Cellular and Lab Techniques

Question 1

Cell cycle regulation

Answer 1

CDKs (always active) — Cyclins (activate CDKs) — Cyclin-CDK complexes (phosphorylate other ptoteins to coordinate cell cycle - p27 inhibits CDK in G1 to inhibit cycle) — Tumor suppressors (p53 and HYPOphoshporylated Rb inhibit G1 to S progression — CDK4 leads to HYPERphosphorylated Rb, releases E2F and enters S phase)

Question 2

3 cell types

Answer 2

Permanent (remain in G0 - neurons, skeletal and cardiac muscle, RBCs) — Stable (Enter G1 from G0 when stimulated - hepatocytes, lymphocytes) — Labile (never go into G0, divide rapidly, most affected by chemo - bone marrow, gut epithelium, skin, hair follicles, germ cells)

Question 3

Endoplasmic reticulum

Answer 3

Rough (secretory proteins, Nissl bodies in neurons make NTs, free ribosomes make cytosolic and organellar proteins - mucus cells of small intestine have a lot of RER) —- Smooth (steroid synthesis and detoxification - liver hepatocytes and steroid hormone producing cells of adrenal cortex have a lot of SER)

Question 4

Golgi functions

Answer 4

Modifies N-oligosaccahrides on asparagine, adds O-oligosaccharides on serine and threonine, adds mannose-6-phosphate to proteins for trafficking to lysosomes

Question 5

I cell disease

Answer 5

Defect in N-acetylglucosaminyl-1-phosphotransferase –> failure of golgi to phosphoryalte mannose residues –> proteins secreted extracellularly rather than to lysosomes —- coarse facial features, clouded corneas, restricted joint movement, high plasma levels of lysosomal enzymes

Question 6

Trafficking proteins

Answer 6

SRP (ribosome to RER – mutation causes accumulation of proteins in cytosol) — COPI (Golgi to Golig, cis golgi to ER) — COPII (ER to cis Golgi) — Clathrin (trans Golig to lysosomes, plasma membrane to endosomes)

Question 7

Peroxisome

Answer 7

Catabolism of very long chain fatty acids, branched chain fatty acids, and amino acids

Question 8

Proteasome

Answer 8

Degrades damaged or ubiquitin tagged proteins

Question 9

Cytoskeletal elements

Answer 9

Microfilaments (muscle contraction, cyokinesis - actin) — intermediate filaments (maintain cell structure - desmin, cytokeratin, lamins, neurofilaments) — microbutules (movement, cell division - cilia, flagella, mitotic spindle)

Question 10

Stains for intermediate filaments

Answer 10

Vimentin (Connective tissue) – DesMin (Muscle) – Cytokeratin (Epithelial cells) – GFAP (NeuroGlia) — Neurofilaments (Neurons)

Question 11

Microtubules

Answer 11

Heterodimers of a and B tubulin (each dimer has 2 GTP bound) — Dynein (retrograde to microtubule) and Kinesin (anterograde to microtubule) (KARD)

Question 12

Drugs that act on microtubules

Answer 12

Microtubules Get Constructed Very Poorly - Mebendazole, Griseofulvin, Colchicine, Vincristine/Vinblastine, Paclitaxel

Question 13

Cilia structure

Answer 13

9+2 arrangement — axonemal dynein links peripheral 9 doublets

Question 14

Kartagener syndrome

Answer 14

Primary ciliary dyskinesia - dynein arm defect – infertility, increased risk of ectopic pregnancy, bronchiectasis, recurrent sinusitis, situs inversus

Question 15

Na/K pump

Answer 15

Located in plasma membrane with ATP on cytosolic side – 3 Na go out of cell (phosphorylated) and 2 K come into cell (dephosphorylated)

Question 16

Collagen

Answer 16

MC protein in body - stains with red safranin – Be (So Totally) Cool, Read Books (types of collagen)

Question 17

Type I collagen

Answer 17

MC - Bone, Skin, Tendon (decreased in osteogenesis imperfecta type I)

Question 18

Type II collagen

Answer 18

Cartilage (including hyaline), nucleous pulposus

Question 19

Type III collagen

Answer 19

Reticulin (skin, blood vessels, uterus, fetal tissue, granulation tissue) – deficient in vascular type of Ehlers-Danlos syndrome

Question 20

Type IV collagen

Answer 20

Basement membrane, basal lamina, lens – defective in Alport syndrome, targeted by antibodies in Goodpasture syndrome

Question 21

Collagen synthesis

Answer 21

Inside fibroblasts: Syntesis (RER - translation of preprocollagen, glycine content!) –> Hydroxylation (RER - specific proline and lysine residues, requires vitamin C) –> Glycosylation (RER - forms procollagen with hydrogen and disulfide bones - problems forming helix in osteogenesis imperfecta) –> Exocytosis –> Outside fibroblasts: Proteolytic processing (cleaving disulfide terminal regions and making tropocollagen) –> Cross linking (reinforcement by covalent lysine-hydroxylysine linkage - problems in Ehlers Danlos)

Question 22

Osteogenesis imperfecta

Answer 22

BRITTLE BONE DISEASE – MC form is autosomal dominant with decreased production of type I collagen – multiple fractures with minimal trauma, blue sclerae (choroidal veins), hearing loss (ossicles), dental imperfections (lack of dentin)

Question 23

Ehlers-Danlos syndrome

Answer 23

Bad collagen synthesis – hyperextensible skin, tendency to bleed, and hypermobile joints (mutation in type V collagen) — can also have vascular type (very rare, type III collagen defect)

Question 24

Menkes disease

Answer 24

X-linked recessive CT disease d/t impaired copper absorption and transport (defective Menkes protein) — decreased activity of lysyl oxidase – brittle, kinky hair, hypotonia, growth retardation

Question 25

Elastin

Answer 25

Lots of nonhydroxylated proline, glycine, and lysine residues — no triple helix – cross linking takes place extracellularly and gives elastic properties (involves desmosomes and lysine) – broken down by elastase (normally inhibited by a1 antitrypsin)

Question 26

Polymerase chain reaction

Answer 26

Amplify a desired fragment of DNA — denaturation, annealing (during cooling, primer binding to DNA single strand), and elongation

Question 27

Blotting procedures

Answer 27

SNoW DRoP – Southern = DNA, Northern = RNA, Western = Protein

Question 28

Microarrays

Answer 28

Used to profile gene expression levels - able to detect single nucleotide polymorphisms and copy number variants

Question 29

ELISA (Enzyme Linked Immunosorbent Assay)

Answer 29

Direct (detects a specific antigen) — Indirect (detect a specific antibody or antigen)

Question 30

FISH

Answer 30

DNA or RNA probe binds to specific gene site of interest – look for microdeletions at molecular level

Question 31

Cloning methods

Answer 31

Isolate eukarytoic mRNA –> exposre to reverse transcriptase to produce cDNA –> insert cDNA into bacterial plasmids –> transform recombinant plasmid into bacteria –> surviving bacteria produce cloned DNA

Question 32

Hydrophobic amino acids

Answer 32

Glycine, Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan, Methionine, Proline — found in transmembrane or membrane associated domains