extra info from Review Sessions Flashcards
what do CDC6 and CDT1 do?
both essential for DNA replication and formation of pre-RC. they act as vehicles to assemble the pre-rc. Once loaded, you can assembly primase, helicase, etc..
cdc6 - loads MCM (helicase) proteins onto the DNA. regulates cell cycle checkpoints. activity regulated by cyclinA
cdt1 - knockouts for cdt1 are lethal - cells undergo mitosis but not dna replciation
what enzymes are needed to unite the lagging strand?
Fen-1, DNA pol delta, ligase
error in helicase?
meier-garlin
error in clamp loader (r-27)
autoimmune disease, chronic inflammation, cancer
why does replicative DNA poly have high fidelity
it’s 3-5 exonuclease activity
enzyme has low kM for strand when mismatch occurs. it slows down rate that DNA unravels, allows the mismatch to be sequestered far away and be repaired
what encourages tight binding of DNA polymerase?
the narrow channel. More error prone DNA polymerases have larger crevice
describe nucleotide pool imbalance
as a result of accumulation of a particular nucleotide, a mistake if quickly skipped over because of the annealing of high concentration nucleotides occurs so quickly
what can stall the fork?
anything that gets in the way
centromeres - lots of protein
telomeres - lots of capping proteins
dna lesions or bulky adducts
premature activation/misregulation of the pre-RC can lead to
nucleotide pool stress
environmental factors that can mess up replication
sunlight casuing DNA damage (TT dimers)
massive amounts of damage - cell undergoes apoptosis. triggered by ATM stimulating p53 via the DDR
describe the error prone response
trasnlesion DNA polymerase uses non-template double strand.
when you have massive damage, sometimes you can’t repair it. translesion polymerases skip the damage by including any random nucleotide. If it can keep the replication fork from moving without being stalled, that is one way to deal with massive DNA damage
repetitive elements can cause..
loops out of DNA sequences. another type of error in DNA replication.
-Error that occurs when Polymerase slips
once the stable hairpin structure forms, it becomes an impediemnt to replication
errors in XPA-G? what pathway?
errors in the NER pathway. Xeroderma pigmentosum
what is the common set of steps of repairing DNA damage
- damage recognition by an enzyme specific to that damage type
- enzyme will remove damage (just base, entire nucleotide, large segment) and leave a gap
- DNA poly syntehsizes DNA to fill in
- Ligase ligates
what causes HNPCC?
mismatch repair errors
what causes aicardi gouteries?
error in RER Excision repair, RNAse
in BER, the lesion is removed by?
DNA glycosylase = cuts sugar/ cuts base/ cuts bond between sugar and base, cuts glyosidic bond
In NER, gap is filled by?
DNA polymerase. Dna poly does make the phosphodiester bond with previous nucleotide, but need ligase for next one to seal
what is the biochemical mechanism that can be used to regulate actin assembly into microfilaments?
energy output and input involved. By using ATP as energy, can control rates of assembly.
fast growing end can do work through the assembly process
fillapodia
fast gorwing ends towards the membrane
stress fibers
bundles where each + end is against membrane. in middle you get overlapping - filaments
lammelapodia
broad edge of cell being pushed out. You have branched filaments. ART binds to ends to existing filaments and creatse a place to nucleate and assemble
contractile ring
similar to stress fibers - lots of filaments go around cell and overlap with each other. Can have myosin between filaments pulling them, involved in cytokinesis.
myosin I
Myosin 1- monomeric, standalone vesicular motor. Sngle ATP binding site. Tail associates with membrane, does not have long tail, has short tail. Vesicular transport if free, or binding of mysoin to plasma membrane to move the filaments relative to the PM.
myosin II
where in non-muscle?
Myson I I - contracile arrays. In muscle and non muscle.
- where in non muscle? Epithelial sheet that has to change shape during a process.
