MCBM 3

How to use RNA for cloning? cDNA library formation

*Start with RNA not DNA Every cell in the body has same genome, but the mRNAs are variable -mRNA (coding sequences of messages) from a cell can be isolated by taking advantage of its polyA tail **Looking for the expressed component of the genome/cell that is expressed in the tissue 1. Isolate the mRNA processed has polyA tail 2. Hybridize with Oligo-dT (poly T) primer 3. Intubate with reverse transcriptase RNA dependent DNA polymerase 4. Have the hybrid can degrade the RNA with RNase H 5. Synthesize complementary DNA stand using DNA polymerase: RNA fragments act as primer 6. End up with double stranded cDNA that is a copy of the original mRNA 7. Generate cDNA library then how do you clone the particular fragment you want use hybridization

Trisomy 21: Down Syndrome

*aneuploidy caused by nondysjunction

Single strander conformational polymorphism (SSCP)

*indirect genetic testing 1. DNA is denatured into single stands 2. Single strands fold: shape is altered by mutations 3. Mobility of mutant and normal strands differ in gel

Non-disjunction at meiosis II

- 1 disomic daughter cell will be trisomic (2n+1) at fertilization; May be viable - 1 nullisomic daughter cell will be monosomic (2n-1) at fertilization; Not viable unless sex chromosome - 2 monosomic daughter cells will be euploid (2n) at fertilization

Sphingolipidosis

-Sphingolipids degraded in lysosomes -Requires lysosomal exoglycosidases to remove sugars -Deficiency → accumulation of sphingolipids in lysosome -Nervous tissue most affected bc of high sphingolipid • Dx requires measuring enzyme activity in leukocytes, skin fibroblasts or amniotic cells (for prenatal diagnosis) • Autosomal recessive (except for Fabry disease - X linked recessive)

Treatment for Inborn Errors of Metabolism describe 4 ways?

1) Avoidance of stimulus that promotes disease episode 2) Dietary restriction of substrate that cannot be metabolized (most effective if implemented immediately after birth; might need to be be life-long) 3) Exogenous replacement of missing metabolite 4) Diversion of accumulated metabolite into alternative pathways to promote excretion

Indications for carrier (asymptomatic) screening:

1) When there is family history of a genetic disorder 2) When parents have had an affected child (parents could be carriers) 3) When marriage is consanguineous (higher likelihood of homozygosity for autosomal recessive loci)

Molecular changes in chronic Burkitt's lymphoma and CML. What is used to treat CML?

1. ABL (tyrosine kinase) gene is fused to BCR. This fusion becomes active means an over expression of oncogene product --> excess proliferation of cells via tyrosine kinase turning on 2. Myc gene is placed in a chromatin domain that is highly active in B-cells, that antibody producing cells (IgH locus) **Gleevec, an inhibitor of Abl, is used to treat CML.

Which complex in ETC has ATP synthesis?

1. ATP synthase is complex V 2. H+ through F0--> rotation of C-ring -> catalytic domain activated 3. F1 active domain: ATP synthesis and H+ gradient dissipates

What else can SNP microarrays detect?

1. Absence of heterozygosity (AOH) or loss of heterozygosity (LOH) 2. Regions of homozygosity (ROH) can be cause by: -consanguinity, incest -common haplotype block shared by parents who are of same ethnicity (haplotype: distinctive chromosome segment associated with a region or ethnicity) -Uniparental isodisomy (both copies of a chromosome are identical and are inherited from the same parent) 3. ROH can identify likely loci for an autosomal recessive trait -look at areas of ROH that affected siblings in consanguineous families have in common "autozygosity" gene mapping

Describe the formation of ketone bodies from FA? What are the 3 ketone bodies?

1. Acetacetate is reduced to β-hydroxybutyrate or decarboxylated to acetone 2. Rate of ketone body formation is regulated by the rate of β-oxidation 3. NAD+/NADH ratio during FA oxidation determines how much 3-hydroxybutyrate is formed Ketone bodies: 1. Acetoacetate 2. Acetone 3. 3 - Hydroxybutyrate (test ordered for ketone bodies)

Acetyl CoA Carboxylase regulation inactive and active form?

1. Active acetyl CoA carboxylase self associated to form multimeric filmanetous complexes used to convert acetyl CoA to malonyl CoA 2. Inactive form ACC is a protomer (dimer) the enzyme undergoes allosteric activation by Citrate which causes dimers to polymerize form active polymer, activated via insulin 3. Allosteric inactivation by long-chain fatty acyl CoA, inactivated by AMPK 4. Requires Biotin

Entry point pyruvate what 4 aa?

1. Alanine 2. Serine 3. Cysteine 4. Aminoacetone from Threonine

Alcohol & Gluconeogenesis

1. Alcohol metabolism reduces the pool of cytoplasmic NAD+ 2. Malate cannot be oxidized to oxaloacetate in cytoplasm 3. Gluconeogenesis is inhibited

Metabolism of non Pro-drugs

1. Already in active form when the patient takes it

Integration TCA cycle into biosynthesis pathway

1. Amphibolic (anabolic and catabolic functions) 2. Interfaces with biosynthetic pathway via intermediates 3. Cycle intermediates are used for the synthesis: -neurotransmitters -heme -aa -nucleotides -FA -glucose 4. intermediates removed need to be replenished via anaplerotic reactions

RFLP analysis for Detection of Mutations

1. Amplicons from patient DNA (by PCR) 2. Determine size of digested fragment and infer genotype of patient 3. Utilize the restriction site via restriction enzyme via base pair change removed or added a restriction site

Examples of deformation? example?

1. Anomaly caused by an abnormal mechanical force that alters the shape of, but does not destroy, an otherwise normal organ without halting morphogenesis. -Presence of an abnormal force -Absence of a normal mechanical force 2. Oligiohydramnios sequence-Potter sequence -renal agenesis -oligohydramnios -pulmonary hypoplasia -in utero contraint -genu recurvatum -rocker bottom feet 3. Breech position in utero -term baby -facial asymmetry -alveolar ridges approximate on right side first -pressure from foot in utero distorted mandible 4. Compression & extra-uterine gestation -placenta attached to exterior of uterus -fetus compressed by mother's abdominal organs -pulmonary hypoplasia from low amnionic fluid -windswept toes 5. Micrognathia, facial compression due to a transverse lie

OAA entry point which AA?

1. Aspartate -transamination to OAA 2. Asparagine -Hydrolysis to Aspartate + NH4 -Transamination to OAA

Genetic Risk and Hardy-Weinberg Application?

1. Assume that an autosomal recessive condition has an incidence of 1/10,000 2. Say, the frequency of normal and mutant alleles are p and q (p + q =1) 3. AA(p2); Aa (2pq); aa (q2) q=1/100; p = 99/100 carrier frequency = 2pq = 2(1/100)(99/100) = 1/50 • Affected child's parent producing another affected = parent's carrier risk x new spouse's carrier risk x 1⁄ 4 (recessive disease) = 1 x 1/50 (population frequency) x 1⁄4 = 1/200

Chromosomal Rearrangement and the formation of antibody diversity?

1. At anytime B-cells are capable of producing 10^15 different antibodies, which is several orders of magnitude higher than the number of genes 2. The process is accomplished by involving a recombination scheme and recombinase enzymes: combinatorial arrangement

Malformation syndrome: Adams Oliver syndrome

1. Autosomal dominant trait variable expression and incomplete penetrance 2. Transverse terminal defect - Ectrodactyly 3. Scalp and skin defects 4. Symmetric lesions 5. Check parents for small scalp defect 6. Can be mistaken for amniotic band sequence, symmetry of lesions rules that out * high recurrence risk (intrinsic)

Odd Chain FA oxidation?

1. Beta oxidation proceeds until a 3 Carbon fragment is left 2. Propionyl CoA -the only FA that can be considered gluconeogenic 3. 3 new enzymes are needed to metabolize propionyl coA 4. ultimate product is succinyl CoA which is an intermediate in TCA cycle (conversion propionyl coA requires (ATP, CO2, Biotin, B12 are necessary) 5. succinyl CoA can form malate which can be converted into glucose in the liver via gluconeogenesis

Describe the role of gluconeogenesis in the homeostasis of blood glucose levels?

1. Blood glucose levels are maintained through -Glycogenolysis in liver (24 hr supply) -Gluconeogenesis in liver and kidney 2. After 24 hr fast glycogen stores are depleted and glyconeogensis is only source of blood glucose

Preparing Karyotype

1. Can stain with Giemsa (G-banding) or Quinacrine stain (Q-banding) 2. Different stains yield different pattern and different resolution 3. Cytogeneticists count # of chromosomes & examine banding patten 4. ID numerical and structural abnormalities Not very high resolution, prenatal screening

Urea cycle explain steps? How many ATP used and how many high energy phosphate bonds are cleaved?

1. Carbamoyl phosphate is transferred to ornithine which forms citrulline 2. Citrulline is transported to cytosol 3. These 2 amino acids are not coded for in proteins 4. Aspartate combines with Citrulline • Forms arginosuccinate • Used one more ATP 5. Aspartate formed from Oxaloacetate 6. Arginine is formed from arginosuccinate 7. Arginine is transformed into ornithine and a urea molecule is liberated 3 ATP used but 4 high energy phosphate bonds are cleaned

Gene therapy : Severe Combined Immuno deficiency (SCID)

1. Caused by impaired functions of B and T cells 2. Patients are unable to mount any immune response. Could result in life-threatening infections 3. Autosomal recessive SCID patients have adenosine deaminase deficiency 4. X-chromosome linked SCID is most common ** X-SCID is due to IL2R (interleukin-2 receptor) deficiency. Bone marrow CD34+ cells from X-SCID are treated ex vivo for correcting the IL2R deficiency IL2R is needed for cytokine mediated used development development bone marrow cells

Difference between cerebrosides and ceramides and sphingolipids

1. Cerebrosides: (sugars attached) important cerebral tissue - Glucose or galactose connected to terminal OH grp by glycosidic bond 2. Ceramide - Sphingosine w/ single acyl group attached via amide linkage -Altered by addition to terminal OH grp 3. Sphingolipids -Sphingosine formed from serine as backbone and FA 4. Sphingomyelin -Sphingomyelin is the sphingosine version of phosphatidyl choline, with a phosphate- choline residue connected to the terminal hydroxyl group by an ester bond

Type I Von Gierke's disease which enzyme deficient?

Glucose-6-Phosphatase

Major Genetic Diseases 4 types?

1. Chromosomal disorders (involved in growth/mental retardation; spontaneous abortions) • Deletion or addition of an entire chromosome • Down syndrome (trisomy 21) 2. Single gene disorders • Hypercholesterolemia (1/500) • Sickle cell anemia (1/400 blacks): glu to val • Cystic Fibrosis (1/2000 whites) 3. Polygenic diseases (complex disorders) • diabetes mellitus, hypertension, schizophrenia 4. Somatic Cell Genetic Disorders • arise only in specific somatic cells • Cancer

α-thalassemia describe chromosome that alpha is on? Name the conditions with number of α's present in each?

1. Chromosome 16 has two copies of normal α- globin genes, a pseudo α-globin and many blocks of repetitive DNA. 2. Unequal crossovers between misaligned X- or Z-repeats can produce chromosomes with 1-3 active α- or no active α-globin. 3. Recombination between two chromosomes occurs the chromosomes align abnormally then crossover or recombination can remove one or two of the alphas ** don't have alpha get alpha-thalassemia Normal = 4 α Silent carrier = 3 α α-thalassemia trait = 2 α HbH Disease = 1 α Hydrops Fetalis = 0 α (high degree of mortality)

CYP2D6 and Codeine

1. Codeine is a pro-drug & is metabolized to morphine (active) by CYP2D6

Coupling of ETC to ATP synthesis

1. Complexes use E from e- transfer to pump H+ to inter membrane space-> creation of H+ gradient and electrical gradient (electrochemical potential) 2. H+ re-enter mitochondrial matrix through ATP synthase and drive ATP synthesis

Beta-Oxidation Defects : Carnitine deficiency

1. Compromise tissues which rely heavily on oxidation of FA for energy -Skeletal muscle -Liver (during fasting period) 2. Carnitine deficiency: in the liver leads to hypoketotic hypoglycemia during period of extended fasting -during fasting FA oxidation is needed to generate acetyl CoA for ketogenesis and ATP for gluconeogenesis -Carnitine deficiency compromises both pathways

ATP synthesis and the role of mitochondria?

1. Contain own circular genome and ribosomes 2. Maternal inheritance (egg provides cytoplasm ) 3. Exercise induces mitochondrial biogenesis 4. e- from cytoplasmic NADH (glycolysis) -> transported into mito via shuttles (malate-aspartate shuttle)-> from cyto NADH to mitochondrial NADH ~glycerol-3-P shuttle -> from cytoplasmic NADH to mitochondrial FADH2

Gene Therapy 2 ways to correct a defect? Gene therapy methods 2 ways?

1. Correction of a defect -Germ-line correction (ethical concerns) -Somatic cell correction can be achieved by: a. Gene augmentation-introduce functional copy: for loss-of function diseases b. Gene replacement (in situ correction) - for gain-of function diseases c. Targeted inhibition of activated oncogenes or pathogens d. Targeted killing- cancer treatments Ex vivo: Correct the defect in patient's cells outside of the body and then inject the corrected cells In vivo: Directly inject the cloned gene

Inhibition of PG Synthesis

1. Cortisol - inhibits phospholipase A2 → less arachidonic acid avail -inhibits COX-2, but NOT COX-1 2. ASA asprin (and indomethacin and phenylbutazone) inhibit COX-1 and COX-2 → inhibits PGH2 synthesis => COX-1 inhibition → stomach and renal cell damage → ASA toxicity 4. COX-2 inhibitors (celecoxib) - maintains physiologic function of COX-1 while reducing inflammation

Triacylglycerols characteristics? Utalization TG?

Glycerol and 3 FA ==> TG 1. The FA at carbon #2 is frequently arachidonic acid 2. The key is to get non polar compounds to function in polar environment 3. Usually taken in with diet with aqueous solution and dumped in acidic environment of the stomach --> problem FA are insoluble

Southern Hybridization Lab workup? Southern hybridization what is the target? what probe is used? what are you looking for to determine?

1. DNA stained and separated gel-electrophoresis 2. Intubate with stain dye so it can fluoresce 3. Transfer onto nylon membrane 4. Look for fragment via hybridization with labelled probe, where there is annealing or hybridization have an impression of that fragment Gene structure can be determined by how long is the gene, restriction map in the genome gene has 1. Electrophoresis of the Digested DNA 2. Hybridizing with a gene specific Probe that contains a radioactive or fluorescent label (probe can be DNA or RNA) Target: DNA

Beta oxidation what are the four reactions? How many ATP's per turn?

