CSF block 4

the small subunit binds tRNA and mRNA, the largesubunit forms peptide bonds

binds to large subunit, blockspeptide bond formation, indirectly blocks movement from A to P sites

Ribosomes have Protection proteins (RPP’s) which protectagainst antibiotics. e.g. Tet(O). Tet(O) binds large and small subunits. kicks tetracycline out of A site

Protein degradation required more than one ubiquitinto be added. They are added in a chain. A minimum of 4 ubiquitins needed for protein degradation. (initial one added to lysine of substrate

remove ubiquitin molecules (also called “Deubiquitinatingenzymes” DUBS). They can cleaveubiquitins one, or more than one at a time

nucleus,mitochondria,plasmamembrane,Golgi,peroxisomes, lysosomes, or are retained in the endoplasmic reticulum. Other proteins leave the cell through vesicular transport from the golgi.

H bonds between bases and stacking forcesbetween adjacent base pairs on the spiral stair case of the helix

1. coiling of the series ofnucleosomes into a helical array to form a fiber of ~30nm. Packing ratio of 40

high predictive value, analytic technique has tobe reproducible, cost must be low, test must support decisions on treatment

22 proteins constitute 99% of total plasmaprotein mass, concentrations below limit of detection byspectrometers. Deplete of majorproteins and improved sensitivity of spectrometers is needed.

Pinocytosis: cell drinking

Receptor mediated endocytosis

Phagocytosis: largeparticles taken in (e.g. bacteria), involves major membrane and cytoskeleton rearrangementsand NRG.

it binds to the collar ofa coated pit (which has lots of clathrin) and pinches off endocytizedvesicles from the plasma membrane. Ituses GTP.

flask shaped pits distinct from coated pits andoccupy ~10% of the surface of endothelial cells. Vesicles also budoff from caveolae during endocytosis but more slowly than from coated pits.

1. early endosomes bud offmembrane to their interior to generate multivesicular bodies. MVBs mature into late endosomes that are distinguished by having lower pH and are denser.

1. Late endosomes fuse with primary lysosomes by a processemploying Rab proteins

1. Early endosomes recycle back to the cell surface either directly orby passing through the TGN

• enzymes missing or defective (gauchers, tay-sachs)
  
• problem with attachment of mannose-6-phosphate to lysosome bound enzymes (I-cell)
  
• lysosomaltransporters defective, accumulation of nutrients inside lysosome (Niemann-Pick)
  

• defect in dystrophin gene
  
• X-linked recessive: female carriers, males affected, 1/3 of cases are new mutations, 2/3 have carrier mothers
  
• Insidence 1/3000

• Gene: 2.4 Mb and 79 exons (large size makes genetic testing hard)
• Protein: Rod shaped (structural), stabilizes muscle fiber during contraction, links actin cytoskeleton to extracellular matrix

• loss of function of RNA-binding protein that is important in maturation of synapses in neurons
• amount of CGG repeats at 5' untranslated region determines severity
• full mutation means no protein produced (gene is hypermethylated, not transcribed)

• severe movement probs: chorea (quick movement of feet/hands, dance-like), dystonia (sustained muscle contraction), cognitive decline.
  
• Adult onset, 25% don't present til 50, a few til 70, (rare juvenile onset 10%)
• prevelence: between 3 and 7 per 100,000

• CAG repeat that makes abnormal protein with lots of glutamine (CAG) that doesn't interact normally with proteins and accumulate certain neurons
• CAG amount correlates disease: >40 repeats= disease
• Problem due to GAIN of protein function
• autosomal dom.

• Inhibits EGFR (epidermal growth factor) dimerization (activation), which is needed for tumor formation in colorectal cancer.

1. -Metacentric = p and q of equal length

   -Submetacentric = p slightly shorter than q

   -Acrocentric = centromere near end of short arm

   (P= short arm, Q= long arm)

• G-banding: pattern light and dark bands (widely used, sharp delineation of chromosome regions)
  
• Q-banding: fluorescent banding ranging in intensity, good for showing heteromorphisms on acrocentric chromosomes, can follow chroms through generations

• onset: puberty for males, third month of life for females
  
• spermatogensis continuous and last 64 days, egg production discontinuous and required fertilization to complete
• four games produced for males, one for females

• segregation of alleles: each gamete only contributes one copy of a particular gene (allele)
  

• transfer of one chromosome segment to another chromosome.
  
