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Résumé Nitzan Altima
Résumé Nitzan Altima
Experimental models:
Microscopes
LM = wave length 0,4-0,7um (purple-red), living cells not visible unless phase contrast microscope
EM = 1000x (0.1nm), wavelength decreases as its velocity increases (0,004nm), 100.000V, aberration
of the electron lens, heavy metal salts fixation needed, staining with dense material
Dark field microscope : side light, cell = bright objet on dark ground
Fluorescence microscope : fluo molecule absorb light at a wavelength and emits it at another longer
WL, filter allows only the emited WL to pass
Cell membrane
Lipids through hydrophobic barrier, hydrophilic via protein specialized- transport, recognition/comms
via carbohydrates prot, energy conversion/enzymatic reactions via prots
Phosphatidylserine = neg, only inner, if outer = macrophage induction (phago dead cell)
Individual mols (lipids and prots) are free to rotate, move laterally, flip/flop
Chol and sphingolipids (sphingomyelin + glycolipid) form discrete lipid rafts (small semi solid patch),
necessary for endocytosis
Extracell part of mb prots are generally glycosylated, same for carbs part of glycolips
Forms glycocalyx = carbs coat, formed by oligosaccharides of glycolips and transmb glycoprots,
protects the cell surface, also oligosaccharide serve as markers for cell-cell interactions
Mb prots :
TRANSPORT :
Reg cell vol through osmotic effect, nerve impulse, assists in active transport of mols (glucose,
symport), secondary active pump. Indirect hydrolysis of ATP, usage of energy concentration gradient
created by primary active transport
ABC (ATP binding cassette) transporters : family of mb transp with ATP binding cassette
MDR prot (multidrug res) = remove toxic forign compounds, found in cancer cells
CFTR (cystic fibrosis transmb regulator) = Cl- channel in epith cells, CF = often single point mut,
interferes with prot folding + Cl- transp, recessive, thick mucus, respi + gastroint epith, CL- remains
inside cell, forms thick mucus with water, breathing problems, infections, sealed pancreas ducts,
salty sweat (diag)
RBC mb = best study cells (no nucl/organelles -> mb/mb prots easily isolated), 52% prots, 40% lips,
8% carbs, pale empty cells = RBC/white ghosts = hemolysis, disease
SDS
SDS-PAGE = separation of charged mols by their charge, sodium dodecyl sulfate identifies 15 diff
prots
RBC cytoskel = spectrin ++, in tetramers, bind with actin for spectrin-actin network (cortical cytoskel),
linked to mb via ankyrin wich binds to transmb prot band 3. Another link is prot 4.1 (spectrin/actin
junction – cytoplasmic domain of glycophorin), mut spectrin = spherocytosis, sphere, less surface
tension, less flexible, band 3 defficiency (transp passage bicarb HCO3- and chloride ion Cl-) across
RBC mb, also spherocytosis
Dystrophin = spectrin related prot, actin – muscl cell mb, protects muscle cells during contraction,
mut = Duchenne muscular dystrophy = boys + (X), muscle degen/weakness
2 – Tight junction = occludine prots, seal epith sheets, separate apical/basolateral domains, found in
BBB (blood brain barrier)
3 – Gap junctions = connects adjacent cells cytoplasm, ion/small mols diffuse freely, 6 connexins
(transmb prots), pore = connexon, allows mb potential to pass from cell to cell (cardiac muscle),
cancer cells lack gap
4 – Desmosome junctions = tight junctions between epith/muscle cell, attached to intermediate fil of
keratin in cytoplasm, keeps tissues tight, pemphigus = autoimm disease against desmosomes
RER = network of tubules and sacs enclosed in a mb, continuous with nuclear mb, covered with
ribosomes, signal sequence in newly formed polypep directs engaged rib towards ER mb (if no signal,
cytoplasm), signal seq binds to SRP (Signal recognition particle) that leads him to SRP receptor near
translocator channel prot that permits translocation to the ER lumen
Prot Glycosylation : addition of carbs, vast majority of ER synthesized pots are glycoprots, as soon as
a polypep enters, a common oligosacc synthesized in the ER on a lipid carrier (dolichol phosphate) is
transferred on an Asn
Prot Folding = polypep chain enters unfolded, is folded in ER lumen, then transferred folded and
partially glycosylated in vesicle to Golgi
Golgi = up to 100/cell, membrane enclosed sacs (cisternae) and associated vesicles, factory where
prots from ER are further glycosylated, sorted (via addition of different carbs) and packed for
transport to : lysosome, plasma mb, secretion; they go from cis to trans face, RER is in constant
contact with golgi via 2 coatomer coated vesicles : COP II coated vesicles (ER to Golgi), COP I (along