1. Dehydrogenation (generating FADHs) 2. Hydration 3. Oxidation (generating NADH) 4. Thiolysis releases acetyl CoA each step is catalyzed by enzymes with chain-length specificity There four reactions then repeated FADH2 = 2 ATP (in ETC) NADH = 3 ATP ** Each turn of the cycle yields 17 ATP's (5 from NADH and FADH2 and 12 from the acetyl CoA)

Potential Therapies Using Pluripotent Stem Cells (ES or iPS)

1. Diabetes (200 million patients with cost of 10-15% of health care budget) • Concept is to form differentiated beta cells in vitro, and introduce through hepatic portal vein (previously done using organ donors) to lodge into liver (where cells will sense and respond to glucose levels in blood). • iPS cell line can be a genetic match, alleviating need for immunosuppression. • Limited applicability for type 1 disease, which involves immune attach. • Slow progress as existing therapies are largely effective. 2. Parkinson's Disease (1-2% Lifetime Risk with Loss of Dopaminergic Neurons) • Challenge is the neurons be replaced in proper context positions in tissue with proper connectivity's (tissue grafts do not form neuronal connections). • Safety concerns involve persistence of a few pluripotent cells in graft that may form teratomas (tumors). • Alternative therapies involving administration of L-DOPA and deep brain electrical stimulation may prove more effective. 3. Spinal Repair • Clinical trials involved goal of remyelinating cells to treat spinal trauma (2009). • Tumor risks resulted in study suspension. • Future potentials remain promising but unclear.

Bile acids purpose what do they metabolize?

1. Dietary triglycerides are metabolized by bile acids 2. Red meat = more fat = more bile acid 3. Net result is likely tumor promotion manifesting as colon cancer 4. Bile acids emulsify dietary TG and then pancreatic lipase works on what is left of TG

Mutation heterogeneity? examples?

1. Different mutations (alleles) in SAME locus (gene) produce disease phenotypes of different severity

Ketogenesis happens when?

1. During fasting FA are liberated and are the major source of fuel 2. Acetyl CoA generated by beta oxidation is used by the liver and converted into ketone bodies 3. Ketone bodies are oxidized for energy by tissues such skeletal muscle, BRAIN, kidney, intestinal mucosa 4. Produced in the liver when the amount of acetyl coA present exceeds oxidative capacity of the liver

How are FA released? What is the signal? fasting or fed? What cleaves the Fa from tricacylglycerol?

1. During fasting cAMP levels rise in adipose cells due to the decrease in insulin and rise in glucagon and EN 2. Stimulates lipolysis 3. PKA phosphorylates hormone sensitive lipase to produce the active form of the enzyme 4. Hormone sensitive lipase cleaves a FA from triacylglycerol 5. Other lipase complete the process of liberating the FA and glycerols into the blood

Standard reduction potentials and e- flow through ETC?

1. E0=standard reduction potential (tendency of a redox pair to lose e-) 2. E0 <<<<0 e- donor in redox pair loses e- easily 3. E0 >>>>0 e- acceptor in redox pair accepts e- easily Path of e- down ETC from E0 <<<0 (-0.32V) to E0>>>0 (+0.82V) NADH has a E = negative means can lose e- well O2 has E = positive can accept e- well

Effect on cSNPs on protein function: silent cSNP and synonymous define effect on protein?

1. Effect of cSNP on aa sequence determined by its codon position (1st, 2nd vs 3rd; 3rd codon position more likely to be a silent cSNP due to degeneracy of genetic code) 2. If not silent cSNP, effect determined by nature of sequence change (synonymous vs non-synonymous) -synonymous : substitution with same type aa, ex. acid with acidic 3. Non-synonymous results in substitution with different type of aa, acidic with hydrophobic etc)

Type V Mc Ardle's which enzyme deficient?

Glycogen phosphorylase

FA elongation steps?

1. Elongation in the smooth ER 2. Palmitate can be activated to palmityl coA which can be elongated 3. Malonyl CoA is the 2 Carbon donor 4. Requires NADPH which supplies e- 5. Similar to SA synthesis except the Fatty acyl chain is attached to coenzyme A instead of phosphopantetheinyl residue of ACP 6. The major product is Stearic acid

Changes in chromosome Ploidy? Euploidy? Aneploidy? Polyploidy?

1. Euploidy is the normal chromosome number - 2n (46 chromosomes) for somatic cells - n (23 chromosomes) for gametes 2. Aneuploidy is a change in the # of a single chromosome • can be gain or loss of a chromosome (autosome or sex chromosome) • chromosome # is different than 2n (2n-1 for loss; 2n+1 for gain) ** LOSS of autosome is NOT viable (spontaneous abortion) ~ GAIN of autosome can be compatible with life (trisomies 13, 18, 21 are viable) • loss or gain of sex chromosomes is viable (X0, XXY) • Aneuploidies => gametes: 1. Nullisomic (n-1: lack of a chromosome) 2. Disomic (n+1: extra copy of a chromosome) 3. Polyploidy is the gain of an entire set of chromosomes - chromosome # is 3n, 4n ... - not viable (spontaneous abortion

What parts of the AA are used for each carbon skeleton? the amino group? What products can be formed from aa?

1. Excess aa cannot be stored 2. Suplus aa are used for fuel 3. Carbon skeleton is converted to -Acetyl-CoA -Acetoacetyl-CoA -Pyruvate -TCA cycle intermediates: -Succinyl-CoA -Fumarate -OAA 4. The amino group Nitrogen is converted to urea and excreted 5. Glucose, FA and ketone bodies can be formed from AA 1. Proteins are degraded into AA 2. Protein turnover is tightly regulated 3. Fist step in protein degradation is the removal of nitrogen 4. Ammonium ion is converted to urea 5. Carbon atoms are converted --> major metabolic intermediates

Peroxisomal Oxidation, what kind of FAs are oxidized? When does it stop? Is carnitine required for this type of oxidation?

1. Exclusive site of very long chain FA oxidation (24-26 carbon) -the CoA esters of eicosanoids -2-methyl-branched fatty acyl-CoAs -CoA esters of the bile acid intermediates di- and trihydroxycoprostanoic acids (cholesterol products) 2. Oxidation stops when a 4-6 carbon fragment is generated 3. The long chain FA acyl CoA synthetase in the peroxisomal membrane does not require carnitine to translocate acyl CoA derivatives across the membrane!

FA carbonyl group pKa? What do double bonds to the melting point of different FA? Such as stearic acid? Oleic acid? Linoleic acid? Which FA are essential?

1. FA carbonyl group has pKa 4.8 2. Double bonds decrease the melting point of FA, does not allow for good stacking (unsaturated fat) 3. Double bonds are usually in cis configuration -Stearic acid Tm=70 C -Oleic acid Tm= 16 C -Linoleic acid Tm = -5 C (essential) omega-6 FA and arachadonic acid (omega-6 FA) -alpha-linoleic acid Tm= -11 C is an essential omega-3 FA

what are some other FA derivates? FA are precursors for?

1. FA from the diet are a major precursor of glycerophospholipids (GPL) and sphingolipids 2. GPL are components of lipoprotein, bile, and lung surfactant 3. GPL is also the source of PUFA's (polyunsaturated FA) especially arachidonic acid which serve as the precursors of eicosanoids 4. Either GPL's have an alkyl group joint to Carbon 1 of the glycerol backbone (platelet activating factor)

Malformation: Isolated Cleft lip/Palate and Van der Woude syndrome

1. Failure of premaxilla to fuse with lateral facial processes at 7 weeks gestation 2. Usually inherited as a multifactorial trait 3. Can occur in isolation or as part of syndromes ** don't forget to look lower lip: van der woude syndrome ** Usually multifactorial trait • Autosomal dominant trait -in complete penetrance • IRF6 gene mutation -50% • One of the few syndromes expressed as cleft palate alone or cleft lip/palate • Cleft palate • Cleft lip • Lip pits, salivary gland fistulas in lower lips • 70% who inherit the mutation have a cleft that requires surgery

Coordinate Regulation of PDH & Pyruvate Carboxylase in Glycolysis and Gluconeogenesis when fasting/starving?

1. Fasting & starvation => increase in fatty acids from lipolysis 2. Fatty acids=> AcCoA (via β-oxidation)=> increase in hepatic [AcCoA] 3. AcCoA is allosteric effector for PDH & Pyruvate Carboxylase 4. AcCoA => inhibits PDH => diverts pyruvate away from TCA cycle 5. AcCoA => activates Pyruvate Carboxylase => directs pyruvate to glucose synthesis

Describe the Carnitine Shuttle?

1. Fatty acyl CoA crosses outer mitochondrial membrane 2. Fatty acyl CoA then exchanges the CoA for carnitine by the action of the enzyme carnitine palmitoyltransferase I (CPTI) 3. The complex then enters the mitochondrial matrix thanks to facilitated diffusion by carnitine-acylcarnitine translocase. Fatty acyl carnitine is translocated into the matrix as carnitine moves out via Carnitine translocase 3. Fatty acyl carnitine converted to fatty acyl coA via Carnitine palmitoyltransferase II and can continue to Beta-oxidation 4. Carnitine then diffuses back across the membrane by carnitine-acylcarnitine translocase into the mitochondrial intermembrane space. This is called the carnitine shuttle system. 5. Which one translocated the Fatty acyl carnitine into the matrix? Carnitine acylcarnitine translocase

Ectodermal Dysplasia: X-linked Ectodermal Dysplasia

1. Females can be mildly affected with thin hair, poor teeth and patchy sweating • Males have missing or needle-like teeth, absent sweating, dry skin, alopecia

Morphogen

1. Folic acid -neural tube defects, cleft lip, cardiac anomalies Morphogen - A factor required for normal morphogenesis • Folic acid is a water soluble B vitamin: Vitamin B9 • Supplementation with folic acid reduces the incidence of many birth defects, including neural tube defects and diabetic embryopathy. • The CDC recommends that all women of reproductive age take a folic acid supplement of 400 micrograms, daily. • After the birth a child with spina bifida or anencephaly, a woman should take 4 milligrams daily to reduce the chance of a neural tube defect in the next pregnancy. • The average American female consumes about 140 micrograms of folic acid/day from dietary sources alone

Penetrance, Fully penetrant versus Incompletely penetrant? Expressivity?

1. Fraction of individuals with SAME genotype that shown expected phenotype -Fully penetrant: phenotype ALWAYS expressed when genotype is present -Incompletely penetrant: phenotype expressed in a Fraction of individuals when genotype is present -Measured across individuals of same genotype NOT WITHIN an individual -Expressed as a percentage of individuals that express expected phenotype (ex. 80 % penetrant for incomplete and 100% for complete) 1. Range of phenotypes produced by same genotype 2. Phenotype is variable among individuals of same genotype

What is a free radical?

1. Free radicals are molecules with highly reactive unpaired e- that can exist independently (not as part of an enzyme) 2. Very detrimental/toxic to cells 3. Involved in a number of disease (cancer, Parkinson's, diabetes, aging)

Formation of TAG's from Glycerol-3-P

1. G3P reacts with fatty acyl CoA form phosphatidic acid 2. Dephosphorylation of phosphatidic acid produces a diacylglycerol 3. Another fatty acyl CoA react with the diacylglycerol to form a triacylglycerol 4. TAGs are produced in smooth ER of the liver 5. They are packaged along with cholesterol, phospholipids, and proteins to form VLDL 6. Microsomal triglyceride transfer protein (MTP) is required for VLDL assembly 7. MTP is required for chylomicron assembly

DNA testing: genomic, genetic, epigenetic

1. Genomic: -Low resolution--> chromosome analysis -High resolution --> a. Microarray analysis (SNP, oligo) b. Whole genome sequencing c. Whole exome sequencing 2. Genetic -Indirect testing --> linkage analysis SSCP -Direct testing --> known mutation: ASO a. Unknown mutation: gene sequencing b. Deletions, duplications-Southern Blot, heteroduplex 3. Epigenetic --> Methylation

Reciprocal Regulation of Glycolysis and Gluconeogenesis? High insulin which pathway turned on glycolysis or gluconeogenesis? Glucagon which pathway is turned on?

1. Gluconeogenesis & glycolysis are competing pathways (substrate of one is product of other) 2. Competing pathways are reciprocally regulated 3. Reciprocal regulation => same signal keeps one pathway ON and competing pathway OFF 4. If competing pathways are both on simultaneously futile cycle occurs 5. Reciprocal regulation prevents futile cycling ** Liver 1. Insulin turns ON glycolysis and OFF gluconeogenesis 2. Glucagon turns OFF glycolysis and ON gluconeogenesis

What happens to the G-6-P after glycogenolysis by glycogen phosphorylase and conversion G-1-P to G-6-P via phosphoglycomutase?

1. Glucose-6-phosphate CANNOT be transported out of hepatocytes 2. Glucose-6-phosphatase produces glucose from glucose-6P 3. Glucose-6-phosphatase ONLY present in liver & used in glycogenolysis & gluconeogenesis

Deamination of AA? What happens to the ammonium ion in protein degradation?

1. Glutamate dehydrogenase -Used NAD/ NADP 2. In the Mitochondria where the toxic effect of ammonium ion can be isolated Urea

Antioxidant Defense Enzymes: Glutathione & Glutathione Peroxidase

1. Glutathione & glutathione peroxidase detoxify H2O2 2. Prevent H2O2 from generating OH· via Haber-Weiss & Fenton runs 3. Glutathione becomes oxidized (G-S-S-G) in the process & needs to be recycled by glutathione reductase (NAPDH needed from pentose phosphate pathway) 4. Glutathione peroxidase need selenium 5. Glutathione & glutathione peroxidase found both in cyto. and mito.->detoxify H2O2 produced outside of peroxisomes

Glycerol converted to what in gluconeogenesis? Alanine is converted to what via which enzyme?

1. Glycerol is converted to dihydroxyacetone in 2 steps 2. Alanine directly converted to pyruvate through alanine aminotransferace (ALT)

Entry of glycerol into gluconeogenesis?

1. Glycerol is phosphorylated to glycerol-3-P by (glycerol kinase ONLY present in LIVER) 2. Glycerol-3P oxidized to DHAP by glycerol phosphate dehydrogenase

Describe Bypass I Gluconeogenesis?

1. Glycolysis Phosphoenolpyruvate (pyruvate kinase)--> pyruvate 2. Gluconeogenesis Pyruvate (pyruvate carboxylase)-> OAA (PEPCK)-> PEP

Devenport Diagram

1. Graphical tool that helps a physician to diagnose the causes of acidosis or alkalosis. 2. Because a specific area in the diaphragm represents a specific disorder 3. Blood sample from a healthy individual in a chamber the PaCO2 being held at 40 mmHg 4. HCO3- and pH experimentally determined a titration curve for the sample is produced 5. The curve a CO2 isopleth, represents all possible combinations of HCO3- and pH at PaCO2 of 40 mmHg 6. Points on the line and outside of the pH range 7.35-7.45 represent metabolic acid-base disorder.

