Smoothened
*area of interest- treating male pattern baldness- Hh makes stem cells in our hair shafts so our hair regrows

215

What is the difference between canonical and non-canonical Wnt signaling pathways?
Describe canonical pathway
What diseases are associate with this pathway?

1) Canonical involves B catenins, non-canonical doesnt
2) Wnt binds to Frizzled and Arrow co-receptors -- >prevents B catenin from being degraded by desctruction complex--> B catenin goes into nucleus and displaces groucho --> binds with TCF to activate
3) Colorectal cancer- due to APC mutation that activates B catenin
Tetra-amelia- both Wynt copies lost --> absence of all 4 limbs
FEVR- eye disorder caused by mutations in Frizzled co-receptor (dominant- only need one bad copy to ruin the receptor)

216

Where is the neural crest? What regulates its formation? How is it affected by levels of BMP? What are major neural crest derivatives?

1) Neural crest at boundary of neural plate and ectoderm
2) at neural crest: Wnt, FGF, intermediate BMP
Induce border specifier genes and snail and Sox10
snail --> inhibits cadherins --> causes cells to become mesenchymal
Sox10 TF--> induces c-Kit and C-Ret tyrosine kinase receptors (work through ras to inhibit apoptosis)
2) Lower levels of BMP at the midline --> neural tube
intermediate levels --> neural crest
high levels --> ectoderm --> epidermis
3) Trunk derivatives- pigment cells, sensory neurons, adrenal cells
Cranial derivatives- cranial neurons, cartilage and bone, connective tissue in the face

217

Describe neural crest migration in general. Describe migration in the gut and its clinical relevance. Describe neural crest migration in the pigment cell pathway.

1) Lose class of cadherins due to snail -- > epithelial structure becomes mesenchyme --> cells migrate through hyaluronic acid-filled space --> localize and differentiate depending on where they end up
2) GDNF ligand acts as chemoattractant when bound to C-ret receptor, sequentially first in gut then down the intestine
Hirschsprung's disease- migration of neural crest cells is not complete, some cells lack enteric ganglion --> cannot relax and past stool --> creates bowel obstruction and megacolon, usually C-ret mutations
Ret mutations can cause C-ret to be constitutively active- causes MEN2
3) Steel factor ligand chemoattractant for C-kit receptor for germ cells, hemapoetic cells, and pigment cells (sterile, low blood count, pigment problems); steel factor can be alternatively spliced to be released and create ligand gradient for cell migration, also prevents apoptosis
Piebaldism- lack of melanocytes; mutations result in pigmentation problem, sterility, anemia
*can have mutation in either ligand or tyrosine kinase receptor

218

What is the phenotype of DiGeorge syndrome? How is that linked to environmental and genetic influence?

1) Phenotype- problems with pharynx but also cardiac outflow tract and thymus (neural crest derivatives)
2) Environment influence: Tbx1 mutations (Tbx found in pharyngeal cells NOT NC cells)--> pharyngeal pouches and arch don't develop properly-->affects environment through which neural crests migrate --> NC cells cant migrate to cardiac, thymus, parathymus --> NC cells do not differentiate or develop there leading to severe problems
3) Genetic influence: Mutation leading to absence of CrkL (involved in NC differentiation)--> affects neural crest cells *but no pharyngeal problems

219

What is the purpose of Tbx1? What are its downstream/upstream regulators?

1) Tbx1: TF on chromosome 22, found in pharyngeal cells
Tbx1 mutations impact pharyngeal development --> Affects environment through which neural crest cells travel --> development problems with cells --> DiGeorge syndrome (pharyngeal, thymus, thyroid, cardiac outflow problems)
2) Sonic hedgehog --> Foxc --> Tbx1 --> FGF signaling

220

What is Fetal alcohol syndrome? What are the causes and affects on neural crest development?

1) Rostral effects- affects nervous system (mental retardation) and neural crest development (smooth philtrum)
2) Ethanol exposure when neural crest cells are starting to migrate --> apoptosis of neural crest cells in rostral part of embryo --> development defects in nasal and jaw

221

Describe aplastic anemia (cause, effects, treatment)

1) Cause: due to autoimmunity, body's lymphocytes attack bone marrow stem cells --> no source of new blood cells
2) Effects: no megakaryocytes--> low platelets --> bleeding
no RBCs--> weakness, shortness of breath
no WBCs --> infection
3) Treatment- suppress body's immune system with antibodies

222

What is the difference between hierarchic and stochastic stem cell development

1) Hierarchic- Division by stem cell --> committed progenitors --> differentiated
*most stem cells e.g. hematopoietic stem cells)
2) Stochastic- can have three outcomes:
one progenitor and one differentiated daughter, two progenitors, or two daughters
*can apply to lymphocytes

223

What is the immunophenotype of hematopoetic stem cells?