• reciprocal= two chromosomes exchange pieces
• balanced = no genetic material lost in translation

single chromosome composed of two identical copiesof a chromosome arm as mirror images separated by a centromere

• two breaks in the chromosome with portions being inverted
  
• Paracentric: two breaks and inversion occur within chromosome arm
• pericentric: one break in short, one in long arm, with inversion piece including centromere

• Caretaker:make proteins that maintain integrity of genome, inactivation= genomic instability
• Gatekeeper: make proteins that directly regulate growth of tumors by inhibiting mitosis and promoting apoptosis. Each cell types has one or a few gatekeepers

• Deregulation gene expression of a structurally normal gene
• Formation of chimeric fusion gene (BCR-ABL in chronic myeloid luk)

• Brings ABL (chrom 9) and BCR (chrom 22) together
• codes for overactive tyrosine kinase
• rearrangement can be monitored by FISH
• for patient monitoring you need both G-banding and FISH

• 4 generations, list all paternal and maternal relatives whether or not they had cancer
• age at cancer diagnosis/age of death
• pathology
• history of oophorectomy (removal of ovary(s)) or hysterectomy, CRC polyps (including # and pathology)

• DNA itself is never toxic
  
• gene product is made within the cell (exogenous protein often can't enter/has short serum half life)
• Continuous production of gene product

• Transient gene transfer: DNA remains outside the chromosome (episomal)
• Stable gene transfer: DNA integrates into genome.

• remember it means the gene is actually incorporated into the chromosome
• advantages: gene will be perpetuated
• Disadvantages: random integration, could be integrated into heterochromatin, could activate oncogenes or inactivate tumor suppressor genes

• identify common sequences that other known genes have like certain promoters, splice acceptors and termination sequences.
• take a protein and infer its sequence to identify the gene
• presence of homologs genes already known in other species looked for

• Moderately repetitive: short sequences repeated 10-1000X
• Highly repetitive: very short sequences repeated many thousands of times. ("satellite DNA, which resides in heterochromatin is an example)

• Transposons (45%)
• processed pseudogenes (.1%)
• simple sequence repeats (3%)
• Segmental duplications (5%)
• Tandem repeats:

• pharmacogenomics: is the study of variations of DNA and RNA characteristicsas related to drug response.
  
• pharmacogenetics: is a subset of pharmacogenomics and is defined as theinfluence of variations in DNA sequence on drug response.

  
  

• probability of getting disease given that you have the disease (all or nothing)
  
• given the same mutation, degree of disease severity
  

• allelic: different mutations in same gene produce same clinical phenotype
• locus: mutations occuring in different genes produce same clinical phenotype
• phenotypic: different mutations in same gene produce distinctly different clinical phenotypes

• germ line: no body really works on this, it would be changing someones gametes so next generation isn't affected
  
• somatic gene therapy: common

• causes thickening of stool and blockage
  
• pancreas gets plugged
• vas deferens doesn't form during development (not known why)

• remember CF autosomal recessive
  
• deletion of single phenylalanine at residue 508 (class II mutations that distrupts folding and prevents new CFTR from exiting ER) (4 different classes of mutations all dealing with folding/trafficking/function of CFTR
  

• digest DNA with restriction enzymes
  
• fraction by electrophoresis
• immobilize them on a membrane
• the labeled probe is then introduced which anneals with it's complimentary sequence on the membrane

• DNA amplification without cloning
  

• bind promoter elements, grabs one strand of DNA and takes it inside it to use it to make a transcript, the other strand of DNA is looped outside of the polymerization site, does not require primer
  