Uncouplers of Oxidative Phosphorylation

1. H+ leak across membrane-> disruption of gradient-> uncoupling of e- flow and ATP synthesis 2. Energy is dissipated as HEAT (fever, hyperthermia) 3. Salicylate is partial uncoupler-> aspring poising (fever & lactic acidosis)

Huntington's Disease and repeats? Normal is? Mutable/premutable? Disease?

1. HD expansion of CAG (polyglutamine repeat) in coding region of gene 2. HD alleles in 3 categories based on # of repeats 3. Normal < 26 4. Mutable/premutation 27-35 (asymptomatic but can expand in germ line & transit to offspring) 5. Disease > 36 (but 36-39 may not develop disease or late-onset disease)

FA characteristics?

1. Highly reduced relative to carbs -hydrophobic hydrocarbon chain with hydrophilic carboxyl group with pKa 4.8 2. Have lots of energy which can be liberated 3. Stored anhydrous which is very efficient 4. Stored as triglycerides (not as free FA which are toxic)

Three types of recombinations?

1. Homologous or general recombination • Reciprocal exchange of DNA sequences. Common during gametogenesis but also observed in somatic cells. 2. Site specific recombination • Observed in viral genome integration. Viral RNA into host DNA 3. Transposition • Carried out by transposable elements (~50% of genome). Responsible for antibiotic resistance in bacteria.

Gene Therapy by Repairing a Mutant Gene or Inactivating a Pathogenic Gene : different ways list 4 ways?

1. Homologous recombination -a mutant gene can be replaced by a normal gene 2. Antisense oligonucleotide -a disease causing gene can be inactivated 3. Ribozyme -the mRNA transcript of a disease causing gene is degraded 4. RNA interference: -target the mRNA of the target gene for degradation

Regulation of Glycogenolysis Fasting?

1. Hormonal regulation: Glucagon activates protein kinase A=> phosphorylates phosphorylase kinase => phosphorylase kinase ON => phosphorylates glycogen phosphorylase => glycogen phosphorylase ON => glycogenolysis ON 2. Allosteric effectors: ATP, glucose-6P & glucose => Glycogen phosphorylase OFF (feed-back inhibition)

Regulation of Glycogenolysis Fed state?

1. Hormonal regulation: Insulin activates protein phosphatase 1=> dephosphorylates glycogen phosphorylase => glycogen phosphorylase OFF => glycogenolysis OFF 2. Protein phosphatase 1 also directly dephosphorylates phosphorylase kinase => phosphorylase kinase OFF => glycogen phosphorylase OFF=>glycogenolysis OFF

Regulation of glycogen synthase when is it turned on which 2 ways can it be regulated by which substrates or products?

1. Hormone regulation -Insulin activates protein phosphatase-1 => dephosphorylates glycogen synthase => glycogen synthase turned on => glycogenesis ON 2. Allosteric effectors: -Glucose-6-P=> glycogen synthase ON (feed-forward stimulation) -Glucose = Protein phosphatase 1 ON (liver only)

How many ATP's are generated for every acetyl CoA made in Beta-oxidation? How many ATP'S per acetyl CoA through the TCA cycle?

1. How many ATP's are generated for every acetyl CoA made in β-oxidation?! => 5 ATP's 2. How many ATP's per acetyl CoA through the TCA cycle? => 12 ATP's **129 ATP for 16-Carbon FA with 7 NADHs, 7 FADH2, and 8 acetyl coA for 18 Carbon = 129+17 ATP = 146 ATP for 14 Carbon = 129 -17 ATP = 112 ATP

Metabolic change in type I diabetes?

1. Hyperglycemia - decreased uptake by GLUT4 in muscle, adipose -increase in gluconeogenesis via loss of PEPCK repression 2. Ketosis (can result in life-threatening ketoacidosis) -Due to increased lipolysis in adipose & subsequent increased hepatic ketogenesis 3. Hypertriacylglycerolemia -excess FAs converted to TAGs & packaged into VLDLs - to elevated chylomicrons &VLDLs because of decrease in lipoprotein lipase activity

Gout

1. Hyperuricemia with recurrent attacks of acute arthritic joint inflammation causes of deposition of monosodium urate crystals 2. Underexcretion of uric acid 3. Allopurinol inhibits xanthine oxidase results with compounds hypoxanthine and xanthine which are more soluble than uric acid

Disruption: Amyoplasia with amnionic band and amniotic band sequence

1. Hypofusion/hypoxia 2. Decreased fetal movement 3. Diminished muscle mass 4. Policeman's tip arm 5. Circumferential constriction -umbilical cord and amnionic band -decreased movement may prevent amputations 1. Asymmetric amputations 2. Circumferential constriction bands 3. Distal swelling 4. Normal anatomy proximal to lesions 5. Distal and proximal amputation 6. Low recurrence risk

Trinucleotide repeats and disease? When does this occur? # of repeats and threshold #

1. Increase in the number of repeats in successive generations 2. Occurs during gametogenesis and expanded repeats transmitted to offspring 3. Slippage by DNA polymerase during replication in gametes 4. If # repeats exceeds threshold # then disease occurs 5. High repeat number leads to earlier onset & severity of disorder (anticipation)

Inborn errors in Metabolism

1. Inherited as recessive traits (autosomal, X-linked) 2. Single gene disorders with Menedelian or mitochondrial inheritance 3. Mutant enzyme not 100% activity 4. Mutations that directly affect enzyme activity (mutations in gene encoding enzyme) - > can affect protein folding/stability, active site, cofactor binding site 5. Different mutations (alleles) in same gene give rise to different disease phenotypes (severity)

What are the inhibitors of ETC? What happens to the carriers before the block are they oxidized or reduced what about after block? What components are blocked with each inhibitor and what is the inhibitor acting on?

1. Inhibit specific complexes of ETC by binding to ETC components and blocking redox reactions 2. Block formation of H+ gradient and ATP synthesis stops 3. Carriers before block are reduced and carriers after block are oxidize

Aspirin

1. Inhibits TXA2 synthesis in platelets by irreversible acetylation (of Ser residue) and inhibition of COX-1 by preventing binding of arachidonic acid 2. Irreversible inhibition overcome by - Endothelial cells w/ nucleus →produce more COX-1 3. Basis for low dose ASA therapy → lower MI and strokes

Hypoglycin A

1. Inhibits acyl CoA dehydrogenase 2. Rare aa (or is it an alkaloid?) is heat labile and found in unripe akee fruit 3. Leads to fasting hypoglycemia 4. Symtoms include vomiting, convulsions and coma 5. Affects people with low levels of acyl CoA DH severely

Regulation of Glycogenolysis during Exercise? What serves as the exercise signal for glycogenolysis in muscle?

1. Insulin & glucagon regulate blood glucose levels and signal metabolic states 2. Muscle DOES NOT respond to glucagon 3. Glycogenolysis stimulated in exercising muscle

Diabetes Mellitus and Gluconeogenesis?

1. Insulin resistance or lack of insulin in diabetes mellitus results in loss of repression of PEPCK 2. Gluconeogenesis is stimulated and glucose is produced, contributing to hyperglycemia in Diabetes Mellitus

What 2 enzymes are needed for metabolizing unsaturated FA?

1. Isomerase : enoyl CoA isomerase (one double bond) for monounsaturated oleic acid 2. Reductase (for polyunsaturated FA) : 2, 4-dienoyl CoA reductase (isomerase + reductase for 2 or more double bonds) for linoic acid

ARMS PCR/Allele-specific PCR

1. Known mutations 2. Allele specific PCR 3. ASP1 and ASP2 and conserved primer 4. Size fractionation by gel electrophoresis and interpretation of amplification pattern to infer genotype of patient

How does lactate enter gluconeogenesis?

1. Lactate is directly converted to pyruvate through lactate dehydrogenase

Mapping genes for multifactorial traits/disorders? Common methods used? Two types of associated studies?

1. Linkage analysis and Association studies 2. Associated studies ==> candidate gene association + genome-wide association • Candidate gene association study: Case-control study to test if specific variants (in candidate genes) have higher frequency in cases vs controls (i.e. associate with disease in statistically significant fashion) • If statistically significant association then variants are involved in disease (causative variants) = susceptibility loci

Reciprocal Regulation of Glycolysis and Gluconeogenesis? Low energy? High energy? which enzymes are turned on and off in glycolysis and gluconeogenesis?

1. Low energy charge=>Low ATP -gluconeogenesis OFF (requires ATP) -glycolysis ON (to generate ATP) 2. High energy charge => High ATP -gluconeogenesis ON (requires ATP) -glycolysis OFF (sufficient ATP) 3. Low energy charge => -PEPCK & PC & F-1,6 Bpase OFF -PFK-1 ON 4. High energy charge=> -Pyruvate kinase and PFK-1 OFF • Fructose-2,6-BP is allosteric effector for: PFK-1 ON Fructose-1,6-Biphosphatase OFF

Chromosome analysis requires dividing cells where are the chromosomes coming from? Steps for chromosome analysis?

1. Lymphocytes - terminally differentiated cells divide in the bone marrow 2. Green top tube - sodium heparin = no clots 3. Mitogens -> stimulate cell division in white cells -T cells => PHA, hytohemaglutinin -B cells => Pokeweed 4. Cochicine - paralyze cells in metaphase 5. Hypotonic solution - spreads out the chromosomes 6. Banding level - 400 bands routine, > 550 high resolution 7. Staining Method - Trypsin and Giemsa (G banding)

VLDL what is the role of LPL what are the end products? Where is LPL secreted? What controls LPL? What is the major protein in VLDL? What state is the body in? What happens to the glycerol?

1. Major protein in VLDL is apoB-100 2. VLDL is processed in Golgi complex and secreted into the bloodstream by the liver 3. Lipoprotein lipase (LPL) cleaves the TAGS in VLDL (and chylomicrons) forming FA and glycerol 4. LPL is secreted by adipose cells when the insulin/glucagon ratio is elevated (fed state) 5. FA enter adipose cell and form TAGs in the same fashion in the liver 6. Adipose cells lack glycerol kinase and cannot use the glycerol produced by LPL and hence the glycerol is taken up the liver and reused

Bicarbonate Buffer System

1. Major source metabolic acid: CO2 2. Up to 22 mole/day 3. Catalyzed carbonic anahydrase (CA) 4. H2CO3 dissociates spontaneously into H+ and HCO3- ** Plasma pH may be affected by a change in either the bicarbonate concentration of the Partial Pressure CO2 (PaCO2)

Regulation of FA synthase how does malonyl coA play a role?

1. Malonyl CoA inhibits carnitine palmitoyltransferase II 2. Malonyl CoA levels are elevated when acetyl CoA carboxylase is activated thus mitochondrial beta oxidation is inhibited 3. This prevents a futile round of FA synthesis going on while beta oxidation is also on

5 ways cell defense mechanism against oxidative damage?

1. Metal sequestration -Transition metals (iron & copper) bound to cellular proteins (ex. iron with ferritin & hemosiderin; albumin for copper) 2. Antioxidant defense enzymes -> convert ROS to non-toxic products (catalase, SOD, GSH, Glutathione peroxidase) 3. Anti-oxidant vitamins->terminate free radical chain reactions: (Vitamin C, Vitamin E , beta-carotene) 4. Cellular compartmentation->ROS-generating reactions in distinct subcellular compartments with strong defense mechanisms (ex. peroxisomes) 5. Repair (DNA) or replacement (proteins & lipids) of damaged components ~When rate of ROS generation exceeds rate of defense mechanisms ==> oxidative stress

What AA enter TCA cycle via succinyl - coA

1. Methionine 2. Valine 3. Isoleucine ** Methionine forms S-Adenosylmethionine (SAM) role in metabolism 1. Methionine related to Homocysteine levels 2. Ultimately becomes Succinyl coA

Multifactorial/Complex Inheritance explain monogenic and polygenic.

1. Monogenic/single gene disorders & traits: alleles @ Single locus -3 possible genotypes produce 3 distinct phenotypes in population -Traits and disorders involving alleles at 2 loci would produce 9 possible genotypes & 5 distinct phenotypes in population 2. Polygenic: disorders and traits involve alleles @ Multiple loci; genes have additive effect on phenotype -many possible genotypes result in many phenotypes -phenotypes follow continuous and close to normal distribution (height, intelligence, skin color, blood pressure) -Gene X environment interaction results in greater phenotypic diversity ==> multifactorial/complex traits and disorders

How many diseases caused by triple repeat expansion? What is usually the repeat rich in? It can be transmitted as? Give some examples of diseases?

1. More than 20 genetic diseases due to triplet expansion 2. Repeats can be in exon, intron, 5' to 3' UTR of gene 3. Sequence subject to expansion varies but usually repeats are GC rich (CAG, CTG, GCG, CGC, etc) 4. Can be transmitted as autosomal dominant, autosomal recessive, or X-linked traits (all show anticipation) 5. Threshold # repeats for disease manifestation varies -Huntington disease (autosomal dominant) -Spinocerebellar ataxias -Synpolydatyly -Friedrich ataxia -Fragile X syndrome -Mytonic dystrophy

Medium Chain acyl CoA DH Deficiency

1. Most common inherited defect in the Beta-oxidation sequence 2. Affected enzyme catalyzes the first reaction is Beta-oxidation of medium chain (C5-C12) fatty acids 3. Short chain and long chain FA use two other dehydrogenase enzymes for the same reaction 4. Patients present with fasting hypoglycemia during infancy or childhood -Often during an infectious illness when the child eats little -Many cases go undiagnosed and some infants die during their first hypoglycemic attack (under circumstances suggesting sudden infant death syndrome)

Metabolism of pro-drugs

1. Must be converted to active form

Locus heterogeneity? examples?

1. Mutations in Different loci (genes) produced the SAME phenotype/disorder ex. Retinitis pigmentosa, BRCA1 & BRCA2 in familial breast and ovarian cancer, Bardet-Biedl syndrome

Mitochondrial disorders what happens? Who transmits it? Where is the mutation? Characteristics?

1. Mutations in mitochondrially encoded genes 2. Mother to child transmission (all children of affected mother are affected) 3. Father does not transmit 4. Mitochondrial genes involved in energy metabolism 5. Clinical symptoms in muscle, heart, CNS ** most code e- for the transport chain

Polyunsaturated FA (N-6 and N-3) effects on LDL and HDL. Effects of omega-3 FA on the body benefits?