- bipolar arrays cause contraction
assembly of microtubules is…
rate limiting step?
more complicated than filaments, Uses GTP not ATP.
the slow GTPase activity of bound subunits
meier garlin syndrome
helicase error
clamp loader mutation
autoimmune, chronic inflammation, cancer
mechanism that controls DNA replication to once oer cycle
cyclinA/CDK kinase - degrade CDC6 and CDt1 by phosphorylation and removing
microfilaments can form
microvilli
filipodium/lammelapodium - fast growing ends push membrane out
stress fibers - each + end against membrane, overlapping filaments in center to get contraction
lamellapodium - broad edge fo cell fushed out, branched filaments, actin related protein (ART) binds to ends, allows nucleation.
contractile ring - lots of filaments go around cell and overlap. can have myson between filaments pulling, involved in cytokinesis
extending nerve terminus
assembly of actin. get actin subunits to the end, vesciles get transportered to end and “hop off”
secretory and actin
vesicles get close to membrane, actin filamnets bring them up to membrane, allow secretion
mysosin walking across actin filaments
same cycle of ATP bidning, myosin release, ATP hydrolysis, change in confirmation, reattatch, release energy and pull along filament
how is mysoin regulated?
depends where you are
in skeletal/cardiac muscle - directly involves calcium assocation with proteins on thin filament
or phosphorylation of proteins associated with myosin like chains
general contraction/relaxation cycle
Thin filament (actin), mysoin tries to move toward plus ends at Z disk, trying to move in 2 direction at once. The contractile unit (Sarcomere) shortens. This happens when calcium bins to the proteins on actin filaments, moving the tropomysin out of way so myosin can interact with actin. Regulated through sequestration of CA in membrane vesicles of SR that get released and pumped back in.
intermediate filaments
family of proteins that provide structural support, NOT tracks for motility
keratins, vimentin/related, neurofilaments, nuclear lamins (all cells)
microtubules vs microfilaments
microtubules - hollow, more complicated assembly, uses GTP and slow GTPase of beta subunits
microfilaments - use ATP and actin
microtubules involved in
nucleic and cell division, organization of intracellular structure, and intracellular transport, as well as ciliary and flagellar motility
where are microtubules found in cells?
nucleating sites (where - are togheter and cant assemble) associated with gamma tubulin rings to rapid assembly
what moves along microtubules?
membrane associated things
ER and golgi comparments move to different parts of cell
Dynein - pulls things closer to centrosome (-). keeps golgi next to nucleus. nuclear membrane and golgi have dynein associated
ER - spreads throughout microtubule network using kinesin
motile vs non motile cilia
motile - have dyneins
non-motile - lots of sensory receptors on membrane, no dynein
primary places where you see defects with cilia?
not having receptors on non motil cilia - causes polycistic kidney disease where the kidney cilia dont assemble or receptors are mutated
ramachandran plot
glycine?
certain regions are compatible with secondary structures due to bonding angles
glycine has large area in plot suggsting flexibility
can the C-N bond rotate?
no, all four atoms connected are in a plane and rotate together, C-N bond has partial double bond character
Phi Angle?
PSi angle?
Phi - Con
Psi - CC
phi and psi in beta sheets?
alpha helicies?
beta sheets - close to 180
minus 60 - alpha helix
if NH3+? pH must be..
if histidine is protonated?
if carboxyl is deprotonated?
below 8
must be below 6.5
must be above 4
Alpha-1-antitrypsin
serine protease, example of disease cuased by protein misfolding
leading to aggregation or loss of function
Correct function of alpha1-antitrysin
reactive center loop where neutrophil elastasce likes to cleave (cheese in mousetrap).
Bites the loop, forms covalent bond to loop. Loop cleaves and now inserts into central beta sheet (it is complementary)
- Loop drags neutrophil elastase down to lower section of alpha-1-at, and the interaction cuases neutorphil elastase to misfold
- other proteases now degrade neutrophil elastase
what can do wrong with alpha-1-antitrypsin?
2 things
-suggested by lock of similar antithrombin enzyme
when reactive loop inserted into beta sheet, it sprung all by itself (more stable, cant go back). Can no longer inhibit. Happens too much due to mutation - can no longer inhibit neutrophil elastase
second thing that can go wrong - insertion happens without cleavage, it mis inserts into neighboring molecule. Abundant in liver, causes a cascade of polymerization that builds up in liver
alpha helix amino acids
helix formers - mainly alanine, but also arginine and leucine
if the N-terminal is in the internal lumen of the ER…
, then when it is sent to the plasma membrane, the N-term will be on the external face.