• made 5' to 3' like DNA

• Core promoter: made of TATA box, initiator sequence (Inr), downstream prom elem. It recruits transcription factors so RNA pol II binds
• Silencers/enhancers: determine strength/activity of promoter. thousands of NT's up or down of promoter, or in intron

• poly A tail is about 80 to 250 adenylate residues
• 5' cap is made of 7-methylguanosine and methylribose (a protein hooks on to the 5' cap and delivers it to the ribosome)

• 70 works like a clamp. when ATP is hydrolyzed HSP 70 clamps down and has ADP bound, it's very stable now. When ADP leaves and ATP binds again, the clamp opens
• HSP 60 is the barrel

• Type 1: C terminus outside (made by signal sequence plus stop transfer sequence)
• Type 2: N terminus outside (made by a signal anchor sequence)

• regulates ER size and lumenal chaperone content.
  
• It is type 1 membrane protein, that has lumenal "sensing" domain, cytosolic kinase and RNase domains

• regulated by XBP1 transcrip fac, which gets cleaved by cytosol RNase domain of IRe1, protein is re-ligated then translated to give active protein, which up regulates ER chaps( BIP, etc.).
  
• Also increases ER lipid synth, size, folding capacity of ER

• ER quality con type 1 membrane prot.
  
• decreases protein load delivered to ER
• Works by sensing amount of unfolded prot in ER, dimerizing, autophosphorylating, then phosphorylating eIF2 alpha (i.e. shuts down prot translation)

• Helix-turn-helix, zinc finger (e.g. steroid hormone receptor family), leucine zipper, helix-loop-helix, etc.
  
• These proteins make base-specific contact with DNA (could be almost any attractive force: H bond, electrostat, contact phosphate backbone, etc)

• Co-activators: some have enzyme activity that promote transcription (histone modifying enzymes, chromatin remodeling)
  
• Co-repressors: often uses chromatin remodeling etc in opposite ay of co-activators
  

• Acetylation: makes chromatin active (euchromatin). thought to mark chromatin regions so chromatin remodeling enzymes can work on it
• Hypoacetylated makes silent chromatin (heterochromatin). (histone and DNA methylation also silences

• bind DNA as dimers to either inverted or direct repeats of DNA
• They can either activate or repress gene expression
• They are target of some common therapeutic agents: thyroid hormone, vitamin D, estrogen, etc.
• in absence of a ligand they repress transcrip

• like a southern blot scaled up to measure thousands of DNA sequences at once
• each array has many thousands of probes
• DNA or RNA samples hybridize with the probes on the array and their pattern is examined

• Expression profiling: complimentary sequences on the microarray that test for transcriptome. mRNA converted to cDNA and labeled before test. 2 or more samples compared to see diffs in expression
• genotyping: arrays scan for over 1 million SNP'

• allows sequencing of many different DNA strands simultaneously
• drastically reduces time to sequence individ genomes

• massively parallel sequencing of clonallyamplified DNA molecules that are fixed and spatially separated in a flow cell.

• contain about 40 enzymes
  
• most require pH of 5
• lysosomes range in size a lot (.05 to .5 microns)

• E1: activates ubiquitin
• E2: conjugating enzyme
• E3: ubiquitin protein ligase (has one of 2 domains: HECT and RING.)
• Ub transfered in 2 ways: directly from E2 catalyzed by RING of E3. Or via HECT of E3
• It's E2/E3 complex that recognizes substrate!

• retrograde translocation to the cytosol
• ubiquitination machinery, transport machinery and degradation machinery are linked

microsomal triglyceride transfer protein) lives in ER, transfers lipids from inner membrane to incoming lipoprotein, helps them fold. MTP inhibition lowers cholesterolcause Apo B isn’t made.

binds small ribosome subunit that has a mettRNA bound. whole complexbinds mRNA and moves along til reaches start codon. eIF2 alpha has GTP bound that hydrolyzes whenit reaches start codon

• all amyloid diseases lead to amyloid fibrils (huntingtons, parkinsons, etc.).
  