True Stem cell- C-kit+, Lin-, CD34-
Multilineage progenitor - C-kit +, Lin -, CD34 + (mediates adhesion of stem cells in bone marrow)
*hematopoietic stem cells - have CD34 marker

224

What is a stem cell niche and how does it relate to hematopoiesis? How is it regulated?

1) Niche- nurturing environment that supports growth and proliferation of stem cells to maintain body's reserve
Want more Ca2+, less 02 in niches e.g. osteoblast lining
2) Regulated through chemokines produced by ostebolasts (make bone marrow) that attract stem cell receptors along a concentration gradient
*bone metabolism and hematopoiesis are interrelated -transcription factors and cofactors activate genes that lead to hematopoiesis and bone development

225

What is the dependence of hematopoietic stem cells on environment e.g. C-kit? Through what experiment did we understand this?

1) Steel factor on stromal cells in bone marrow attaches to C-kit ligand on stem cell --> how stem cells are maintained, C-kit also keeps the stem cells in niche
2) Jack Sprat experiment: direct cell to cell contact --> activation --> necessary for maintaining functional stem cells for blood production
w/w (no c-kit): can't engraft a normal mouse but can accept graft
sl/sl (no steel factor): can engraft normal and w/w mice but cannot accept graft

226

What is the difference between chemokine and cytokine? What are their functions?

1) Chemokine- chemoattractant cytokine that attracts other cells along concentration gradient e.g stem cell niche
e.g. CXCL4- attracts cell into bone marrow niche
2) Cytokine- cell signaling protein--> promote growth and maturation along a specific line of maturation, regulate number of divisions and cell cycle time e.g. CSF (colony stimulating factor)- e.g. EPO, C-kit/Steel factor (C-kit mutation causes leukemia)
*hematopoietin is a blood cell cytokine (can be endocrine- organ affects organ- paracrine- cell affects cell or autocrine- cell affects itself)

227

What is EPO? How does it work? Why can it be controversial?

EPO- cytokine produced by the kidneys, influences maturation of hemapoietic stem cells --> mature erythrocytes
EPO induces JAK/STAT 5 signaling --> transcriptional events enhance growth of red blood cells, suppress apoptosis
Can be controversial- bikers too EPO to increase number of RBCs and improve their oxygen during races

228

What are the formed elements of blood, their morphology, and function?

1) Red blood cell/erythrocyte - 02 delivery, acid-base balance
2) White blood cells/leukocyte - immune response (lymphocyte, monocyte/macrophage, eiosinophil, neutrophil)
3) Platelets - help blood clot

229

Describe the purpose of these factors on hematopoiesis:
1) Runx/CBFbeta
2) Vegf and tel
3) Erythropoietin
4) CSF

1) TF/cofactor that bind to target genes for differentiation, cell cycle regulation, p53 pathway *mutation can cause leukemia
2) delivery of blood cells to periphery
3) factor that induces maturation of red blood cells through JAK/STAT signaling
4) cytokine that mobilizes stem cells and causes them to mature (erythropoiesis) e.g. M-CSF- maturation of macrophages

230

What are the main components of the extracellular matrix? What is the principle cell?

1) Fibers (e.g. collagen, elastin) and ground substance
2) fibroblasts- produce both fibers and ground substance

231

What is the difference between tendons and ligaments?

Tendons- muscle to bone, not that much elastic
Ligaments- bone to bone, lots of elastic

232

Difference between loose and dense connective tissue

Loose- fewer fibers, more cells, abundance of - charge molecules in ground substance so can hold Na+ ions and water --> resist compression
Dense- more collagen fibers, fewer cells

233

White fat cell:
-function
-morphology

-energy storage, provide protection (eye socket, around kidneys)
-large lipid droplet inclusion
*reside in connective tissue

234

Brown fat:
-function
-morphology

-thermogenesis
-smaller and more numerous lipid droplets per cell
lots of mitochondria
*reside in connective tissue

235

Neutrophil:
-function
-morphology

1) first responders in phagocytosis, most abundant type of WBC, migrate through blood vessels following Interleukin chemokine signals
2) multi-lobed nuclei, lighter cytoplasm color
*granulocyte (white blood cell with secretory granules in its cytoplasm)

236

Eiosinophil:
-function
-morphology

1) immune response - phagocytosis
2) lobed nuclei
lysosome granules look like hamburgers
*granulocyte (white blood cell with secretory granules in its cytoplasm)

