Biochem FA - p46 - 56 Cellular/Labs Flashcards
Explain p53’s role in cell cycle?
p53 induces p21, which inhibits CDKs –>hypophosphorylation (activation) of Rb –> inhibition of G1-S progression
How do cyclin-cdk complexes affect other proteins to continue the cell cycle?
phosphorylation
Growth factors bind to ______ to move cell cycle from ____ –> _____ phase
Growth factors (eg, insulin, PDGF, EPO, EGF) bind tyrosine kinase receptors to transition the cell from G1 to S phase
What makes permanent cells unique?
Remain in G0
How to permanent cells regenerate?
from stem cells
Ex of permanent cells
neurons, sk musc, cardiac musc, RBCs
What is a stable cell?
Can enter G1 from G0 when stimulated
Ex of stable cells
hepatocytes, lymphocytes, PCT, periosteal cells
Ex of labile cells
Bone marrow, gut epithelium, skin, hair follicles, germ cells.
What does the RER add to lysosomal proteins?
N-linked oligosaccharide
Which cells are rich in RER?
Mucus-secreting goblet cells of the small intestine and antibody-secreting plasma cells are rich in RER.
What is a Nissl body and what is its function/
Nissl bodies (RER in neurons)—synthesize peptide neurotransmitters for secretion
Where are steroid hormones produced?
smooth ER
What is the other function of smooth ER
detox of drugs and poisons
What cells are rich in smooth ER?
Liver hepatocytes and steroid hormone– producing cells of the adrenal cortex and gonads are rich in SER
Function of Golgi apparatus?
distribution center for proteins and lipids from the ER to the vesicles and plasma membrane.
What are additions that the Golgi makes to proteins?
Modifies N-oligosaccharides on asparagine. Adds O-oligosaccharides on serine and threonine. Adds mannose-6-phosphate to proteins for trafficking to lysosomes.
Disease with defect in N-acetylglucosaminyl-1-phosphotransferase?
I-cell disease (inclusion cell disease/mucolipidosis type II)—inherited lysosomal storage disorder
What is the underlying issue of I cell disease?
failure of the Golgi to phosphorylate mannose residues (dec mannose-6-phosphate) on glycoproteins –>proteins are secreted extracellularly rather than delivered to lysosomes.
Sx/ of I cell disease
coarse facial features, gingival hyperplasia, clouded corneas, restricted joint movements, claw hand deformities, kyphoscoliosis, and high plasma levels of lysosomal enzymes
Difference between COPI and COPII?
COPI: Golgi –> Golgi (retrograde); cis-Golgi –>ER. COPII: ER –>cis-Golgi (anterograde)
Function of peroxisomes
β-oxidation of very-long-chain fatty acids (VLCFA)
α-oxidation (strictly peroxisomal process)
Catabolism of branched-chain fatty acids, amino acids, and ethanol
Synthesis of cholesterol, bile acids, and plasmalogens (important membrane phospholipid, especially in white matter of brain)
Sx of Zellweger syndrome?
Hypotonia, seizures, hepatomegaly, early death.
Zellweger - what gene and mode of inheritance?
autosomal recessive disorder of peroxisome biogenesis due to mutated PEX genes.
Sx of Refsum disease
scaly skin, ataxia, cataracts/night blindness, short 4th toe, epiphyseal dysplasia
R - reptile (scaly) skin E- epiphyseal dysplasia F - fourth toe short S - sight issues U - uh-taxia? M - treat with PlasMAphoresis (A = alpha oxidation)
Underlying cause and MOI of Refsum?
—autosomal recessive disorder of α-oxidation –> phytanic acid not metabolized to pristanic acid.
XLR disorder of B-oxidation is? mutation?
Adrenoleukodystrophy due to mutation in ABCD1 gene - leads to VLCFA build up in adrenals and white matter of brain and testes
Defects in the ubiquitin-proteasome system is assoc with what disease?
Parkinsons
ex of micro filaments and function?
Muscle contraction, cytokinesis ex/ Actin, microvilli.
Fxn and ex of intermed filaments
Maintain cell structure
ex/ Vimentin, desmin, cytokeratin, lamins, glial fibrillary acidic protein (GFAP), neurofilaments.
Fxn and ex of microtubules
Movement, cell division
ex/ Cilia, flagella, mitotic spindle, axonal trafficking, centrioles.
Structure of microtubules
Cylindrical outer structure composed of a helical array of polymerized heterodimers of α- and β-tubulin
Each dimer of microtubules has what energy molecule attached to it?
GTP
Drugs that work on microtubules?
s (Microtubules Get Constructed Very Poorly): Mebendazole (antihelminthic) Griseofulvin (antifungal) Colchicine (antigout) Vincristine/Vinblastine (anticancer) Paclitaxel (anticancer)
Molecular motor proteins - what direction?