• PROCESS of misfolding and not the particular protein that causes probs
• normal protein folding relies on side chain interactions, amyloids on peptide backbone interactions

• PrP is normal protein in body, PrP(c) can undergo slow changes to harmful PrP(Sc) protein ("scrapies" form). This causes chain rxn where more are converted.
  
• PrP(Sc) form fibrils that build up in neurons, kill them, leave holes that look like sponge

• nucleation polymerization: small oligomer acts as seed for recruiting and changing PrP(c) into PrP(Sc)
• template assisted polymerization: a mysterious chaperon X changes PrP(c) into intermediate that uses PrP(Sc) as template to change

• Standard antiviral and bacterial approches useless
• almost negligible immune response

• senile plaques due to accumulation of B-amyloid and abnormal NF tangles
  
• APP present on cell surface and is processed to B-amyloid
• Tau protein is abnormal

• amyloid precurser protein: very hydrophobic, which is a major force for aggregation

• Their expression goes down, this might help explain health decline with aging
  
• Crystallins: only alpha-crystallin has chaperon activity and is constitutively on in muscle, heart and eye. alpha-crystallin efficiency decreases with age

• Galactokinase deficiency: associated with cataract formation, which isbelieved to be due to the accumulation of galactitol in the lens
  

• Epimerase deficiency: benign condition. accumulation of UDP-gal. impact on glycoproteinbiosynth

• Galactose is required for the synthesis ofglycoproteins and glycolipids.
  
• abnormal glycosylation of FSH causes ovarian failure
• abnorm. glycosylation can cause cognitive impairment in white matter

• Newborns: Jaundice, failure to thrive, liver dysfunction, renal tubulr dysfunction (fanconi syndrome), and gram negative sepsis.
  
• Children: learning probs, speech dyspraxia, low bone dens, ovarian failure

• deficiency in the aldolase B enzyme that converts fructose-1-phosphate to DHAP and glyceraldehyde.
  
• lack of aldolase B makes fructose-1-phosphate accumulate, which inhibit fruktokinase leading to buildup of fructose in blood

• Gluconeogenesis is reduced. accumulation of fruc-1-phosphate inhibits two gluconeogenesis enzymes (glucose-6-phosphate isomerase and aldolase).

• ALT (alanine amino transference) and AST (aspartate aminotransferase) areliver enzymes that are normally found at low levels in blood
  

• elevation of these in HFI is result ofthe metabolic probs that occur after eating fructose

• increase in LDL, builds up in arteries, tendons, skin
• autosomal dominant: heterozygotes: two fold increase. Homozygoes: extremely elevated
• defective LDL receptor
• Population incidence of 1/500.
  

• HMG-CoA reductase inhibitor (statin) can achieve upto about 50 % reduction in LDL cholesterol. Statin-typedrugs are reasonably effective in heterozygous

  
  

• Statins not affective for homozygotes

• Total= <200 mg/dl
• LDL <190
• HDL >35

• Several enzymes form a pathway of synthesis, It's mostly regulated by first enzyme (ALA synthase) by feedback inhibition

• all nucleated cells have machinery to synthesize heme. Heme pathway is most active in bone marrow (where RBSc's made) and liver (CY P450's made)
  
• Synthesis happens in cytosol and mitochon. Formation of porphyry ring happens in cytosol

• Acute intermittent porph/variegate porphyria: ab pain, neuro probs
• Porphyria cutanea tarda/hereditary coprophyria: lesions in latter stages of heme synthesis, photosensitivty

Acute, cutaneous, and mixed. Acute is with abdominal pain, constipation,vomiting, and neuropsychiatric disorder. Cutaneous is photosensitivity only.

Some of his relatives showed skin sensitivity based upon medical accounts oftheir physicians. The second article now implies that heavy metal poisoning,particularly arsenic, might have contributed to George’s symptoms.

Lead poisoning inhibits a number of enzymes in the heme biosynthetic pathway,particularly the ferrochetolase, the last step. Heavy metals inhibit enzymes bind to andinactivating sulfhydryl groups, the inhibition would benoncompetitive

Jaundice is not a porphyria. Jaundice is a condition (not a disease) that iscaused by heme breakdown. Have the students describe jaundice to you.