237

Monocyte/Macrophage:
-function
-morphology

1) immune response - phagocytose foreign materials, present antigens to lymphocytes
2) horseshoe shaped nucleus
-pseudopods around PM
-heterogeneous ingested particles
*becomes a macrophage when it migrates INTO connective tissue

238

Plasma cell:
-function
-morphology

1) Formed from lymphocyte, produces antibodies
2) nucleus off to one side and is soccer ball pattern, clearly formed ER and golgi

239

Lymphocyte:
-function
-morphology

1) responds to inflammation/infection- makes B and T cells
2) small darkly stained cells clustered together
small nucleus
found in lamina propria (respiratory, GI, mouth), can also be in epithelium

240

Mast cell:
-function
-morphology

1) immune response -histamine for vasodilation, found in small blood vessels
2) lots of histamine granules in cytoplasm
*granulocyte (white blood cell with secretory granules in its cytoplasm)

241

Fibroblast:
-function
-morphology

1) make ground substance and fibers
2) long and tapered, oval nuclei
hard to see cell boundaries
*reside in connective tissue

242

Mesenchymal cells:
-function
-morphology

1) undifferentiated- potential to develop into fibroblasts, smooth muscle cells, adipocytes, etc
2) hard to ID- large nucleus, little cytoplasm with few organelles
*reside in connective tissue

243

Basophil:
-function
-morphology

1) circulating white blood cell - attract WBCs to point of infection, has histamine like mast cells (v similar to mast cells)
2) bi-lobed nuclei, darker cytoplasm color than neutrophils
*granulocyte (white blood cell with secretory granules in its cytoplasm)

244

Which cells reside vs travel through connective tissue? How do these cells get to the tissue?

Reside- adipocytes, fibroblasts, mesenchymal cells
Travel- white blood cells
Diapedisis- move through the endothelium-- squeeze right through the cell

245

CML:
-how to clinically ID
-clinical features
-specific karyotype and abnormal chromosome
-features of the mutation that results in disease
-molecular diagnostics
-drug development

1) High white blood cell count - esp lymphocytes and neutrophils
blood smear --> increased number of myeloid progenitors prematurely released from the bone marrow
2) fatigue, weight loss, but can appear normal
large spleen- because HSCs leave bone marrow and circulate til they get to spleen
3) Philadelphia chromosome- 22 appears smaller
balanced reciprocal translocation between 22 and 9 (so 22 shorter, 9 longer)
46XY, t(9; 22)(q34;q11)
4) because of break- BCR gene on 22 becomes BCR-abl
BCR-Abl tyrosine kinase can now dimerize--> kinase can activate itself--> constitutively active --> no regulation
Abl trapped in cytoplasm now, cant go to nucleus (cannot help with DNA repair response) --> more mutations --> leads to blast crisis
*see increased myeloid and lymphoid WBCs but mutation/probl1em is in progenitor cells
5) use RT-PCR to confirm presence of fused chimeric protein (808 bp BCR + ~300bp Abl) -- see 2 bands in gel lane
6) Gleevec/imatinib is a tyrosine kinase inhibitor - prevents BCR-Abl from using ATP to phosphorylate and activate substrate

246

Outline steps of normal coagulation and list complexes involved in each step. What are the thrombin amplification effects?

1) Local vasoconstriction- due to release of endothelin
2) Primary hemostasis- formation of platelet plug
-vWF + FVIII stretch out at exposed subendothelium
-platelets bind to vWF and collagen and are activated
-platelets change shape (flatten) and release granules of more vWF and agonists
-recruit other platelets and aggregate
3) Secondary hemostasis- cross-linked fibrin to seal plug
*coagulants bind to Ca2+ bound to platelets
-Tissue factor on endothelium binds and activates FVIIa
-TF + FVIIa bind and activate FVIXa (also, intrinsic activation by FXIa, which is bound to XIIa on a negative surface)
-TF + FVIIa + FIXa + FVIIIa activate FXa
-FXa + FVa activate FIIa ie thrombin
-Thrombin cleaves peptides from fibrinogen and activates fibrin which self-assembles
-FXIIIa cross-links the fibers
4) Thrombin amplification:
-activates FVIIIa and FIXa to make more FXa
-activates FXa and FVa to make more thrombin
-activates FXIa to propagate more clot formation

247

What are the Vitamin K dependent coagulation factors? Explain the role of Vitamin K in factor synthesis and how warfarin interferes with this

1) Factors: VII, IX, X, II, Protein C
2) Vitamin K needed to carboxylate these factors and make them more negative
being more negative is important because platelets are (-) and have Ca2+ bound - so they need to be (-) to bind to the Ca2+
3) Warfarin inhibits Vit K reductase --> no more VIt K in active form--> cannot carboxylate the Vit-K dependent coagulation factors --> ANTI clotting result
*babies given Vitamin K shots at birth