Dynein—retrograde to microtubule (+ −).
Kinesin—anterograde to microtubule (− +).
Cilia structure?
9 doublet + 2 singlet arrangement of microtubules
What does axonemal dynein?
ATPase that links peripheral 9 doublets and causes bending of cilium by differential sliding of doublets
What is the basal body, what’s the structure?
Base of cilium below cell membrane - consists of 9 microtubule triplets B with no central microtubules
What disease involves an issue in embryologic heart looping?
Primary heart tube loops to establish left-right polarity; begins in week 4 of gestation.
Defect in left-right Dynein (involved in L/R asymmetry) can lead to Dextrocardia, seen in Kartagener syndrome
What is the primary malfunction in Kartagener syndrome? Mode of inheritence?
immotile cilia due to a dynein arm defect; AR
Why are there fertility issues in Kartageners?
Results in DEC male and female fertility due to immotile sperm and dysfunctional fallopian tube cilia, respectively; inc risk of ectopic pregnancy
Sx of Kartagener?
Can cause bronchiectasis, recurrent sinusitis, chronic ear infections, conductive hearing loss, and situs inversus (eg, dextrocardia on CXR
Dx screening test of Kartagener?
Dec nasal nitric oxide (used as screening test).
How does Na/K pump work?
Na+-K+ ATPase is located in the plasma membrane with ATP site on cytosolic side. For each ATP consumed, 3Na+ go out of the cell (pump phosphorylated) and 2K+ come into the cell (pump dephosphorylated).
Drugs that work on Na/K pump?
Ouabain (a cardiac glycoside) inhibits by binding to K+ site. Cardiac glycosides (digoxin and digitoxin) directly inhibit the Na+-K+ ATPase, which leads to indirect inhibition of Na+/Ca2+ exchange --> Inc[Ca2+]i --> inc cardiac contractility.
Collagen type I is found where?
Bone (made by osteoblasts), Skin, Tendon, dentin, fascia, cornea, late wound repair.
Collagen type II is found where?
Cartilage (including hyaline), vitreous body, nucleus pulposus.
Collagen type III is found where?
Reticulin—skin, blood vessels, uterus, fetal tissue, granulation tissue.
Collagen type III is deficient in what disease?
Type III: deficient in the uncommon, vascular type of Ehlers-Danlos syndrome (ThreE D).
Collagen type IV is found where?
basement membrane , lens
What kidney disease shows issues with collagen type IV?
Targeted by auto antibodies in Goodpasture syndrome
Defective in Alport syndrome;
HLA type of Goodpasture?
HLA DR2
What type of hypersensitivity reaction in Goodpasture?
HS - II
How does Goodpasture look microscopically?
LM—crescent moon shape Crescents consist of fibrin and plasma proteins (eg, C3b) with glomerular parietal cells, monocytes, macrophages
Linear IF due to antibodies to GBM and alveolar basement membrane
What is the pattern of amino acids in precollagen?
usually Gly-X-Y (X and Y are proline or lysine). Glycine content best reflects collagen synthesis (collagen is 1⁄3 glycine).
What residues are hydroxylated in collagen? Req which vitamin?
Proline, Lysine. Req vitamin C
What is the next step after hydroxylation in collagen synthesis?
glycosylation of pro-α-chain hydroxylysine residues and formation of procollagen via hydrogen and disulfide bonds (triple helix of 3 collagen α chains).
What disease has issues forming collagen triple helix?
osteogenesis imperfecta
What converts procollagen into insoluble tropcollagen?
cleavage of disulfide-rich terminal regions of procollagen –>insoluble tropocollagen.
What disease shows issues with procollagen cleavage?
Ehlers Danlos syndrome
What vascular issue is seen with EDS?
berry and aortic aneurysms
How does tropocollagen become collagen fibrils?
reinforcement of many staggered tropocollagen molecules by covalent lysine-hydroxylysine cross-linkage (by copper-containing lysyl oxidase) to make collagen fibrils
Disease with issues with cross linking of collagen?
Problems with cross-linking –>Ehlers-Danlos syndrome, Menkes disease.
Disease caused by defect in COL1A1 and COL1A?
Osteogenesis imperfecta
What type of collagen is affected in osteogenesis imperfecta most commonly?
Most common is AD dec production of collagen type I
Why is the sclera blue in osteogenesis imperfecta?
due to the translucent connective tissue over choroidal veins
How to reduce fracture risk in OI?
treat with bisphosphonates
Sx of OI?
Patients can’t BITE: Bones = multiple fractures I (eye) = blue sclerae Teeth = dental imperfections Ear = hearing loss
List the 3 types of EDS - incl. which is most common, which genes.
Hypermobility type (joint instability): most common type. Classical type (joint and skin symptoms): caused by a mutation in type V collagen (eg, COL5A1, COL5A2).