Biosynthesis of glycosaminoglycan chains on proteoglycans occurs primarily in
golgi
When a protein destined for the plasma membrane is synthesized, it is inserted into the ER membrane with its extracellular domain on
the lumenal side of the ER membrane.
to be facing out of the cell, must be facing inside of the ER
lyosome, endosome, peroxisome
lyososome - degradation/recycling
endosome - sorting from endocytosis
peroxisomes - creates h202
2 organelles that do not communicate with other organelles via vesicles
mitochondria and peroxisomes are not a part of the vesicular trafficking pathways
when LDL receptor iis synthesized in the ER..
the LDL binding domain faces the ER lumin, but will face the cytosol once it reaches the PM
the adaptin domain starts off facing the cyotosol, will end up pointing inward
When we put proteins into the ER, we put them in the same way in correct orientation every time. Something that needs to face outward and inward
-something initially faces lumen then faces outside
mitochondrial receptor VS ER receptor
the receptor for the mito signal sequence is on the mito membrane
the receptor for the ER directing signal is in the lumen (Soluble)
scanning EM of golgi vs rough ER
rough ER will have visible studded ribosomes
golgi Vs SMOOTH ER?
golgi- tight pancake stack
lysosome scanning image
very dark, black
peroxisome scan
uniform gray, smooth, very dark spot in center (Crystallization of enzymes)
Phospholipids and Sphingolipids
Phospholipid
ex) phosphoglyeride - 2 fa, glycerol backbone, phosphate alcohol head
Sphingolipid
ex) Glycolipid- sphingosine backbone/fa, carbohydrate
shingomyelin -is a sphingolipid and a phospholipid (sphingosine, fatty, acid, phosphate choline
mito genome is in the
mito matrix
proteins required for Mito genome (RNA poly, DNA poly) come from the…encoded where?
cytosol, in the nucleus
transcribed in nucleus, translated in cytosol, transported into mitochondria
x-linked adenoleukodystrophy
cant import long fatty acids, beta oxidation doesnt occur, they accumulate
familial hypercholesterolemia
LDL can’t get taken up by endocytosis. LDL receptor suffers a loss of function mutation, accumulated in blood
where are the ABO antigens synthesized?
in golgi
3 functions of golgi
n-linked sugars modified
o-linked sugars added to proteins and lipids
GAGS built on proteoglycan core proteins
ABO blood group antigens
degrade glycoproteins and proteoglycans
mucopolysaccharidoses
-defects in the degradtion of GAGS
oligosaccharidosis
defend in degredation of oligosaccharides
sphingolipidosis
defects in degredation of sphingolipids
inclusion-cell disease
defect in generating M6P tag on lyososomal enzymes. lysosomes fill with substrate with nothing to degrade them. the enzymes are sent throguh default pathway which is harmless because they only act at low pH
why don’t the proteases in lysosomes self digest the other enzymes?
the other enzymes are protected by sugars, proteins are heavily glycosylated
all nuclear pores are ______, what determines the direction taken?
equivalent, direction is based on the carrier intermediate
there are vanishing amounts of GTP in the
cytoplasm
Role of Ran GTPase?
provides energy for transport in form of energy stored as chemical gradient
helps form export compleex and accompanies these complexes out of nucleus
homologous recombination mixes up…
parent and grandparent alleles by exhanging arms of chromosomes
we all inheret ______ loss of function alleles
250-300
half of our genome are ________
transposable element sequences
retrotranspons
in humans, move via RNA intermediate with RNA reverse transcriptase
retrotranspons and exon shuffling
Rretrotranspons have unique transposon ends. The enzymes recongize these tranposable ends. Make RNA out of this, reverse transcriptase can recognize the other two, and move entire
Normally the RT would transcribe and transport the small pink, instead it mistakenly cuts out and tranports the larger green gene between the two similar transposons
-this can cause misalignment
creation of unequal gene families by crossing over
an entire gene can get duplicated, or the same thing can occur WITHIN a gene causing a single exon duplication or deletion
multiplex PCR
many PCR in single tube, get multiple products. You’ve got 9 reactions at same time, done diagnostically, how you look for deletions in dystropin
RT-PCR
You can PCR and amplify when template is RNA - use RT enzyme to do first early rounds to turn RNA into DNA, then it’s a normal PCR. It’s a PCR where the first round is
Microarrays
lung cancer example but lots of things. Different kinds of tumors in cancers, how they might respond to different chemo treatments. Microarrays give you a readout of all the genes being expressed in that tumor. Gives you a very detailed view of what that cell is doing and how its doing it. Much more so then under a microscope (Stain, shape) very high res.