1. N-6 (omega-6 FA) -linoleic acid -lowers both LDL and HDL -required for membrane fluidity and synthesis of eicosanoids 2. N-3 (omega-3 FA) -principally alpha-linolenic acid -little effect on LDL and HDL Varied effects: -Reduction of BP -Treating infection -Treating psoriasis -Anti-inflammatory - Antithrombotic

Complexes of ETC what is the path of NADH and FADH2?

1. NADH Complex I-> Co Q (Ubiquinone) -> Complex III -> Cytochrome C -> Complex IV 2. FADH2 Complex II -> Co Q -> Complex III -> Cyto C -> Complex IV Complex I (pumps H+) Complex II Complex III (pumps H+) Complex IV (pumps H+) catalyzes reduction O2 to H20 Cytochrome C - move freely carry e- from III -> IV Ubiquinone (Co Q) (non-protein component) - move freely carry e- from I -> III or II -> III ** bulk of oxygen in the body is used ETC

Non-disjunction meiosis I

1. Non-disjunction at meiosis I => all daughter cells are affected -Disomic daughter cells will be trisomic (2n +1) at fertilization; May be viable -Nullisomic daughter cells will be monosomic (2n-1) at fertilization; Not viable unless sex chromosome

How is phytanic acid oxidized?

1. Normally a minor pathway 2. Up regulated in MCAD deficiency

ω-Oxidation

1. Normally a minor process in metabolism seen in MCAD deficiency that limit fatty acid Beta-oxidation 2. FA are oxidized at omega end by endoplasmic reticulum enzymes 3. Process utilizes cytochrome P450, molecular oxygen and NADPH to oxidize the omega carbon to an alcohol 4. A dehydrogenase then converts the alcohol into a carboxylic acid 5. Omega oxidation produces dicarboxylic acids which can be utilized in beta oxidation

Oxidation of Unsaturated FA?

1. Not all FA's are ideally structured 2. Consider an unsaturated FA such as oleic acid (18:1,Δ9) commonly found in olive oil or linoleic acid (18:2,Δ9,12) 3. In β-oxidation a trans double bond is created between the α and the β carbons 4. For unsaturated fatty acids to undergo β- oxidation the naturally occurring cis bonds must be isomerized to trans double bonds which will end up between the α and the β carbons OR the bond must be reduced

Mendelian vs Multifactorial Inheritance

1. Number of genes and alleles contributing to phenotype makes ID of causative genes/alleles for multifactorial diseases a challenge 2. ID of causative genes & alleles = mapping disease genes and alleles

Mitochondrial Inheritance and Heteroplasmy and Homoplasmy?

1. Number of mutant mitochondria within a cell has to exceed a threshold (80%) for disease manifestation -Homoplasmy ==> all mitochondria within a cell have identical sequence (WT or mutant) -Heteroplasmy ==> mixture of wild-type & mutant mitochondria within a cell ** Heteroplasmy contributes to wide variability of symptoms in mitochondrial disorders

Where does Beta-oxidation occur in body? What do the mitochondria enzymes do?

1. Occurs both in mitochondria and peroxisomes 2. Mitochondria catalyze the β-oxidation of the bulk of short-, medium-, and long- chain fatty acids derived from diet -constitutes the major process by which fatty acids are oxidized to generate energy

Glycogenesis and Homeostasis of Blood glucose levels?

1. Occurs in fed state in liver & muscle 2. Helps prevent hyperglycemia because it allows sequestration of blood glucose 3. Glucose blood => => Glycogen liver, muscle

Oxidation of FA where does it occur? FA cannot get across the membranes and FA must be activated how do they get across?

1. Occurs in the mitochondrial matrix 2. Transport system : Carnitine Shuttle

Oleic Acid

1. Oleic acid content is responsible for reduction in blood pressure induced by olive oil 2. Olive oil intake may increase levels of oleic acid in membrane -This may regulate membrane lipid structure in such a way as to control G protein mediated signaling, causing a reduction in blood pressure 3. No effect with oleic acid analogues elaidic and stearic acids

Eicosanoids

1. Origin: 20 carbon long Polyunsaturated FA refers: - Prostaglandins (PG) - Thromboxanes (TX) - Leukotrienes (LT) 2. Cannot be synthesize de novo by humans (i.e. from glucose via palmitate) 3. From dietary plant oils which contain linoleic acid (most abundant consumed) essential 4. In humans linoleum acid can be elongated and desaturated to arachidonic acid --> PG 5. Act locally, produced small amounts, short half life, plasma and nuclear membranes mediate the action of eicosanoids

OAA transport from mito to cyto what is required?

1. Oxaloacetate needs to be transported to cytoplasm to complete reactions of bypass 1 via 2-step transport 2. Oxaloacetate reduced to malate by mitochondrial malate dehydrogenase (mMalate dehydrogenase) 3. Malate is exported into cytoplasm 4. Malate reoxidized to oxaloacetate by cytoplasmic malate dehydrogenase Mitochondrial--> Cytoplasmic Oxaloacetate---> Oxaloacetate

Inhibitors of Purine synthesis? How do they work?

1. PABA analogs -sulfonamides are structural analogs of para-aminobenzoid acid that inhibits folic acid synthesis, slow down the pathway in bacteria NO affect on humans cannot synthesize folic acid 2. Folic acid analogs -methotrexate inhibit the reduction of dihydrofolate to tetrahydrofolate -limit the amount of THF available for purine synthesis and slow down DNA replication, useful in treating rapidly growing cancers toxic to all dividing cells Activators: PRPP Inhibitors: Glutamine:phosphoribosyl pyrophosphate amidotransferase rate limiting step (-) AMP (-) GMP Inhibitors: Purine ribonucleotides (-) PRPP synthetase

PDH complex and lactic acid?

1. PDH issues get a buildup of pyruvate end up increase concentration drive lactate dehydrogenase forward produce too much lactate 2. Lactic acidosis & Neurological symptoms (brain uses lots of glucose) can't produce ATP from glucose

Formation of Superioxide anion by Coenzyme Q?

1. Passage of e- from Complex I -> CoQ and from CoQ -> Complex III involves the radical intermediate Co Q- 2. Co Q- can pass e- from O2 -> O2 - 3. Co Q is a MAJOR source of superoxide anion in cell ~ Antimycin A (Complex III inhibitor) may increase likelihood of superoxide formation ** Superoxide anion cannot diffuse far from site of origin but generate other free radicals

Maternal Triple Serum Screen

1. Performed non-invasively by blood draw • Maternal serum screen (MSS; triple screen) at 15-22 weeks gestation for: 1) alpha-fetoprotein (AFP; neural tube defects) 2) unconjugated estriol (uE3) Trisomies 18 & 21 3) human Chorionic Gonadotropin (hCG) Trisomies 18 & 21 • If MSS is positive ! additional diagnostic testing (CV sampling/amniocentesis)

Peroxisomal regulation? What is their main function? How are they regulated?

1. Peroxisomal alpha and beta oxidation and microsomal ω-oxidation are not feedback regulated 2. They function mainly to lower levels of H20 insoluble compounds which resemble FAs which are toxic to cells 3. Rate is regulated by availability of substrate

Fumarate production from which AA?

1. Phenylalanine 2. Tyrosine

Interpreting Test Results What do each mean: Positive for deleterious mutations, No mutations detected, Uncertain clinical significance?

1. Positive for a deleterious mutation • Once found in first person in family, able to easily detect in rest of family 2. No mutation detected • Mutation previously identified in the family • No known mutation in the family → cannot completely reassure risk is zero possible unrecognized mutation 3. Uncertain clinical significance - single NT change, never been seen or reported b4 • Variants of uncertain clinical significance can be further characterized by type of aa change • Can verify by testing family and see if they possess the mutation

LT Synthesis

1. Produced from arachidonic acid by the action of several lipoxygenases 2. Lipoxygenases are not affected by NSAID's 3. LT's are involved in allergic responses and inflammation - Used in treating asthma in the form of 5- lipoxygenase inhibitors and leukotriene receptor antagonists (Singulair)

Platelets and PG's

1. Produces/activates TXA2 → forms blood clots by - Adherence and aggregation of circulating platelets - Vascular smooth muscle contraction 2. PGI2 (prostacyclin) →inhibits thrombogenesis by - Inhibiting platelet aggregation - Vasodilation

Anticipation and Disease? Number of which repeats?

1. Progressively earlier age of onset and severity of symptoms 2. Correlates with number of repeats

Western Blotting Lab Workup? Western hybridization what are you looking for? What is the probe used?

1. Protein separation by electrophoresis -2D or 1-D separation 2. Transfer blotting of protein on membrane 3. Protein ID by antibody binding -incubation antibody 4. Protein-antibody complex detection by color development Abundance of a specific protein in a cell/tissue is determined by western hybridization - total protein isolated from cell is electrophoreses and immobilized onto a membrane and then probed with antibody

Describe Long Chain Branched FA? Most common? Where are they found? Breakdown product of what? How are they oxidized?

1. Pytanic acid and pristanic acid are the most common dietary LCBFA's 2. Breakdown product of chrolorphyll consumed through green vegetables 3. Not found in animals 4. Oxidized in peroxisomes to an 8 carbon branched FA which is then degraded further in the mitochondria

Summarize the mechanisms of cellular ROS production?

1. Reactive oxygen species (ROS) arise from O2 2. ROS are either free radicals or can be converted to free radicals 3. ROS formed by environmental factors, enzymatic or non-enzymatic reactions, mostly as byproducts of normal cellular metabolism

Refsum Disease

1. Recessively inherited defect of peroxisomal α-oxidation 2. Causes the accumulation of phytanic acid -Branched chain FA is derived from phytol -Phytol is a constituent of chlorophyll which accumulates in the fat of ruminants 3. The Beta-Carbon is methylated so alpha-oxidation is needed to shorten phytanic acid by one carbon 4. Round of Beta-Oxidation ---> yields propionyl CoA rather than acetyl CoA 5. Refsum disease characterized by slowly progressive peripheral neuropathy, irreversible neuro damage 7. Patients usually respond to dietary restriction of green vegetables and reduced amounts of remnant milk and meat

What is the role of Cori cycle?

1. Recycling of lactate generated by RBC and exercising muscle via gluconeogenesis in LIVER --> CORI cycle

FA Desaturation

1. Requires molecular oxygen, NADH and cytochrome b5 2. Occurs in the SER via enzymes desaturases that add cis double bonds 3. Most desaturations involve the formation of a double bond between carbon 9 and 10 which produce palmitoleic acid 16:1 4. Another common conversion is stearic acid into oleic acid 18:1 5. Other positions which can be desaturated in humans include carbon 4, 5, and 6 desaturases

Formation of Recombinant DNA Molecules

1. Restriction endonuclease recognize specific 4,6, or 8 Basepair sequences and generate restriction fragments with cohesive ends : 5' - PO4 and 3'-OH, Eco RI makes two cuts between base pairs 1 cut on 1 stand and second cut another stand 2. Ligase (reform the phosphodiester bond) can then join the two cohesive restriction fragments via matching base pairs annealing them and generate the recombinant molecule ** two fragments can anneal to each other based on the same matching ends of 3' and 5' regardless of the base pairs in between (based on the same cohesive ends can joint two different fragments) ** EcoRI (restriction endonuclease) recognizes the sequence GAATTC cleave between GA and AG on the stands

Teratogens

1. Retinoic Acid - Accutane -absence of external auditory canal -ear, aortic arch, brain defects, Sab 2. Thalidomide -limb defects, phocomelia 3. Tobacco -growth retardation, miscarriage 4. Alcohol (fetal alcohol syndrome) -poor grown, microcephaly -mental retardation, short palpebral -fetal alcohol spectrum affects up to 1% of population in US -heart defect, cleft lip

Liability/Threshold Model in Multifactorial Disorders? Define risk? Define liability? How does this relate to threshold?

1. Risk --> disease susceptibility based on genes alone 2. Liability --> ALL factors affecting disease development: genes and environment (G X E) 3. Liability distribution of population is continuous variable with normal distribution 4. Discrete liability threshold for disease development 5. Disease development is result of "bad" genes and "bad environment Key points of model: - Liability of majority of population is below threshold - Relatives of affected individual have more risk genes & higher incidence of disorder than general population (familial vs population incidence) - Risk increases the closer the familial relationship (1st vs 2nd vs 3rd degree) because amount of DNA shared increases

The uses of SNPs in molecular medicine?

1. SNP/cSNP ->disease causing mutations via candidate gene association/study 2. SNP can be linked with (close to) genes => can serve as markers for associated studies to ID disease genes: these SNPs are non-coding **SNPs are evenly & abundantly distributed throughout genome (appr. 1/kb) - Use I=> as markers for association studies to identify susceptibility loci - Use II=> can be causative agents for disease or causes of non-disease phenotypic variation (differences in drug metabolism, drug response); these are cSNPs

Antioxidant Defense Enzymes: SOD & Catalase

1. SOD -inactivates O2- -important defense because O2- initiated chain runs -exists as 3 isozymes -> cyto, mito, and extra cell -Cu needed active site 2. Catalase -detox H2O2 -prevent H2O2 from generating OH radical via Haber-Weiss & Fenton runs -found in peroxisomes where most H2O2 is produced

Dietary Lipids what are the three classes?

1. Saturated FA -myristic acid and palmitic acid elevate cholesterol levels more 2. Monounsaturated FA -lower both cholesterol and LDL with modest or no effect on HDL -olive oil high in oleic acid 3. Polyunsaturated FA -linoleic acid is a major dietary PUFA for humans ** unsaturated fats primarily interfere with plaque formation, interfere with inflammatory states, lower BP, regulate G-proteins

Single Nucleotide Polymorphisms (SNPs)

1. Single bp changes & bi-allelic (major and minor alleles0 2. Minor allele frequency >1 % 3. Most common form of sequence variation in human genome (90% of observed sequence variation) 4. Spaced @ 1 SNP/kb 5. Occur in coding & non-coding regions (coding regions SNPs are cSNPs) 6. cSNPs further classified by effect on aa sequence: silent, synonymous, and non-synonymous

Alternate modes of FA oxidation

1. Some FA can be oxidized by pathways other than Beta-oxidation 2. The function is to convert as many unusual FA into usable fuel or biosynthetic precursor 3. The remainder is to be excreted in bile or urine 4. Even large hydrophobic carboxylic acids resembling FA can be utilized in this manner

How is DNA cloned? What are the possible starting materials what are they called?

1. Starting material: DNA/RNA • If chromosomal DNA is used as a starting material it is called--> Genomic Cloning - Clones will contain genes with exons and introns. • If RNA is used as a starting material, which is converted into complementary DNA (cDNA) by reverse transcriptase this is called--> cDNA cloning - Clones will contain exon sequences, which can translated into proteins. • cDNAs can then be cloned in special vectors for making proteins, not every cDNA can be used to make proteins: - Desired hormone, protein or vaccine.