Vascular type (fragile tissues including vessels [eg, aorta], muscles, and organs that are prone to rupture [eg, gravid uterus]): type III procollagen (eg, COL3A1).
Mode of Inheritence of Menkes disease?
XLR
Which enzyme has dec activity in Menkes? why?
Leads to dec activity of lysyl oxidase (copper is a necessary cofactor)
Underlying issue in Menkes?
caused by impaired copper absorption and transport due to defective Menkes protein
Genes in Menkes vs Wilsons
ATP7A, vs ATP7B in Wilson disease
sx of Menkes
Results in brittle, “kinky” hair, growth retardation, and hypotonia.
What makes elastin different from collagen?
Rich in nonhydroxylated proline, glycine, and lysine residues, vs the hydroxylated residues of collagen.
What inhibits the breakdown of elastin?
alpha 1 antitrypsin , which normally inhibits elastase
Changes in elastin with aging?
Changes with aging: decdermal collagen and elastin, dec synthesis of collagen fibrils; crosslinking remains normal.
Genetic issue in Marfan?
AD FBN1 gene mutation on chromosome 15 (fifteen) results in defective fibrillin, a glycoprotein that forms a sheath around elastin
Sx/ of Marfan?
tall with long extremities; pectus carinatum (more specific) or pectus excavatum; hypermobile joints; long, tapering fingers and toes (arachnodactyly); cystic medial necrosis of aorta; aortic incompetence and dissecting aortic aneurysms; mitral valve prolapse.
Difference between subluxation of lens in Marfan vs homocystinuria?
Subluxation of lenses, typically upward and temporally (vs downward and medially in homocystinuria).
murmur in Marfan sounds like what?
holosystolic murmur with mid systolic click
How does squatting and standing up affect the murmur in Marfan?
Squatting delays mid systolic click, standing is opposite (dec preload)
What other genetic diseases we went over has the same cardiac issue as Marfans
Fragile X or EDS
What does PCR amplify?
DNA
3 steps to PCR, temp for each one
- Denaturation - heated to 95* C
- Annealing - cooled to 55*C
- Elongation - temp inc to 72*C
What is added at the annealing stage
. DNA primers, a heat-stable DNA polymerase (Taq), and deoxynucleotide triphosphates (dNTPs) are added
Fxn of RT-PCR?
Detects and quantifies mRNA levels in a sample. Uses reverse transcription to create a complementary DNA template that is amplified via standard PCR procedure
What is CRISPR/cas9?
A genome editing tool derived from bacteria
Parts of CRISPR?
Consists of a guide RNA (gRNA) , which is complementary to a target DNA sequence, and an endonuclease (Cas9), which makes a single- or double-strand break at the target site
Potential fxn of CRISPR?
Break imperfectly repaired by nonhomologous end joining (NHEJ) –> accidental frameshift mutations (“knock-out”) , or a donor DNA sequence can be added to fill in the gap using homology-directed repair (HDR) .
Fxn of Southern vs Northern vs Western vs SW blot
Southern - DNA
Northern - RNA
Western - protein
SW - DNA binding proteins
Fxn of flow cytometry?
Laboratory technique to assess size, granularity, and protein expression (immunophenotype) of individual cells in a sample.
How does flow cytometry work?
Cells are tagged with antibodies specific to surface or intracellular proteins. Antibodies are then tagged with a unique fluorescent dye. Sample is analyzed one cell at a time by focusing a laser on the cell and measuring light scatter and intensity of fluorescence
Microarrays can detect?
Able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) for a variety of applications including genotyping, clinical genetic testing, forensic analysis, cancer mutations, and genetic linkage analysis.
ELIZA used for?
used to detect the presence of either a specific antigen or antibody in a patient’s blood sample.
Detection in ELIZA
Detection involves the use of an antibody linked to an enzyme. Added substrate reacts with enzyme, producing a detectable signal.
In Karyotyping, _______ is added to cultured cells to halt chromosomes in ____ (what phase of cell cycle)?
Colchicine is added to cultured cells to halt chromosomes in metaphase.
Gene issues that could be detected with FISH?
microdeletion, translocation, duplication
Steps of molecular cloning of recombinant DNA molecule in bact host?
- Isolate eukaryotic mRNA (post-RNA processing) of interest. 2. Add reverse transcriptase (an RNA-dependent DNA polymerase) to produce complementary DNA (cDNA, lacks introns). 3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes. 4. Transform (insert) recombinant plasmid into bacteria. 5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA).
What does the Cre-lox system do?
Can inducibly manipulate genes at specific developmental points
RNA interference?
dsRNA is synthesized that is complementary to the mRNA sequence of interest. When transfected into human cells, dsRNA separates and promotes degradation of target mRNA, “knocking down” gene expression.