- tells subtype of cancer - what mutations might be present - what pathways elevated - will tell you what chemo will or wont work
FISH vs Microarray
fish - looking at whole chromosome, you see large changes in chrosomomes. Cancer cell karyotype. Shows approximate lesions of chromosome and how its been moved
typical microarray - analyzes mrna to determine expression (what and how much) of a gene. Tells more than fish, lets you get to see exactly where translocation is
spirochetes under a microscope can appear
either spiral or invisible (pallidum). need darkfield
What do we do antibiotics resistance testing on?
the enterobacteriaecae - they are so abx resistant due to horizontal gene transfers that we need to do agar plate + abx disks
what would a culture of rickettsia on agar look like?
rickettsia is intracellular, plane of agar would be blank. must culture in tissue
we do not do antibiotic resistance testing on…
environmental microbes, such as clostridium
to establish intracellular infection (4)
need type 3SS - important for host cell takeover/living inside of them
why don’t we use the TB vaccine (bcg)?
our incidence of TB is low, and we primarily screen people as necessary with PPD. Giving people the vaccine would cause all PPD to come back positive, new way to screen would require chest xray
Childhood infection due to bacteriophage, has been declining ever since
scarlet fever
N.meningitidis
gram negative diplococcus that oxidizes maltose and has a a capsule
N.Gonorrhea
gram - diplococcus that does not oxidize maltose, has pili
most common disinfectant used for living tissue?
alcohol, is also a crossover agent used for countertops/objects
quaternary ammonium compounds
most common use of sterilization and disinfecting and detergent in hospital of surfaces. Quats have residual activity even after wiped off.
Augmentin, ceft, vancomycin all…
inhibit peptidoglycan synthesis
the beta lactams act as PBP substrate analog
vancomycin (glycoproteins) - bind to the terminal d-ala strands to block PBP
penicillinase/beta lactamase resistant penicillins
used against organisms that product beta lactamase
MRSA
has found a way to become resistant to all beta lactams and beta lactamse inhibitors
MRSA changed its PBP2 to PBP2a
cephalosporin generations
move up food chain, become more and more resistant to beta lactamase
carbepenams
the most powerful beta lactams, resistant to ALL beta lactamases, sensitive to carbepenemases
CRE
carbepenam resistant enterobacteriaca - stared off as just kleb, produces carbepenamases
why might a patient not respond to cephalosprin treatment?
the organism is producing beta lactamase to “break the floor of the garage”
treatment of simple betalactamases expressing organisms?
can use antibiotic + b lactam inhibitor (augmentin_
treatment of sophisticated beta lactamase expressing organisms?
produced by the ESBLS, can no longer use 3rd and 4th gen cephalosporins. use carbepenam
CRE’s are ____
plasma encoded superbugs. plasmids contain genes for all mechanisms of resistance. we only have colistin left
treatment of CRE’s?
colistin
most common immunodeficiency?
lack of IgA - get more colds and gi infections
why doesnt cholera have a high infant death rate?
IgA are present in breast milk
why is IL1B in a prophase?
so it is already made, and can quickly be cleaved by the inflammasome to be realeased to begin inflammation
enzyme for immunoglobulin class witching and somatic hypermutation?