Criteria to establish stem cell line?

1. Stem Cell line Forms an Embryoid Body (similar to embryo) 2. Forms a Teratoma-type Tumor When Implanted Into Immunodeficient Mice. 3. Forms a Chimera when Implanted into Early Embryo.

Laboratory Dx and Screening of Cystic Fibrosis

1. Sweat testing: measure sodium and chloride levels 2. Immunoreactive trypsin test (IRT) 3. DNA analysis

Fluorescence in Situ Hybridization (FISH) on chromosomes Lab workup?

1. Take intact chromosomes hybridize chromosomes with gene-specific probes which are labelled with fluorescent molecules 2. Then see hybridization in various regions of the chromosome find there gene location this way • Takes advantage of hybridization of complementary nucleic acid sequences • Uses pt. metaphase chromosomes and fluorescently labeled probes • Visualized using fluorescence microscope

Peroxisomal Oxidation first step? What is the enzyme? Energy production? What is the end product?

1. The first enzyme is FAD-containing oxidase which directly transfers e- to O2 ---> hydrogen peroxidase 2. No energy produced in the first step 3. Different genes encode for the enzymes which proceed with steps similar to beta oxidation after initial step ** Formation of H2O2 hydrogen peroxide means there is peroxisomal oxidation happening 4. Peroxisomal β -oxidation cycle of straight-chain fatty acyl-CoA

How is nitrogen removed from AA? What happens to the α-Amino groups in the steps of protein degradation?

1. The first step in AA degradation is the removal of nitrogen -The Liver is the major site of protein degradation 2. Deamination produced α-keto acids which are degraded to other metabolic intermediates ~ α-Amino groups are converted to ammonium ions by the oxidative deamination of glutamate * glutamine most important nitrogen carrier protects the cell from the harmful effects of ammonium, ammonium levels are very toxic in the brain

NADPH produced by which two pathways?

1. The pentose phosphate pathway (also known as the hexose monophosphate shunt) (2 NADPH/glucose molecule) 2. Malic enzyme (which converts malate into pyruvate) produce NADPH most important for FA synthesis **NADPH is the reducing power in the cell, e- carrier

Serine and Threonine how are they deaminated? What is the end product?

1. The β-hydroxy amino acids, serine and threonine, can be directly deaminated into Pyruvate

What is the role of transaminases in protein degradation?

1. These enzymes funnel amino groups to α - ketoglutarate -Aspastate transaminase -Alanine transaminase ** enzymes used to measure the metabolic integrity of the liver, liver function test (LFT) determine any injury in the liver elevation of these enzymes means a hepatic injury

Production of Malonyl coA

1. This is the rate limiting step 2. Overall reaction is spontaneous 3. Prosthetic group is biotin linked to a lysine residue 4. Forms a flexible arm

Trans FA issues? What levels are raised in the body?

1. Thought to promote CV disease 2. Raise LDL levels 3. Mechanism not clear 4. Now banned in some cities

Debranching enzyme

1. Transfer 3 glucose segment from end of branch to non-reducing end of linear chain 2. Catalyzes hydrolysis of α(1,6) bond of branch point glucose & releases glucose 3. After branch removal, glycogen phosphorylase starts new cycle

Triacylglycerols precursor? What is the intermediate? where is the precursor made in the body?

1. Triacylglycerols produced by a pathway which has phosphatidic acid as an intermediate -Produced in liver, muscle, and adipose tissue 2. Glycerol-3-P is the glycerol backbone for triacylglycerol synthesis 3. G3P comes from different sources depending on the tissue type 4. In liver G3P is produced by glycerol kinase which acts on glycerol 5. In adipose tissue G3P can only be made from glucose via dihydroxyacetone phosphate -adipose tissue lacks glycerol kinase

Glycogenesis what is the substrate? Where are the substrates added on what end of glycogen?

1. UDP-glucose (not glucose) is substrate for glycogenesis 2. Sequential addition of UDP-glucose to non-reducing ends of glycogen 3. Mechanisms for chain elongation (creation of α(1,4) bonds) & for branch formation (creation of α(1,6) bonds) 4. Glycogen synthase creates α(1,4) bonds & branching enzyme creates α(1,6) bonds

Ammonia Transport?

1. Urea most important disposal route for ammonia 2. Glutamine -nontoxic storage and transport form of ammonia -very important for removal of ammonia in brain

Sanger DNA sequencing

1. Use when mutation is unknown and ID novel mutations 2. Can ID SNP/point mutations, deletions, insertions 3. Expensive and time consuming but highly accurate (used in BRCA1/BRCA2 testing)

Microarray Hybridization

1. Used primarily to ID novel mutations/changes (can also be used for known) 2. Screen entire genome or trancriptome with single assay 3. Expensive 4. Used for a variety of applications: detect chromosomal abnormalities (array CGH), gene expression changes (expression arrays), genotyping/GWAS (SNP arrays)

Exome Sequencing

1. Used to ID novel mutations 2. Sequence all human exons with single assay 3. Expensive 4. Used to ID genes for syndromes (Kabuki syndrome)

Southern Blotting

1. Used when mutation is known 2. Used for the detection of relatively larger rearrangements( insertions, deletions, duplications) 3. Used frequently for triplet repeat expansion disorders -Huntington's

Methods of delivering genes into cells?

1. Viruses -adenovirus -adeno-associated virus -lentivirus -HSV 2. Nonviral -lipososmes -injection/bombardment -receptor mediated

2 nitrogens in urea come from to enter the urea cycle?

1. ammonia 2. aspartate

Heteroduplex analysis (CSGE)

1. amplify and denature DNA 2. single-stranded DNA 3. Cold to reannealed the DNA 4. Run in gel

Adenosine Deaminase (ADA) deficiency

1. autosomal recessive deficiency causes severe combined immunodificiency (SCID) involving T-cell, B-cell, and NK-cell depletion (lymphocytopenia) 2. Untreated children die before 2 years of age from overwhelming infections

What array CGH cannot detect?

1. deletions too small to be detected with the probes in the array 2. balance chromosome rearrangements -reciprocal translocations -inversions 3. Triploidy 4. Low level Mosaicism 5. Mitochondrial DNA deletions 6. Gene mutations

α-Ketoglutarate Entry Point which AA?

1. glutamine 2. proline 3. arginine 4. histidine

Northern Hybridization Lab workup? Northern hybridization what is the target? what probe is used? what are you looking for?

1. mRNA transferred onto membrane 2. Look for the gene in the tissue use a probe corresponding to the gene 3. Hybridize with a labelled probe then wash and selectively retain probes looking for expression of the mRNA in various tissue Abundance, how big of a specific mRNA is a tissue/cell in determined by northern hybridization Target: RNA -target mRNA is hybridized to gene-specific RNA or DNA probe

Chronic myeloid leukemia which chromosomes are involved? What is the name of the chromosome that is formed?

1.Chronic myeloid leukemia (CML) patients have a small chromosome, Philadelphia (Ph) smaller than chromosome 22 native 2. Recombination-->Translocation joins the ABL oncogene on chromosome 9 with the 5' part of BCR gene on chromosome 22.

e- flow through ETC Schema

1/2 O2 + 2H---> H20

How many NADPHs are used per acetyl CoA during FA synthesis?

2 NADPHs/acetyl CoA

ALA ultimately becomes Protoporphyrin IX give some key products? which cycle?

2 molecules of ALA condense to form porphobilinogen 4 molecules of porphobilinogens condense to form hydroxymethylbilane ---> Reactions leading to coproporphyrinogen III (cytosol) ultimately Protoporphyrin IX heme synthesis cycle in the cytoplasm

How many ATP are produced for each NADH and FADH2 that enter ETC? Energy yield from glucose oxidation total?

3 ATP per NADH 2 ATP per FADH2 ~ fewer ATP per FADH2 because FADH2 bypasses Complex I • Approximately 50-60% of energy in proton gradient is not captured as ATP (heat and other reactions ) 36-38 ATP / glucose

Genome sequence

3.1647 billion base pairs ~ 25,000 genes function of a majority of genes not known genes in G+C rich regions many genes as families genes concentrated in random areas pseudogenes ** small fraction of genes are coding while mitochondria genome majority is coding

Estimated number of nucleotides and genes in the human genome.

3.3 billion NT

Klinefelter Syndrome

47, XXY

Dysplasia define.

A pattern of malformations affecting the same tissue type.

Association

A pattern on anomalies that occur together more often than expected by chance alone, but not in any single pattern and not necessarily with a single cause.

Syndrome define

A recognizable pattern of anomalies with a single cause

Describe what happens with respiratory acidosis? Hyper or hypoventilation? What Happens with CO2 levels? What are the consequences and how does the body compensate?

A retention of CO2 generally caused by respiratory problems (hypoventilation) Consequences: pH decreases, PaCO2 increase, HCO3- normal Compensate: PaCO2 remains high, pH normalized, HCO3- is raised; kidney retains HCO3-

Ubiquitin and protein degradation?

AA used for synthesizing proteins are obtained by degrading other proteins 1. Proteins destined for degradation are labeled with ubiquitin 2. Polyubiquinated proteins are degraded by proteosomes 3. AA are also a source of nitrogen for biomolecules

Y-linked

Affected males only, all affected males have affected father, all sons of affected male are affected

Reciprocal Regulation of Glycogenesis & Glycogenolysis: FED?

After ingestion of dietary glucose => glycogenesis ON very rapidly Glucose allosterically activates Protein phosphatase-1 and off glycogen phosphorylase and turns on glycogen synthase

Recombinant Technology what can be used? Which are used for short fragments, larger pieces, million basepairs?

Allows Cloning and stable propagation of the fragments by using Vectors, putting two pieces of DNA together and clone them in a variety of vectors: • Plasmids (cloning short fragments- a few kilo basepairs) • Phages (cloning of larger pieces- 20 kilo basepairs) • Yeast artificial chromosome vectors (cloning of million basepairs)

Disruption define? Disruption: Maternal and Vascular

Anomaly caused by an environmental or extrinsic factor that destroys otherwise normal tissue and interrupts normal morphogenesis. ** Genetic predisposition can be a factor in teratogenesis. • Maternal Disease -Diabetes -Phenylketonuria (PKU) • Maternal Infection -Rubella -Cytomegalovirus • Vascular accidents -Hypotension, placental hemorrhage/abruption - Thromboemboli - Amniotic Rupture

Malformation define? Malformation: Isolated Neural tube defect

Anomaly caused by intrinsic factors that result in abnormal tissue or defective morphogenesis 1. Failed closure of neural tube at 28 days 2. Multifactorial trait 3% recurrence risk 3. Folic acid can prevent 70% when taken daily, beginning prior to conception

Coordinate system ID position of the chromosomes?

Arms ==> regions==> bands Arms (p,q) subdivided into regions Regions subdivided into bands Bands sometimes subdivided into sub bands # centromere (1) to telomere (n) ** Specific banding pattern depends on the stain used Chromosomes counted and banding pattern from pt. sample analyzed to identify cytogenic abnormalities (trisomies, deletions, inversions, translocations etc)1

The significance of the BRCA1, 2 genes as well as relation to cancer?

BRCA1-2 Mutations Increase Risk of Cancer More Than Other Factors • mutations found thruout BRCA 1-2 genes make detection difficult • linked to familial ovarian/ breast cancer, up to 56-87% penetrance • Sequence Analysis of BRCA1 and BRCA2 Can Find Needle in the Haystack • the inheritance pattern (e.g. dominant) - Dominant X-linked (females) • the relative risk associated with a mutation in one of these genes as well as how it relates to the risk in the general population. BRCA 1-2 mutations INCREASE risk of early on-set breast cancer • the relationship of these genes to ovarian cancer. BRCA 1-2 mutations INCREASE ovarian cancer risk

What complex is needed for acetyl CoA carboxylase?

Biotin, ATP, CO2

Type IV Andersen's which enzyme deficient?

Branching enzyme

Chemical buffer system where do they occur ICF or ECF? How do they buffer? What are the name?

Buffer systems ICF: 1. Phosphate buffer system 2. Protein buffer systems, including: -hemoglobin buffer system (RBC only) -aa buffers (all protein) -plasma protein buffers Buffer system ECF: 1. Carbonic acid-bicarbonate buffer system 2. Protein buffer system

Describe Bypass 2 and 3?

Bypass 2 Glycolysis: Fructose-6P (PFK-1) -> F-1,6BP Gluconeogenesis: F-1,6P (F-1,6-Bpase)-> F-6P ~Fructose-6-P => glucose-6P by phosphoglucose isomerase Bypass 3 Glycolysis: Glucose (glucokinase)-> glucose-6P Gluconeogenesis: glucose-6P(g-3P phosphatase)-> glucose

The mutations seen in Fragile X patients and why Southern blotting is used to detect this disease as well as? Limitations of PCR for fragile X syndrome? Is the fragile X premutation really asymptomatic?

CGG Repeat in Fragile X Syndrome • X-linked, caused by expansion of tandem repeats • Normal range: 6-54 repeats • Premutation range: 52-200 repeats • Full mutation range: 200-> 1000 repeats • Alleles with >200 repeats are hypermethylated, transcriptionally repressed • Use southern blotting because of different sizes of DNA with different repeats. ~ full mutation gives not PCR product because polymerase falls off before able to amplify • Recent reports of premature ovarian failure in female premutation carriers • Late-onset tremor-ataxia-dementia syndrome in male premutation carriers • May be due to mRNA interference with expression of normal FMR1allele or of other genes

Alleles in relation to allele frequency? Hardy-Wrinberg distribution? Mating and allele frequency? What is the frequency in the first generation?

Hardy-Weinberg distribution Relationship between gene and genotype frequencies: - Allele frequencies do not change from generation to generation in large populations with random mating - This distribution is violated if mating is not random (that means there is inbreeding) • In such situations carrier frequency will be overestimated. Assume the frequencies of alleles A and a are p and q, respectively. • (p + q = 1) • Mating of Aa X Aa= AA; aa ; 2Aa • Frequencies in first generation = p2 ; q2; 2pq

Role of polymorphisms and genetic testing. The four major types of polymorphic testing and pros/cons of each

CLASSES OF DNA POLYMORPHISMS: 1. RFLP = Restriction Fragment Length Polymorphisms - restriction endonuclease recognition sites that are present in some people but not in others 2. VNTR = Variable Number Tandem Repeats - strings of nucleotide sequences (usually of 16 or more nucleotides each), of different repeat number in different people, existing between fixed restriction endonuclease sites; also called minisatellites 3. STR = Short Tandem Repeats - strings of oligonucleotide sequences that are shorter than VNTR (usually of 2, 3, 4, or 8 nucleotides each), of different repeat number in different people, not necessarily lying between restriction endonuclease sites; also called microsatellites 4. SNP = Single Nucleotide Polymorphisms - single nucleotide differences between individuals that are not associated with restriction endonuclease sites

For Respiratory and Metabolic disorders in acidosis and alkalosis what happens to the CO2 and the pH what is it called respiratory academia or alkalemia? When HC03- increases what happens to the pH and is it called metabolic alkalosis or acidosis?