248

Describe the function of the following proteins in the coagulation process:
1) Tissue Factor
2) Von Willebrand Factor
3) Thrombomodulin
4) Protein C and Protein S

1) Tissue factor is normally not produced, but it is produced by monocytes/endothelial cells when there is injury and triggers first step in extrinsic pathway of coagulation by binding/activating FVII
2) vWF in primary hemostasis: vWF + FVIII travel in bloodstream and attaches to subendothelium - facilitates platelet tethering and facilitates collagen binding to platelets
vWF in secondary hemostasis: vWF increases half life of FVIII which is needed with FVIX to amplify FXa
3) Thrombomodulin- receptor for thrombin which inactivates its activity --> anticoagulant
4) Protein C and S (non enzymatic cofactor)- activated when Thrombin binds Thrombomodulin
inactivates FV and FVIII (the other enzymatic cofactors) --> anticoagulant

249

ID the coagulation proteins whose defects/deficiencies result in excessive bleeding and which ones are involved in excess clotting

1) Excessive bleeding:
-Hemophilia A- FVIII deficiency
-Hemophilia B- FIX deficiency
-Hemophilia C- FXI deficiency (intrinsic)
*need VIII and IX as amplification to get more X
-Ebola- hijacks tissue factor --> increased consumption of coagulation factors --> body cant make more --> leads to hemorrhage
-Sodium citrate- inhibits Ca2+, so you don't have the platelet-Ca2+-coagulation factor sandwich and blood doesn't clot
-von Willebrand disease- most common hereditary bleeding disorder
2) Excessive clotting:
Factor V Leiden mutation- resistance to activated protein C - cant inactivate FV --> increased clotting

250

What is the role of platelets?

1) Important in hemostasis- normally not active in circulation
2) low platelet count results in bruising (petichiae) which indicates that platelets helps endothelial cells bind together at junctional complexes

251

How is coagulation regulated?

1) Thrombin binds to the thrombomodulin receptor --> which inactivates it
2) Thrombin binding thrombomodulin activates Protein C into APC; APC + Protein S inactivate FV and FVIII
3) Thrombin and Tissue plasminogen activator (TPA) convert plasminogen --> plasmin
plasmin breaks up fibrin clots and degrades them

252

What are the three classes of genes in which mutations contribute to cancer? Describe types of mutations. Why can recessive mutations seem dominant?

1) Caretaker genes (tumor suppressor)- repairs DNA e.g. MLH, BRCA1/2
loss-of function, recessive, increases change of cancer
2) Gatekeeper genes (tumor suppressor)- regulate cell division/apoptosis e.g. Rb, p53
loss of function, recessive, will cause cancer
3) Oncogenes- promote cell growth and division
gain of function, dominant e.g. bcr-Abl, c-myc (translocated next to enhancer and causes Burkitt's lymphoma)
4) Recessive - already have one gene knocked out, so faster loss of heterozygosity which makes it appear dominant though the first inherited copy was inherited recessively

253

List one type of cancer associated with mutated caretaker genes in these pathways:
1) Mismatch repair
2) Nucleotide excision repair
3) dsDNA break repair

1) MLH- Lynch syndrome
2) NER-ERCC1/XP- Xeroderma pigmentosum
3) BRCA1/2- familial breast/ovarian cancer

254

Describe genetic effects underlying retinoblastoma. What are some routes to loss of heterozygosity?

1) Rb- binds and inhibits EF2 and is a gatekeeper gene in regulating entry past G1 into S phase
Sporadic rb- accumulate two defective genes, tumors in one eye
Familial rb- usually earlier onset, with tumors in both eyes
"Two Hit hypothesis"
2) chromosome loss, uniparental disomy, recombination, deletion, second mutation

255

What is Waardenburg syndrome? What protein mutations cause it? Why is it dominant? How do you see if there are any downstream proteins affected?

1) MITF: activates anti-apoptosis factors (BCL2), activates genes for melanin production
C-kit/Steel factor and Wnt--> increase amount of MITF
2) Mutations in MITF: Increased apoptosis --> absence of NC-derived melanocytes that produce endolymph --> no hair cells in the ear --> hearing problems
Pigmentation problems --> blue iris, white forelock
3) Dominant because of haploinsufficiency (gene dosage)- if you're heterozygous, not making enough normal protein so exhibiting phenotype
4) Downstream protein is Slug
could do knockout microarray, more specific is promoter binding assay to see how activity is affected
CHiP- look at DNA/protein interactions (cross-link, shear, co-IP with antibody, sequence)