AID
RAG1 and RAG 2 are needed to
recombine the b cell chain DNA
TDT
adds n-regions during splicing to give random diversity
N regions exist primarily in
heavy chains, leading to more diversity
someone without TDT
has antibody and t-cell receptors, but doesnt make as high affinity antibody because they dont have the same amount of diversity to start with
_______ is never present in T-CElls
AID
what stimulates AID in b-cells?
t-cells
t-independant antigen wil not
class switch or do hypermutation
western blot and elisa both show….
difference?
how much antigen there is
western blot takes longer and limits how much can be run at once
Western blot does let us know the molecular weight of the antigen
advantage of western blot?
lets us know molecular weigth of the antigen
-sometimes there is a mixture of things that are reactive, but we want to know the particular protein that is causing the reaction
_____ provides better info about size
______ is much more quantitative
western blot - size
elisa - quantitiative, shows amount
which can tell concentration of antibody, western of elisa?
both can, simply titrate until you don’t have reaction anymore
Fc receptor on macrophage binds the
IgG constant region
what is an allergen?
an antigen that binds IgE
epitope
part of antigen that single monoclonal antibody binds to
the is no Fc receptor for…
IgM
complement can enhance phagocytosis due to
prescence of receptors for c3b on macrophages and neutrophils
____ is the main target of major complement receptors
c3b, used to opsonize a target to be cleared out of the body
b-cell differentiation timeline
heavy chain, then light chain. Heavy chain gets made first and each chromsome gets only one chance to rearrange its VDJ successfully.
Light chains get multiple cahnces to rearrange their DJ regions.
hapten experiment
carrier coated with hapens
t-cells respond to carrier
b-cells respond to hapten and carrier
switching the carrier - no secondary response by the B cells against the hapten
-B cells and T-cells need to be on the same page to have a secondary response
toxoids are similar to hapten molecules. TF
True
we get less crossreactivity with a monoclonal because
this is a laboratory artifact, we screen for crossreactives and they are not used
mito matrix contains
mito genome, beta-oxidation enzymes, TCA cycle enzymes
mito inner membrane contains
components of oxidative phosphorylation, ETC proteins
mito outer membrane contains
porin molecules
intermembrane space contains
cytochrome c - important for cell death signaling
all bacteria have…
internal plasma membrane
describe exotoxins
polypeptides secreted from pathogen or injected into host cell via t3ss
- superantigenicity
- actin depolarization
- interference with signal transduction
unknown anaerobes can be identified by
gram stain, biochemical tests, gas chromatography analysis of waste products
major pathogenic anaerobes
Clostridium, GNAB, and actinomyces
Gnab are gram -
clostridum and actinomyces are gram +
GNAB, actinomyces, and C.diff are normal flora
remainer of clostridum are soil organsism (tetani, botulinum) that depend on spore forming ability
anaerobic virulence depends on
exotoxin expression and tissue degrading enzymes to create abscesses
metabolism of staph?
facultative anaerobes - wall themselves off
treatment of anearobic infections?
antibiotics against the bacteria, antitoxin against the toxin, and wound draining/debridement
spirochetal trasmission
sexual, vector, environmental
common spirochete themes
cross into bloodstream quickly/easily
virulence primarily due to immune evasion (not antigenic but also immunomodule the host) - no vaccines
diagnosis challening - treponema are too small to see, cause various odd symptoms. Lyme tests are difficult
treatment easy
often get herx reaction
vaccines against spirochetes effective?
no vaccine, spirochetes are not immunogenic enough
syphilis
3 stages
painless chancre to variable rash/flu
to either early or late latency
to dangerous cardiac or CNS involvment. “Gummas”
lyme stages
early localized - rash
early disseminated - meningitis, facial paralysis, rashes, cardiac symtpoms
late disseminated - arthritis, encephalitis
treatment of lyme
doxycycline
appearance of vibrios
reservoir?
curved, gram - rods.
ocean dwelling, infected humans, halophiles (thrive in high salt concentrations)
most vibrios can cause
fecal oral gastroenteris primarily
may infect wounds exposed to ocean water
H.pylori causes peptic ulcurs
v.cholera
complex planktonic lifecyle outside of human host
pathogenic strains have O1 antigenic marker of colonization by lyosgenic phage that carries virulence factors
trasmission is fecal oral, usually killed in stomach via acid
virulence of v.cholera
encoded by lysogenic phage (has O1 marker)
- secretes mucinase to attatch/colonize intestine
- secretes choleragen - AB enterotoxin that interferes with signal transduction = massive watery diarreah
treatment of v.cholera
antibiotics may not be necessary, self limiting in otherwise healthy host
provide hydration and electrolytes
intracellular bacteria may be facultative-
facultative - enter has part of life cycle, can replicate outside on agar
-neisseria, enterics, mycobacter, bacilli, legionalla
or obligate, must be grown on tissue
-rickettsial, chlamydia
how do they get around?