CO2 = acid (respiration) CO2 increases = pH decreases (respiratory acidemia) CO2 decreases = pH increases (respiratory alkalemia) HCO3- = Base (metabolic) HCO3- increases = increase pH (metabolic alkalemia) HCO3- decreases = decrease pH (metabolic acidemia)

Vitamin D Deficiency & Toxicity

Causes of Deficiency: • Inadequate dietary intake (no foods of animal origin; vegans are at risk) • Inadequate exposure to sunlight • Renal disease & liver disease (decreased formation of active form) • Fat malabsorption (due to gut resection or celiac disease) Deficiency: • Causes low plasma Ca2+ levels & impaired bone mineralization (soft & pliable bones) - children => rickets - adults => osteomalacia • Causes hypocalcemia & hypophosphatemia (low plasma calcium & phosphate) Toxicity • Most toxic vitamin • Causes hypervitaminosis D - nausea, vomiting, muscle weakness, thirst - hypercalcemia & Ca2+ deposition on organs (metastatic calcification)

Iron Deficiency Anemia

Causes of Deficiency: • Increased requirements (pregnancy, growth, menstruation & premature babies) • Inadequate dietary intake (vegans are at risk) • Blood loss (including menstrual loss) • Malabsorption (absence of vitamin C or gastrectomy) Deficiency: • Causes iron deficiency anemia => decreased production of hemoglobin & decrease in oxygen reaching tissues - pallor - tiredness - shortness of breath - palpitations • Epithelial abnormalities - angular stomatitis (cracked corners of mouth) - glossitis Management & Treatment: • Oral supplements • IV if malabsorption is concern

Copper Deficiency & Overload

Causes of Deficiency: • Menke's kinky hair syndrome (rare X-linked disorder) caused by defective absorption of copper from SI (life expectancy < 2 yrs) • Clinical features - depigmentation of hair - arterial degeneration - neuronal degeneration & mental retardation - growth failure & anemia Causes of Overload: • Wilson's disease (rare autosomal recessive disorder) resulting in failure of liver to excrete copper in bile, leading to copper accumulation • Copper deposition in - liver (cirrhosis) - brain (severe progressive neurological disability, mental deterioration) - eyes (yellow-brown rings around corneal limbus) • Treated by daily chelation therapy with d-penicillamine

Vitamin E Deficiency & Toxicity

Causes of Deficiency: • Severe fat malabsorption • Seen in premature infants Deficiency: • Can cause hemolytic anemia in newborns (due to increased oxidative stress in RBCs) • In children & adults causes - muscle weakness - ataxia - peripheral neuropathy - nystagmus Toxicity: None - least toxic of fat-soluble vitamins

Vitamin K Deficiency & Toxicity

Causes: • Decrease in bacteria in gut (long-term antibiotic therapy) • Insufficient dietary intake • Newborns cannot synthesize because they have sterile gut Deficiency: • Causes poor clotting & bleeding disorders • May result in hemorrhagic disease of newborn (prophylactic administration of Vitamin K to all newborns) Toxicity: • Can cause hemolytic anemia & jaundice in infants

Embryonic stem (ES) cell

Cells arising from in vitro culture of the inner cell mass of a mammalian blastocyst stage embryo. Depending on the tissue culture media, ES cells can grow without limit, or can be caused to differentiate into ALL the cell types found in the normal body embryonic stem cells (pluripotent) Inner Cell Mass of Blastocyst Gives Rise to Embryonic Stem (ES) Cell Line

Mode of Action (of PG)

Compared to hormones but differ: • Small amounts in all tissues • Act locally • Not stored • Short half life (metabolized to inactive products) • Plasma and nuclear membranes mediate the action of eicosanoids Inactivated mostly in lung

DNA sequencing ribose, deoxyribose, dideoxyribose

DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule In vitro synthesis in the presence of chain terminators -ribose has 2' as well as 3' position occupied with OH -deoxyribose has only 3' positions occupied with OH -dideoxyribose has no OH at 2' and 3' positions ** Chain terminators are used to find specific sequences in DNA ** 5'PO4 and 3' OH is the phosphodiester bond if DNA is synthesized in the presence of terminators it will get incorporated and it will terminate the DNA synthesis at specific points of that DNA 1. Need template and primer 2. Denature DNA and anneal with primer 3. Intubate with excess dATP, dTTP, dCTP, dGTP 4. Divide the reaction into four tubes with one tube ddATP and DNA polymerase, tube 2 ddTTP and DNA polymerase and so on 5. Make fragments ending with the A for ddATP, T for ddTTP, C for ddCTP, G for ddGTP 6. Run the fragments on gel, DNA read bottom up read as a sequence increase by one nucleotide

Types of Cellular Damage Induced by ROS?

Damage to: 1. Lipids-> lipid peroxidation -> lipid degradation ->membrane damage (mitochondrial, cell, nuclear, ER ) 2. Carbs-> advanced glycation end-products (AGEs) occur as a result of hyperglycemia in diabetes mellitus 3. Proteins -> pro, his, arg, cys, met particularly vulnerable to OH· attack->protein cross-linking, fragmentation 4. DNA -> backbone cleavage, stand breaks, base alterations by OH radical attack -> mutations and apoptosis

Type III Cori/ Forbes which enzyme deficient?

Debranching enzyme

Biotin deficiency

Deficiency & Causes: • Extremely rare but can occur by consuming large (>20) amount of egg whites (contain avidin which binds biotin & prevents absorption) • Can occur as a result of long-term antibiotic therapy (kills intestinal bacteria) • Deficiency may cause dermatitis

Vitamin B2 deficiency?

Deficiency: • Causes ariboflavinosis - glossitis - cheilosis (fissures at corners of mouth) - dermatitis - angular stomatitis - cataracts

Vitamin B3 (Niacin) deficiency

Deficiency: • Causes pellagra (skin, GI & nervous system symptoms) - photosensitive dermatitis - diarrhea - dementia (apathy, confusion, disorientation, lethargy) - can lead to death Causes of Deficiency: • Decreased dietary intake • Protein deficiency (inadequate intake of tryptophan) • Vitamin B6 & PLP deficiency (cofactor required for conversion of Tryptophan to niacin) • Hartnups disease (failure to absorb dietary tryptophan)

Folic acid deficiency?

Deficiency: • MEGAloblastic Anemia: - Decrease in purines & TMP leads to decrease in nucleic acid synthesis & affects cell division - Rapidly dividing cells are affected (bone marrow & gut) - Large immature RBC precursors accumulate (megaloblasts) • Neural tube defects in developing fetus Causes of Deficiency: • Increased demand (pregnancy & lactation) • Decreased dietary intake • Alcoholism • Poor absorption (pathology of SI) • Treatment with dihydrofolate reductase inhibitors (methotrexate)

Vitamin B12 Deficiency

Deficiency: • Results in accumulation of odd-numbered fatty acids which may be incorporated into cell membranes of nerves • Causes Secondary Folate deficiency leading to MEGAloblastic anemia Causes of Deficiency: • Insufficient dietary intake (rare but vegans are at risk) • Disease of the ileum (deficiency of IF; e.g., Crohns disease) • Surgical resection of the gut (deficiency of IF) • Pernicious anemia (deficiency of IF caused by autoimmune destruction of gastric parietal cells • Pancreatic insufficiency (inability to digest protein-bound B12)

Prader- Willi syndrome

Deletion of paternal 15q11 1. Hypotonia 2. Poor feeding in infancy 3. "almond shaped" eyes 4. hypo pigmentation compared to relatives 5. hypogenitalism in males 6. decreased pain sensation 7. Obesity and lack of satiety in childhood 8. no vomiting 9. mild to moderate intellectual disability

FADH2

Difference between FADH2 and NADH is the prosthetic group on NADH is diffusible can travel in cyto or mito the FADH2 is attached to enzymes

Apolipoprotein AI milano factor

Differs from native Apo AI in that arginine is replaced by a cysteine residue at position 173 • Allows a disulfide linked dimer to be formed • Thought to be the result of increased reverse cholesterol transport from plaques to liver for reprocessing • If confirmed in larger trials this will change post MI care ** regressed the amount of plaque in the vasculature Apolipoprotein A-I is the major protein component of high density lipoprotein (HDL) in plasma. Chylomicrons secreted from the intestinal enterocyte also contain apo A-I but it is quickly transferred to HDL in the bloodstream.[3] The protein promotes fat efflux, including cholesterol, from tissues to the liver for excretion.

What is a recombinant fraction? 20 centiMorgans = how many basepairs?

Distance Between Disease Gene and Marker 1% Recombinants = 1 centiMorgan • Say, you have investigated 100 individuals of a pedigree. • Assume that you noticed 20 meioses as being recombinants. • That means the recombinant fraction is equal to 20/100 or 20%. - The recombinant fraction is expressed as genetic distance in units of cM (centiMorgan) • 20% = 20 cM ~ 20 million basepairs of physical distance

AA classification which are glycogenic, glycogenic and ketogenic? ketogenic?

Essential: HMTV -Histidine -Methionine -Threonine -Valine PIT (gluco and keto) * tyrosine (nonessential) Phenylalanine Tryptophan Isoleucine LuLy (ketogenic) Leucine Lysine

Describe what happens with respiratory alkalosis? Hyper or hypoventilation? What Happens with CO2 levels? What are the consequences and how does the body compensate?

Excessive loss of CO2 generally caused by hyperventilation ==> anxiety, hysteria Consequences: pH increases, PaCO2 reduced, HCO3- normal Compensate: PaCO2 remains low HCO3- is reduced; HCO3- is removed by kidney pH is nromalized

FA synthesis what are the three enzyme systems?

FA synthase FA elongase FA desaturase 1. Cytosolic acetyl CoA is carboxylated into malonyl CoA which serves as the immediate 2 carbon donor to the growing chain 2. Acetyl CoA carboxylase requires biotin and ATP to synthesize malonyl CoA 3. This is the rate limiting step of FA synthesis. -occurs in liver, lactating mammary glands, and adipose tissue

Gene Knockout Schematic with mice hair length? What is the gene involved for hair growth?

FGF5 gene, a negative regulator of hair growth, is knocked-out. Hair will grow uninterrupted.

Vitamin E

Family of 8 compounds called tocopherols α-tocopherol is most active • Antioxidant • May be protective against heart disease Absorption & Transport: • Requires dietary fat for absorption & transported in chylomicrons • Transported from liver to adipose via VLDLs • Stored in adipose

In situ hybridization?

Find the location of a particular gene in the chromosome via in situ hybridizing a gene-specific probe to chromosomes in situ. 1. Intact chromosomes denatured and hybridized with a particular probe **gives the relative position of the gene in specific protein

How are FAs transported into cells what are they bound to?

Free fatty acids are bound to albumin in serum and transported into cells. Release of fat for energy is highly regulated.

Type VI Her's which enzyme deficient?

Hepatic Glycogen Phosphorylase

Biological roles of homologous recombination?

Homologous recombination is involved in - genetic re-assortment during gametogenesis (Meiosis). - DNA repair. - clearing replication hurdles. • Recombination can also lead to deleterious effects such as - deletions, inversions, translocations, and other rearrangements- some leading to cancers.

What inhibits glycogenesis? what inhibits glycogen synthase in what way?

Hormonal regulation: Glucagon activates protein kinase A => phosphorylates glycogen synthase => glycogen synthase OFF => glycogenesis OFF

Full turn TCA cycle what is produced? Fraction of turn?

Full turn: • 8 reactions & 8 enzymes • Acetate (from AcCoA)=> 2 CO2 • Energy production => 1 GTP, 3 NADH & 1 FADH2 To ETC • No net production or consumption of intermediates • Occurs in mitochondria Fractions of turn: • Intermediates removed for biosynthetic pathways as needed • Intermediates replenished via anaplerotic reactions

Biotin

Functions: • Coenzyme for carboxylation reactions (activated carrier for CO2) 1) Pyruvate carboxylase (gluconeogenesis & TCA anaplerotic reaction) 2) Acetyl CoA carboxylase (fatty acid synthesis) 3) Propionyl CoA carboxylase (β-oxidation of odd numbered fatty acids) 4) Branched-chain aminoacid metabolism

Vitamin K

Functions: • Coenzyme for γ-carboxylation of glutamate residues of clotting factors II, VII, IX, X & prothrombin • Activates clotting factors & clotting cascade can help with clot formation specifically for the gamma carboxilation of glutamine residues important for clotting

Zinc

Functions: • Cofactor for enzymes - dehydrogenases (ex. lactate dehydrogenase) - peptidases - superoxide dismutase (ROS) - carbonic anhydrase • Component of zinc finger DNA binding domains in certain transcription factors Deficiency: • Caused by malabsorption in SI in acrodermatitis enteropathica (rare autosomal disorder) • Acrodermatitis enteropathica characterized by - growth retardation - hypogonadism - delayed wound healing - lesions around orifices & on hands/ feet • Completely cured by zinc therapy

Vitamin B3 (Niacin)

Functions: • Cofactor for oxidase & dehydrogenase enzymes (active forms are NAD+ & NADP+) • Repair of UV light damage in DNA in skin • Treatment of hyperlipidemia in high doses (nicotinic acid inhibits lipolysis & leads to decrease in VLDL synthesis) • NAD+ is a Mobile Electron Carrier for Catabolism (Breakdown). • NADP+ is a Mobile Electron Carrier for Anabolic (Biosynthesis).

Vitamin B2 (Riboflavin)

Functions: • Cofactors for oxidase & dehydrogenase enzymes (active forms are FAD & FMN)

Iron

Functions: • Oxygen transport: - component of heme (hemoglobin) 60-70% of total body iron - component of myoglobin 3.5% of total body iron • Component of iron-containing enzymes 0.2% of total body iron - cytochrome P450 (drug metabolism) - iron-sulfur centers (ETC) - catalase (breakdown of H2O2) Disease: - Estimated 2 billion cases of anemia worldwide (reduced cognitive performance, impaired iodine and Vit. A metaboism, increased mortality). - Approx. 50% of pregant women in developing world.