Unclear, but this is a good time to emphasize to students that a differentialdiagnosis is difficult to perform in the absence of a patient

increased oxygen, intracellular HbS concentration, and thepresence or absence of HbF.

• gel electrophoresis and takesadvantage of the difference in mobility of Hb caused by the replacement of anegatively charged residue with a non polar one.
  

• High in West African (20-30% carry the gene), AfricanAmerican

These fall into four categories: Inhibition of HbS polymerization, reduction ofintracellular Hb conc., induction of HbF, and, of course, transfusion in severecases.

• X-linked recessive disorder of Cu deficiency
• neuro degeneration, kinky depigmented hair, connect tissue probs (weak bones, skin laxity
• die before 3 yo

associated with deletions, chromosomal rearrangements, or mutationsin the gene ATP7A. causes reduced transport of dietary copper across enterocytes to hepatic portal circulation. Traps Cu in GI tract so it can't get to other places

• autosomal recessive
• accumulation of Cu in liver and other tissues
• causes liver failure, chronic hepatitis, cirrhosis
• Cu can also build upon brain causing neuro/psych probs (abnormal behavior, depression, tremors etc)
• 
  

• mutations in gene ATP7B (Menkes is ATP7A).
• mutates proteins needed for Cu excretion.
• Cu accumulates in Liver, brain

Menkes disease protein (ATP7A) and Wilson’s disease protein (ATP7B) are the copper-transporting ATPases. large transmembrane proteins that use energy of ATPhydrolysis to transport copper from the cytosol across cell membranes

Both proteins are located in thetrans-Golgi network (TGN) where they transport copper from cytoplasm into the lumen ofTGN for incorporation of copper into secreted copper-dependent enzymes

1. HER-2 overexpression predicts responsiveness to trastuzumab.The trastuzumab antibody binds to the ectodomain of HER-2 and modulatesHER-2 actions. Tumor cells that express low levels of HER-2 don’t respond totrastuzumab

Humanized antibodies are effective as therapeutics because hypersensitivityreactions are diminished when most of the antibody consists of human-specific amino acid sequence. antibodies also show very high affinity and specificity

• autosomal recessive
• inherited mutation in enzyme glucocerebrosidase, causes accumulation of substrate glucocerebroside in lysosomes
• Enzyme found in all cells, but most important lysosomes of phagocytes in bone marrow, spleen, liver
  

Gaucher’s is autosomal recessive. Four specific mutations are responsible for most cases in the Ashkenazi Jewish population. Thesemutations are less prominent in other populations. Phenotype does not correlate withgenotype.

abnormal hemoglobin levels, Enlarged liver and/orspleen is often seen. Bone marrow biopsy would show gaucher cellsengorged with glycolipid. enzyme assays for glucocerebrosidase would showno or little activity. MRI and CT

Unlike the case with Tay-Sachs disease, Gauchers carriers can not be picked up byenzyme assay screen. Heterozygotes show normal to greatly reduced activity.

• ERT is expensive, continuous administration and monitoring required.
  
• another option is removing spleen, bone marrow transplant, or gene therapy.
• substrate reduction therapy has been suggested

• phenotype under a particular environmental condition, is identical to the one of another individual whose phenotype is determined by the genotype.
  
• both cause limb reduction, cardiac defects, scoliosis. Holt-oram caused by TBX5 mutation, autosomal dom

• Genes + environment
• no distinct pattern inheritance
• risk much lower for 2nd deg. relatives than 1st, but declines less rapidly for remote relatives
• recurrence risk higher when more than one family member affected.
• consanguinity increases risk

• early onset of bone and soft issue sarcoma, adrenocortical cancer, and other tumors
• rare, autosomal dominant, related to to P53 mutation
• by age 30 50% of p53 mutation carriers have cancer diagnosis

• Mitchondria: oxidative phos, TCA cycle, acetyl-Coa production, fatty acid oxidation (beta oxidation)
  
• cytoplasm: glycolisis, fatty acid synthesis, pentose phosphate path, steroid synth