pathogens use infected macrophages for transport around body, avoid humoral
treatment of intracellular bacteria?
tetracyclines except pregnant
Reactive arthritis may be caused by
chlamidia, shigella, or eneterohemmogenic e.coli
listeria
causes gentroententeritis, escapes endosome after endocytosis and uses actin based motility to spread between cells (ActA)
chlamyida
use t3ss to enter and create paranuclear inclusion body for the larger reticulate bodies to multiple in
enterobacteriaca
two groups
foodborne - shigella, e.coli, salmnella, yersinia
oppotunistic ICU bugs - kleb, enterobacter, proteus, providencia, serrata, morganella
describe the testing results of enterbacteraicace
gram -, non spore forming, straight rods, facultative anaerobes
catalase +, oxidase -, glucose fermenters
problem with the enterobacteriaceae
promiscuous with DNA, extreme abx resisitance is common. must do sensitivity testing
virulence factors of enterbacteriaceae?
pili for attatchment, t3ss for injecting enterotoxins and subverting gut macrophage.
allow themselves to be sampled by M-cells in peyers patches
Y.enterocolicita and macrophages
use subverted macrophages as trojan horse to local lyph nodes (false apendicitis)
s.typhi
system wide entry using macrophages
HUS
hemolytic uremic syndrome - pediatric complication caused by release of shiga toxin into bloodstream by either shigella or eneterhemmogenic e.coli
patients with human antigen b27 infected with enterobacteriaceae
may develope reactive arthritis (conjunctivitis, urethritis, and arthritis) after infection from Shigella, salmonella, yersinia, campylobacter, or chalomydia
the 6 ICU bugs
defense?
kleb, enterobacter, morganella, proteus, serratus, providencia
growth rate of M.tuberculosis
slow, difficult to culture and defeats some antibiotics
gram stain of M.Tuberculosis?
poorly due to mycolic cell wall
spread of M.Tuberculosis
inhalation of infectious particles, spreads to lymph nodes, long bones, kindney, and CNS by hematogenous spread (trojan horse macrophages)
immunocompetent reaction to TB?
creates strong CMI, can hold latent
cd8 cells respond to tb by
killing infected macrophages and forming granulomas to wall off infection
classic TB
cough, weight loss, fever, bright pink chains of rods in sputum
extrapulmonary TB
ussually reactivations - scrofulas, genitourinary, CNA, skeletal
in pediatrics, TB can cause
miliary and meningitis
main treatment of TB
4 drug coctail with isoniazid, 2 week isolation
aytipical mycobacteria?
infection symptoms?
environmentally acquired, don’t cause TB or leprosy
ussually cutaneous - scrofulas.
in compromised - may mimic TB, especially M.Kansasii
two forms of leprosy
treatmetn?
tuberculoid - strong cmi that causes nerve damage, paucibacillary, ppd +
leproid - weak CMI, causes extreme cutaneous damage, multibaccilarry, PPD -
2 years dapsone/rifampin
TF can be a…
an activator, repressor, or recuitor of chromatin modifier
what can result from enhancer translocation?
t-cell acute lymphocytic ademia - make tons of T-cells, way more than needed
Transcription factors use different motifs to..
contact DNA and provide specificity
typically have an alpha helix that inserts into major groove. multiple contacts gives specificity, does not need to unwind DNA helix
Transcription factors stimulate the general transcription machinery via a
mediator
-bridges RNA pol activators to RNA pol II and general factors
-required for transcription activation
what do chromatin modifiers do?
adds covalent modifications to histone N-terminal tails
acetylation - activation
deacteylation - deactivation
methylation - activation or repression
phsophorlyation - coupold to acetylation