Pantothenic Acid

Functions: • Transfer of acyl groups as component of CoA (acetyl CoA, succinyl CoA, fatty acyl CoA) • Component of fatty acid synthase Deficiency: • Not well characterized & rare • Burning foot syndrome

Vitamin B1 (Thiamine)

Functions: Cofactor for enzymes (active form is thiamine pyrophosphate; TPP) 1) Pyruvate dehydrogenase complex 2) α-ketoglutarate dehydrogenase 3) Transketolase 4) Branched chain aminoacid "α-ketoacid dehydrogenas Thiamine catalyzes bond cleveage formation of alpha ketols

Vitamin B12 (Cobalamin)

Functions: Enzyme cofactor for 2 essential enzyme catalyzed reactions: 1) Methylmalonyl CoA mutase (deoxyadenosyl cobalamin) critical for the breakdown of odd-numbered fatty acids. 2) Homocysteine methyltransferase (methylcobalamin) necessary for methionine synthesis Absorption & Transport: • Dietary B12 released from food in stomach • B12 binds to glycoprotein carrier (Intrinsic Factor; IF) • B12 - IF complex transported to ileum & binds to receptor on mucosal cells • B12 transported into mucosal cell & into circulation with B12-binding proteins • Storage in liver (5 mg; supply for several years)

Retinoic Acid function?

* regulaties gene expression * anti-acne medication I. Hormonal Regulation • Retinoic acid is a steroid hormone & ligand for Retinoic Acid Receptor (RAR; transcriptional regulator) • Liganded RAR activates gene expression of genes involved in growth & differentiation of epithelial cells (e.g., keratin gene) II. Vision (Signal Transduction) • cis-trans isomerization of retinol upon light absorption • G-protein coupled receptor (transducin) is activated • Phosphodiesterase is activated (cGMP is lowered) • Cation influx channels close, resulting in hyperpolarization • Voltage gated calcium channels close, and Glu neurotransmitter release drops.

Absorption & Transport of Iron

**Free (unbound) iron can be very toxic as a result of free radical formation • Dietary (heme) iron is preferentially absorbed • Absorption is facilitated by vitamin C (ascorbic acid) • Transported in bloodstream as complex with transferrin to sites of storage • Storage in bone marrow (hemoglobin synthesis), liver (enzyme synthesis), 50 muscle (myoglobin synthesis) • Stored mainly as ferritin with smaller amount stored as hemosiderin (27% of total body iron)

What is a cDNA Library and cloning describe?

Genomic Library is a collection of clones (vector + DNA/cDNA) of whole genome fragments or cDNAs corresponding to mRNAs. • Cloning is isolation of a specific fragment from the above library.

How is recombination used in mapping disease gene? Frequent and infrequent recombination what are the distances?

If a disease travels with a specific marker allele: - That means, the disease causing mutation is linked to the marker gene. • How close or far the disease gene is from the marker? - That is estimated by the knowledge of how frequently the disease gene recombines with the marker gene • Infrequent recombination- shorter distance • Frequent recombination- far apart

Burkitt's lymphoma chromosomes involved genes?

In Burkitt's lymphoma, MYC oncogene from chromosome 8 is translocated to IGH locus on chromosome 14. ** recombination chromosome 8 and chromosome 14 via translocation

Mechanism of Insulin Signaling

Insulin --> Liver, Muscle, Adipose --> Insulin receptor (RTK)-> active insulin complex -> amplify the signal IRS-1 binds recognize the autophosphorylated receptor -> allows for the complex insulin receptor+IRS-1 and signaling proteins --> downstream Effectics: 1. Gene expression: induction/repression change in enzyme levels 2. Enzyme dephosphorylation: Change in enzyme activity (Reversal of glucagon effect) 3. Glucose uptake: Increase in substrate availability

Kantianism, a Deontological(Duty-Based) and categorical imperative?

Kantianism, a Deontological(Duty-Based) theory asserts there are principles that are universal. These actions are based on the motives and/or intent of the moral agent. Categorical Imperative-> if the action is amplified and committed by all people, is it then still morally allowable?

What delivers cholesterol to the tissues?

LDL

During fasting what varies glucose or ketone bodies?

Levels of ketone bodies in the blood varies during fasting Glucose levels remain relatively constant as do levels of FA * Ketone levels increase markedly to levels which can be used by the brain and other nervous tissue

LOD Score

Logarithm of ratio of likelihoods: - Likelihood of linkage between two loci at a specific recombination fraction / Likelihood that the loci are unlinked • If the ratio is 3 odds are 1000 times in favor of linkage - Two loci are linked when greater than 3 What is the likelihood of a disease linked at a certain recombinant fraction? -disease is linked with the marker

Describe what happens with metabolic acidosis? Hyper or hypoventilation? What Happens with CO2 levels? What are the consequences and how does the body compensate?

Many diseases and medical conditions lead to metabolic acidosis: 1. Diabetic ketoacidosis: accumulation of aceoacetic and hydroxybutyric acids 2. Renal failure: kidney unable to retain HCO3- 3. Diarrhea: loss of solutes including HCO3- Consequences: pH is low, PaCO2 is unchanged, HCO3- is reduced Compensate: PaCO2 is lowered, the lungs excrete more CO2 HCO3- remains low pH is normalized ** must compensate via lungs because the rest of the body systems are having problems functioning

Double Effect

Many embryos obtained for research come from Individuals/Couples who have extra embryos stored after undergoing fertility treatment ->One ethical justification for using these embryos for research is that if not they will be discarded and no medical benefit will arise! Consider the principle of Double Effect

Hematopoietic Vitamins which ones? What happens when the body is deficient in these? What is the diagnosis? What do the Cells look like? Classification of nutritional anemia?

Megaloblastic anemia occurs due to folate and Vitamin B12 deficiency -Cannot synthesize DNA, results in the inability of cells (RBC precursors) to divide Diagnosis as increase in mean corpuscular volume. 1. Classified by size of RBC in patient (mean corpuscular volume) • Most common nutritional anemia is due to iron deficiency 2. Macrocytic (MCV) Megaloblastic anemia: -Deficiency in Vitamin B12 -Deficiency in folate

Why acid-base balance? Values pH body? Level pH acidosis? alkalosis?

Metabolic reactions are sensitive to changes of pH The body fluids are maintained within a narrow range of pH values Intracelluar pH is in the range of 7.0-7.1 Extracelluar pH is in the range of 7.35-7.45 ↓ or ↑ by > 1/10 unit can be fatal pH < 7.35 Acidosis pH > 7.45 Alkalosis

Trisomy 13: Patau Syndrome

Microphthalmia Scalp defects Coarse facies Cleft lip Polydactyly Holoprosencephaly Severe neurologic impairment Lethal in 95% by 1 year

FA Synthesis regulation

Mitochondria contain three FAD-dependent acyl CoA dehydrogenases which act on long, medium and short chain FA's

Why mitochondrial polymorphisms can be useful to geneticists and the pattern of mitochondrial inheritance.

Mitochondria more stable, inherited maternally i. Mitochondria DNA, present in higher copy numbers. Only inherited from mother. It's a circle genome, more stable. It's inherited in maternal line. Can take from ancient DNA, dry bones, no soft tissue, where most DNA gone. 1. Useful ID of diseased skeleton 2. Point polymorphyism SNP.

multipotent, Unipotent, Pluripotent

Multipotent: tissue specific stem cells Stem cells that give rise to a limited number of cell types are Multipotent (e.g., the blood). Unipotent: implies stem cells give rise to a single cell type (e.g., sperm-forming cells of testis). Pluripotent: the ability to differentiate into ALL cell types normally found in the body

Gene Knockout explain?

Mutant gene introduced ->cloned into vector and introduced in embryonic stem (ES) cells. • Mutant gene will homologous recombine with native gene, where specific gene is replaced. • Injection of engineered cells into mouse embryo. Have made ES where the native gene has been disabled. • Chimeric animals when embryo grows. ** Isolate the cells where recombination has occurred and put into the embryo of mouse breed chimeric mice to get mice that completely generated from the generated ES cells

Mycophenolic acid

Mycophenolic acid an immunosuppressant drug used to prevent rejection in organ transplantation. It inhibits an enzyme needed for the growth of T cells and B cells. Mycophenolate mofetil is metabolised in the liver to the active moiety mycophenolic acid. It inhibits inosine monophosphate dehydrogenase, the enzyme that controls the rate of synthesis of guanine monophosphate in the de novo pathway of purine synthesis used in the proliferation of B and T lymphocytes.

What is needed to desaturate a site on a FA?

NADH, O2, and cytochrome b5

Why mutations associated with CF are considered to be optimal for carrier screening?

NATIONWIDE CF CARRIER SCREENING: REVELATIONS FROM "POST-MARKET SURVEILLANCE" 1) Panel mutation less frequent than expected: 1078delT 2) Panel mutation not a mutation: I148T (benign polymorphism) 3) Low OB penetration to provide screening

Describe what happens with metabolic alkalosis? Hyper or hypoventilation? What Happens with CO2 levels? What are the consequences and how does the body compensate?

Net loss of H+ through vomiting; ingestion of soda (NaHCO3) Consequences: pH is high, PaCO2 is unchanged, HCO3- is increased Compensate: HCO3- is raised PaCO2 is raised, the lungs excrete less CO2 pH is normalized

Nitrogen balance when is it positive or negative? What should the typical healthy adult nitrogen balance be?

Nitrogen balance: N consumed - N excreted 0 = healthy adults + Positive • Childhood • Pregnancy • Muscle building • Healing - Negative • Protein malnutrition • Deficiency of essential amino acids ! Stress (fever, surgery etc...)

Nomenclature for cytogenetic abnormalities

Nomenclature for cytogenetic abnormalities del: deletion dup: duplication inv: inversion t: translocation ter: terminal (pter/qter)

PEPCK role in bypass I

Oxaloacetate(PEP Carboxykinase)-> Phosphoenolpyruvate 1. OAA decarboxylated & phosphorylated to phosphoenolpyruvate (PEP) by PEP-Carboxykinase (PEPCK) 2. Requires GTP hydrolysis

Turner's Syndrome

Paternal sex chromosome is missing in 80%. Associated with young maternal age. No significant recurrence risk. High lethality in utero - common in spontaneous abortuses. Coarctation of the aorta, horseshoe kidney, edema, webbed neck, short stature, lack of secondary sexual characteristics, infertile.

Energy Yield TCA cycle

Per full turn: • Acetate (from AcCoA)=> 2 CO2 • Energy production => 1 GTP, 3 NADH & 1 FADH2 • Reduced coenzymes will enter ETC & generate ATP by oxidative phosphorylation Note: 2 pyruvate & 2 AcCoA are generated per glucose

How does the phosphate buffer system work?

Phosphate anion dissociates to generate H+ and the conjugate base, mono hydrogen phosphate with pKa 7.2 ICF buffer

Type VII Tarui's which enzyme deficient?

Phosphofructokinase

Type VIII

Phosphorylase kinase

Allele specific PCR? Perfect match and imperfect match what happens? Any amplification when? What is the purpose of PCR?

Practical Applications of Hybridization/Annealing: Allele Specific PCR looking at two alleles heterozygote 1. Denature and PCR with ASP1 (allele specific primer) for the allele looking for selectively anneal to its allele 2. PCR amplify it -Perfect Match: strand extension and amplification will occur -Imperfect Match: No strand extension and No amplification will occur 3. Denature and PCR with ASP2 primer specific to allele 2 -Imperfect match with allele 1 so no amplification -Perfect match allele 2 elongation and amplification ** find the genotype of the individual -allele specific PCR: distinction between homozygous(one allele copy) and heterozygotes (two different allele copies) for that individual for each allele

What is the current recommendation for CF screening.

Preconception testing - covered by insurance (ex. Kaiser - incidence of CF births reduced by 50%, incidence of unwanted CF births to 0)

Pros and cons on DNA fingerprinting ?

Pros: • Paternity testing • establish zygosity of twins • Determining bone marrow transplant engraftment • Id mislabeled pathology specimens • Pedigree analysis of animals • Establishing id of rapists, murderers Cons - military finger printing; where after the soldiers leave the military, there is ethical concern over what the government will do with the DNA fingerprinting files, since they are not released.

PCR Pros and Cons?

Pros: less expensive way to replicate gene, few hrs, back and forth DNA polymerase copying, allows to get many copies, genetic anaylysis of single DNA, purifies one gene fr rest of genome (which will get diluted out after numerous cycles), non-invasive (can use saliva, skin scraping, hair, urine, buccal mucosa) Cons: contamination, psychosocial/economic consequences bc samples can easily be easily collected and sequenced • Ethical concern: Any cell fr person can be used for genetic testing (eg. urinalysis)

Pyrimidine nucleotide degradation?

Pyrimidine ring is opened and degraded to soluble products - Alanine - Aminoisobutyrate - Ammonia and carbon dioxide is produced

Pyruvate carboxylase what does it convert, what does it depend on and what is the cofactor?

Pyruvate(Pyruvate Carboxylase)--> Oxaloacetate • ATP dependent carboxylation in mitochondria • Biotin as a cofactor • Pyruvate carboxylase reaction is part of gluconeogenesis & also anaplerotic reaction for TCA cycle (replenishes TCA cycle intermediates)

Meiosis I when does recombination occur which state? Genetic mapping what is measured in relation to recombination? Greater or less separation when recombination happens?

Recombination (synaptonemal complex at zygotene stage; crossing-over at pachytene) • Recombination is a measure of separation between two loci. • Linkage between two loci is interrupted by recombination - Consider two loci A and B on a chromosome - Consider two alleles A/a and B/b at these two loci - If no recombination, AB and ab are inherited together. - If recombination- Ab and aB will be inherited together * if loci are very far apart they are more likely to recombine

Pros and Cons Southern blot?