Golgi, Golgi to ER)
Lysosome = enzymes produced through ER/Golgi, break down polymers (prots, AN, carbs, lips), pH 5,
endosome = mb organelle in animal cell, carries material ingested via endocytosis, pass majority to
lysosome (early endosome -lowers pH-> late endosome -fuses with lys. enzyme laden vesicle->
lysosome)
Uptake of transferrin = iron from digestive syst to liver via transferrin, apotransferrin + 2 Fe3+ =
ferrotransferrin
Lysosomes diseases : tay sachs = gen, destruction of nerve cells and spinal chord; gaucher =
glucocerebroside accumulated in cells and organs; silicosis/asbestosis = lung disease; rheumatoid
arthritis = autoimm, joints; gout = inflammatory arthritis
SER = cell with lipid metabolism ++, netlike labyrinth of tubules, continuous with RER, functions :
Lipid biosynthesis = enzyme for synth located on cytoplasm face of ER mb, enzyme flippase transfers
synthesized lips to inner layer if they remain in ER, lips are transferred to other organelles bound to
prots
Prod of cytochrome P450 enzyme = catalyzes a series of reaction to make water insoluble
drugs/metabolites (alcohol) soluble enough to be excreted in urine
Peroxisomes = small, single mb organelle, 500/cell, 50+ enzymes, carry out oxidation reaction that
produce toxic hydrogen peroxide H2O2, decomposed by catalase in 02 and H20, contains oxidative
enzyme that uses oxygen for degradation of AA, purines, methanol, fatty acids; involved in chol and
dolichol biosynthesis; perox prot cyto synth, perox phospholips ER synth, additional entry of both
leads to growth then division; diseases : Zellweger syndrome = no entry of perox prots in perox
(empty), early death; Peroxisomal protein signal sequence mut = Alanine glyoxylate
aminotransferase (AGT) enzyme has a changed signal sequence, directed to mitochondria instead, no
function, formation of kidney stone
Microfilaments : major CS prot in most cells, polymerizes to form microfilaments (e.g myosin),
beneath mb + for shape/movement, myosin = prototype of motor-a protein that converts ATP to
movement (muscle); globular G-actin binds tightly and polarly into filaments, used ++ by bacteria
Microtubules : dimer of α and β tubulin, polym. into microtubules made of 13 linear protofil. around
a hollow core, 25nm diameter, polar, continuous (dis)assembly from – α end to + β end
Intracell. transports (ves. and orgl., kinesin/dynein), mitosis, cilia, flagella; centrosome = 2 centrioles
(each 9 triplets), near nucleus during interphase, replication before cell div. (cancer cell = incorrect
mitosis = 4 centrosomes); Cillia/Flagella : 9 doublets + 2 in the middle, dynein generates mov.; basal
body : cylindrical orgl., base of flagellum/cilium in the cytoplasm, same structure as centriole
Keratins : epith, 15 diff. prots, epidermolysis bullosa = mut. autodom, blistering of the skin at light
stress
Neurofilament : in mature neurons along axon, assemble the nerve cell CS with longitudinal
microtubules
Lamins : nuclear, inner mb of nuclear envelope, important for mitosis, disassemble if phosphorylated
Mitochondria : own DNA, encodes small part of its tRNAs, rRNAs and some mitochondrial prots
Structure : double mb with folds (cristae) around matrix, outer mb = porins + (aqueous for mols <
5000 daltons), mitochondrial lipid synthesis / conversion enzymes ; inner mb = cardiolipin + (double
phospholip. with 4 fatty acid chains), impermeable to ion, transport prots and ATP synthase ; matrix =
hundreds of enzymes, ribosomes, t/r/m/mtRNA
Respiratory chain : C6H12O6 + 6O2 -> 6CO2 + 6H20 + energy (36-40% efficiency)
If absence of O2/mitochondria, fermentation instead (pyruvate -> lactate), Cori cycle (acid lactate
moves to liver to be converted in glucose and etc)
Oxydation of pyruvate into acetyl CoA in the mitochondria : Pyruvate + CoA + NAD+ → Acetyl-CoA +
NADH + CO2(x2)
Citric acid cycle (Kreb) = acetyl CoA oxidated to C02, reduction of NAD+ and FAD to NADH and FADH2
: Acetyl CoA + ADP + Pu + 3NAD+ + FAD -> 2CO2 + ATP + 3NADH + FADH2
Electron transport chain / Oxidative phosphorylation : high energy e- from NADH(2E-) and FADH2 are
transferred through a series of carriers on inner mb, energy from these transfers is used to pump out
H+, H+ comes back through ATP synthase and the energy is used for ADP + Pi -> ATP
Endosymbiotic origin theory for mitochondria and chloroplast, same structures and sensible to same
antibio
Multiple circular DNA coding for 13 prots, 2 rRNA (12S, 16S), 22 tRNA, 16 mRNA ; not sufficient for all
DNA activity
Double mb complicates trans., different target signals for different compartment, most studied TS
are amino-terminal presequences, electrochemical gradient drives the prot to the matrix (presequ +,
inside -), HSP 70 keeps prot unfolded, HSP 60 folds prot properly
1 – Respiratory chain loses 2-3% electrons during their transfer to oxygen -> free radicals -> damages
mtDNA -> mutation
2 – mtDNA maternally inherited -> hundreds of mitochondrial diseases like Leber Hereditary Optic
Neuropathy (LHON) (visual loss, mitochondrial disorder, young males +, mutation of mitochondrial
DNA)
Central Dogma
DNA : sugar phosphate backbone with a nitrogenous base (purine : 2 rings, pyrimidine : 1 ring) in
keto tautomeric held together by H-bonds
Watson and Crick : X-ray diffraction taken by Rosalind Franklin and Maurice Wilkins
Hybridization : 65°C for a prolonged time (works for DNA, RNA and crossed)
Forms of DNA :
DNA replication : multiple origins, bidirectional, 5’ -> 3’, DNA polymerase can’t initiate (≠primases),
Okazaki fragments (1000-3000 pdb)
Helicase unwind DNA via ATP hydrolysis, single-stranded DNA-binding protein stabilizes it (SSB),
primer (short RNA strand) serves as starting point, later removed from lagging strand
In prok. DNA polymerase III is the major responsible of replication, polymerase I/II/III also act as
exonuclease in both direction, removing primers
In euk. DNA polymerase α/δ/ε (γ in mitochondria) take care of replication, RNA is removed by RNase
H and 5’ to 3’ exonuclease, later filled by polymerase δ and joined by ligase
Trancription
Promoter region + RNA polym. + transcription factors (majority of gene expression control)
TBP-TATA binding protein = general transcription factor, allows other TF and eventually RNA polym.
to bind
Terminator sequence
helix-turn-helix
steroid receptors
zinc fingers
leucine zippers
helix-loop-helix
pre-mRNA to mRNA processing : 5’ guanin cap + 3’ polyadenine tail, 150-200 (both protect the
transcript and help trans. to cyt. rib.)
Splicesomes: 5 types of snRNA + 6-10 prots = SNRNPs (small nuclear ribonucleoprotein), central role
in splicing, intron out, exon stays
Translation
mRNAs read from 5’ to 3’, polyp. chains synthesized from amino to carboxy end
tRNA = adaptors between mRNA / AA incorporated into the prot, L shape, end in CCA-adenosine
ribose-AA 3’, anticodon on the other side of tRNA recognises and binds to codon, AA/tRNA binding
mediated by aminoacyl tRNA synthetase
Nucleus
Inner/outer mb joined at nuclear pore complex (50 ≠ nucleoporins, 3-4k pores/cell), bilayer
permeable to small nonpolar mols
Imported prot needs nuclear localization signal (NLS) AA sequence that binds to nuclear transport
receptor (karyopherin +, importin/exportin), ATP/GDP dependent
Some importins (Kap, β) need an adapter karyopherin (Kap α), some are importin/exportin
depending on the protein
Same for export with nuclear export signal In contrast to m/t/rRNAs that function in the cytoplasm,
many small RNAs (snRNAs, snoRNAs) function in the RNA processing of the nucleus ; exported
snRNAs + prots = snRNPS that return to the nucleus
Chromatin becomes highly condensed during mitosis to form the compact metaphase chromosomes
that are distributed to daughter nuclei
Nucleolus : site of rRNA transcription and rib. assembly, genes transcribed by RNA polym. I/III, each
gene is a single transcription unit containing the 18s, 5.8S and 28s rRNAs separated by transcribed
spacers ; the genes themselves separated by nontranscribed spacers ; RNA polym. I = 5.8S, 18S, 28S
(200 gene copies) ; RNA polym. III = 5S rRNA (2000 gene copies) ; imported rib. prot + rRNA in
nucleolus = rib. subunits, then exported
2m of DNA into 5-10um nucleus -> chromatin = DNA + protein, histones + (H1/2A/2B/3/4),
nucleosome = DNA + histone octamer
Condensation of chromatin :
solenoid = 40x
filament in loops
Human chromosome :
acrocentric : near-end centromere, chr. 13, 14, 15, 21, 22 ; satellite chromosome = beside
centromere have a segment separated from main body similarly ; telocentric : centromere at the
end, not found in human
G-banding : dark = heterochropatin, late-replicated and AT rich ; light = euchromatin, early, GC rich
R-banding : opposite
Primary culture : directly from organism tissue, allow proliferation until telomere loss causes
structure unfolding ; cell may detect uncapping and stop growing, enter senescence or self-destruct
Established cell lines : grow indefinitely, useful for long term research (stem cells, tumor cells)
Two main growth conditions : monolayer (adherent structure) or free-floating (suspension) ; usually
pick the closest to physio conditions
PCR : thermal cycling = DNA melting, DNA primer + polym. + free nucleotides etc