Restriction enzyme - binds to DNA strand on recognized sequence of NT and cleaves it → produces same size fragments Procedure: • run DNA frag thru agarose gel • Blot to filter membrane • Hybridize and autoradiograph for targeted sequence Pros: cheaper than DNA sequencing, detect specific DNA sequence in large, complex sample, quantify amt of DNA present, Cons: takes longer, labor intensive, requires large amount of targeted DNA

Nucleotide

Result from linking one or more phosphates with a nucleoside onto the 5' end of the molecule through esterification 1. Mono-, di- or triphosphates 2. Phosphates can be bonded to either C3 or C5 atoms of the sugar • RNA (ribonucleic acid) is a polymer of ribonucleotides • DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides • Both deoxy- and ribonucleotides contain Adenine, Guanine and Cytosine - Ribonucleotides contain Uracil - Deoxyribonucleotides contain Thymine Not all are polymers 1. Nucleoside Triphosphates are important energy carriers (ATP, GTP) 2. Important components of coenzymes: FAD, NAD+ and Coenzyme A

Vitamin B6

Several forms of the vitamin are known, but pyridoxal phosphate (PLP) is the active form and is a cofactor in many reactions of amino acid metabolism, including transamination (urea cycle), deamination (aa catabolism), and decarboxylation (inflammatory response). PLP also is necessary for the enzymatic reaction governing the release of glucose from glycogen. Condensation reactions: porphyrin biosynthesis Functions: • Enzyme cofactor (active form is PLP; pyridoxal phosphate) • Required for glycogen phosphorylase • Required for heme synthesis (ALA synthase) • Required for aminoacid metabolism • Required for conversion of Tryptophan to Niacin amino butyric acid (GABA) • Inhibitory neurotransmitter • Produced by decarboxylation of amino acids in reactions that use pyridoxal phosphate as cofactor -Produced by decarboxylation of glutamate

Karyotype

Staining & display of chromosomes from metaphase spread => karyotype • Chromosomes numbered by length - 1 is largest - 22 is smallest (later found that 21 NOT 22 is smallest but numbering not changed) • 1-22 autosomes • X & Y sex chromosomes • 2 copies 1-22 and 2 sex chromosomes = 46 • Nomenclature changes in chromosome number -total # change -+ sign followed by Chr # indicated autosomal gained (different for sex chr.)

Personalized Pluripotent Stem Cells

Stem cell biology offers the potential to make personalized pluripotent stem cells that are a perfect genetic match to a specific patient, with no immune rejection (i.e., a path to cloning an individual).

Steps of hybridization

Steps for hybridization 1. Denaturation: • Strands falling apart at high temperatures 2. Renaturation: • Complementary strand annealing 3. Probe hybridization: • Search of sequences (identical or related)

Reactions TCA starting from alpha ketogluterate end malate

Substrate-level phosphorylation is a type of metabolic reaction that results in the formation of adenosine triphosphate (ATP) by the direct transfer and donation of a phosphoryl (PO3) group to adenosine diphosphate (ADP) from a phosphorylated reactive intermediate.

Vitamin B6 deficiency

Symptoms of Deficiency (Rare): • Abnormal amino acid metabolism • Secondary Pellagra (due to inability to synthesize niacin) • Microcytic anemia Deficiencies Have Been Observed For: • Women on oral contraceptives • Alcoholics & elderly • Newborns on certain types of formula • Treatment with isoniazid (for tuberculosis infection) inactivates PLP & causes B6 deficiency

Diseases: Tay-Sachs, Gaucher, MEtachromatic Leukodystrophy, Krabbe Disease, GM1 Gangliosidosis, Sandhoff's disease, Fabry Disease, Niemann-pick disease, Farber disease?

Tay-Sachs (SAX): Beta-HeXosaminidase A Gaucher : Beta-Glucosidase Metachromatic Leukodystrophy: Arylsulfatase A Krabbe Disease : Beta-Galactosides GM1 Gangliosidosis : Beta-Galactrosidase Sandhoff's Disease: Beta-Hexosaminidase A and B Fabry: alpha-Galactosidase Niemann-Pick: Sphongomyelinase Farber Disease: Ceraminidase

Maintenance of body pH

The body produces approximately 22 mole/ day of acid from normal metabolism Three important homeostatic regulators of acid-base balance: 1. Chemical buffer systems * 1st to respond * Take < 1 sec * Temporarily "tie up" excess acids or bases * Carbonic acid - Bicarbonate; Phosphate; Protein buffer systems 2. Respiratory mechanism * 2nd to respond * Takes 1-3 minutes * Respiratory center involved * Removes CO2 & therefore H2CO3 3. Renal mechanism * 3rd to respond but most potent * Takes hours to days * Kidneys remove metabolic acids (Pi, uric & lactic acids, ketone bodies)

FA synthase key steps

The multifunctional enzyme Fatty acid synthase produces palmitoyl CoA from acetyl CoA and malonyl CoA with NADPH (from the pentose phosphate pathway) as the source of reducing equivalents.

Therapeutic cloning

The process of creating an embryo by somatic cell nuclear transfer to an oocyte, and then using it to establish an embryonic stem (ES) cell line.

How does the protein buffer system work?

The side chain of histidine in hemoglobin (Hb) has a pKa of 6.7: able to accept a proton Maintains the ICF pH at 7.0

Utilitarianism

This teleological(consequence based ethics) perspective is based on a single guiding principle. The principle of utility states that actions are right if they produce the greatest balance of happiness over unhappines

Tissue-Specific Stem Cells (Identified Using Pulse Chase Labeling)

Tissue Subtypes 1. Post-mitotic (muscle and neuronal) are nondividing cells (formed during embryogenesis or soon after birth) and contain few or NO stem cells. Skeletal muscle contains satellite cells critical for regeneration (not true stem cells). 2. Expanding (liver, kidney, and thyroid) undergo an expansion only during juvenile growth and possess some stem cells that can generate new cells to repopulate tissues (stem cells typically are not clearly identified). 3. Renewal (epithelial lining of intestine, stomach, epidermis of skin, testes, and hematopoietic system) undergo continual replacement (involving transit amplifying cells that cease dividing in basal layers and then progressively differentiate).

Gene transfer via Retroviral vectors? What is needed to package the therapeutic gene? What is needed to initiate packaging? How do you make the viral noninfectious?

To package the therapeutic gene into viral particles need : gag, pol and env proteins are supplied by a packaging cell. Packaging is initiated via a packaging signal: ψ is the viral sequence retained in the therapeutic gene Construct. By substituting gag, pol, env sequences, virus is rendered noninfectious. The ψ sequence, however, are important for packing of viral particles and therefore retained in the vector

Describe ubiquitin? What are the three enzymes involved?

Ubiquitin is activated and attached to proteins using a group of three enzymes required Energy ATP E1 - ubiquitin-activating enzyme E2 - ubiquitin-conjugating enzyme E3 - ubiquitin-protein ligase

Carbamoyl Phosphate Synthetase 1 role?

Urea cycle 1. Forms carbomoyl phosphate needs 2 ATP 2. Ammonia required get from glutamate dehydrogenase 3. N-acetylglutamate is required (positive allosteric regulator), formed by N-acetylglutamate synthase

What carried triglycerides to peripheral tissues?

VLDL

Absorptions, Transport and Storage of Vitamin A?

Vitamin A (Fat Soluble) Family of compounds (retinoids): Includes retinol, retinal, retinoid acid Functions: • Functional forms are retinoic acid, retinol, retinal & β-carotene • Retinal is important for visual cycle • β-carotene is an antioxidant • Retinoic acid is a steroid hormone & is important for epithelial cell differentiation • Retinol & retinal are important in reproduction & fertility (spermatogenesis & placental development) • Growth of bones & teeth

Vitamin D function?

Vitamin D (regulates Ca+2 metabolism) Derivative of cholesterol Cholecalciferol (D3), ergocalciferol (D2) 1,25-dihydroxycholecalciferol is ACTIVE form Sources: Not present in plants • Dietary: Fish (cholecalciferol) • Endogenous synthesis in skin (requires exposure to sunlight) using 7- dehydrocholesterol Functions: • Maintains adequate plasma levels of Ca2+ by: 1) Increasing uptake of calcium & phosphorus from intestine 2) Increasing reabsorption of calcium from kidney 3) Increasing resorption of bone, calbindin promotes the release of Ca+2 from bones ** regulates gene expression: VDR regulates gene expression of genes involved in calcium metabolism

Folic acid

Water-Soluble Vitamins Enzyme cofactor (tetrahydrofolate) • Specifically required for 1 carbon fragment transfers in synthesis of amino acids, purines and thymidine • Prevents neural tube defects in fetus in first trimester (anencephaly & spina bifida) All women of childbearing age (pre-conception)should consume folic acid daily to reduce risk of pregnancy with neural tube defects and other congenital malformations • Pathways to tetrahydrofolate (TFA) are targets of sulpha drugs (sulfonamides) and cancer therapies (methotrexate).

Multiple congenital anomalies

When there is more than one anomaly, the relationship between the anomalies is the key to the diagnosis: • Syndrome -Dysplasia (one tissue type) -Sequence • Association

What happens when acid (excess H+) is added to a buffer system?

[CO2] will increase and will be removed via increase in ventilation if that does help Kidney will remove the H+

Teratoma

a tumor containing a wide mixture of tissue types, often characteristic of those formed form all three embryonic germ layers

the significance of CODIS

a. (Combined DNA Index Sys) o Established thru DNA Identification Act of 1994 o Administered by FBI o Blends forensic DNA technology with computer technology o Pros: common database that uses 13 STR markers o Cons: once in database, will always be there (like "scarlet letter"); not good esp for innocent ppl who were released

What are the three ketone bodies?

acetoacetate, acetone, and Beta-hydroxybutyrate

What is released at the end of each round of Beta-oxidation?

acetyl CoA, FADH, and NADH

Autosomal dominant

affected parent for an affected child; both sexes are affected; Mating of an affected with an unaffected will have 50% change of producing an affected child.

X-linked recessive

affects mainly Males; unaffected parents; carrier mother with affected male relatives. Sometimes affected female has to be carrier female and affected male - NO male to male transmission is absent

Synthesis of Purine Nucleotides?

atoms purine ring contribution from THF which can be broken down to uric acid, purine ring constructed in the liver adding carbons and nitrogens to ribose-5-phosphate made in pentose phosphate pathway 1. Convert Ribose-5-Phosphate (PRPP synthetase~Mg2+ATP)-->5-phosphoribosyl-1-pyrophosphate (PRPP) Steps utilize: Glycine, Glutamine, 2 steps require THF (Formyltransferase), 2. ------> IMP (inosine 5'-monophosphate) base is hypoxanthine 3. IMP --> AMP or GMP 4. IMP conversion to AMP requires Aspartage and GMP 4. IMP conversion to GMP requires Glutamine and ATP and NAD+ ** Purines: 1. Start with sugar + phosphate (PRPP) 2. Add base Purine base production or reuse from salvage pathway. De novo requires aspartate, glycine, glutamine, and THF.

X-linked dominant

both sexes affected; females variably affected; child of affected mother has 50% chance of being affected; affected males produce affected daughters

Induced pluripotent stem cells (iPS cells) "Adult Stem Cells"

cells closely resembling embryonic stem (ES) cells that are made from normal somatic cells by introduction of a small number of genes that encode pluripotent behavior.

Progenitor cells

cells that are dividing and are committed to form a specific cell type. Do not self-renew long term. Include transit amplifying cells and all cells of the early embryo.

From which two substances are phospholipids made?

diacylglycerols and phosphatidic acid

Transit amplifying cells

dividing cells that are derived from tissue-specific stem cells and are committed to differentiate after a certain number of divisions.

Inducing factors

extracellular proteins (growth factors and cytokines) that control embryonic development by causing cells to select a specific pathway of development

Distribution of Carbons from Macromolecules and entry into the TCA cycle?

go from cyto to mitochondrial matrix ETC

Which enzyme hydrolyzes triacylglycerols into free FA and glycerol?

hormone sensitive lipase

Where are triglycerides produced?

liver, muscle, adipose tissue

Reproductive cloning

making an identical genetic copy of an animal, which typically involves somatic cell nuclear transfer (SCNT) into an oocyte, or by injection of iPS cells into an early embryo, following by implantation of the resulting chimeric embryo.

What happens add Iron to Protophoryn IX? Where does this reaction occur? What is the inhibitor of the enzyme?

mitochondria Adding Iron converts the Protoporphyrin to heme Lead inhibits Ferrochelatase

Hematopoietic stem cells (HSCs)

multipotent stem cells resident in the bone marrow that generate all the cell types of the blood and immune system HSCT = hematopoietic stem cell transplantation = bone marrow transplantation. HSCT includes bone marrow and other blood-forming (hematopoietic) stem cells of the graft from non-marrow sources such as peripheral blood or umbilical cord blood. Current practice - HSCT does not destroy the cancer by radiation or chemotherapy. Rather, the main purpose of the graft is to destroy residual cancer cells as lymphocytes in the graft mount a very effective immune attack on tumor cells (graft-versus-leukemia effect). Mechanism poorly understood.

Chromosomes Anatomy

p arm : short arm of chromosomes (petit) q arm : long arm of chromosome Position of centromere ==> meta, submeta, acrocentric Metacentric = centromere located centrally Acrocentric = centromere located terminal position Submetacentric = centromere located in intermediate position

Kwashiorkor

protein deprivation result high carb diet instead of protein in the diet especially in children

Somatic cell nuclear transfer (SCNT)

replacement of the nucleus of a secondary oocyte by that of a somatic cell. Animal Cloning • Nucleus from Somatic Cell Transferred into Enucleated Eggs. • Early Embryo Can Be Used to Create Embryonic Stem Cells (Genetic Match to Donor). • Offers Pathway to Therapeutic Cloning or Propagation of Livestock.

What intermediate allows propionyl CoA to enter into TCA cycle?

succinyl CoA

Define gluconeogenesis?

synthesis of glucose from non-sugar precursors (mostly liver but also kidney) glucogenic aa, lactate, glycerol get converted to glucose When starving the only way to produce glucose when glycogen stores run out

What regulates the rate of ketone body formation?

the rate of Beta-oxidation

Autosomal recessive

unaffected parents of affected child; parents are carriers; both sexes are affected; 25% chance produce affected child

DNA sequencing (Gold std) define?

using gel like southern blot, uses capillary electrophoresis that differ by NT using florescent markers, goes thru laser beam and get a peak for NT → reads out sequence for entire genome; more expensive, more information, used for known/unknown mutations

Invasive versus Noninvasive sampling?

~ Invasive sampling (risk for miscarriage) 1. Chorionic villus sampling removal of CV cells (extra-embryonic/placental cells); 10th-12th week of pregnancy) 2. Amniocentesis = removal of amniotic fluid & isolation of fetal cells (15th-18th week of pregnancy) 3. Cordocentesis (Percutaneous Umbilical Blood Sampling; PUBS) -> removal of fetal blood cells from umbilical cord (after 17th week of pregnancy; can also detect infections) 3. Pre-implantation genetic diagnosis!removal of single cell from 6-8 cell blastomere (1 day post fertilization) after in vitro fertilization (IVF) for genetic testing; Only unaffected embryos implanted ~ Non-invasive sampling (no risk for miscarriage) 1. Isolation of fetal cells from maternal circulation & separation of fetal cells (MaterniT21Plus)

How is UDP-glucose formed?

~ form PPi that are removed by pyrophosphates drives formation of UDP-glucose forward

What are the gluconeogenic precursors?

~ glucagon activates 1. Glycerol (lipolysis-12%) --> glucose 2. AA (degradation muscle proteins-60%) --> glucose 3. Lactate (from anaerobic glycolysis RBC-28%)-> glucose

FA Synthase Complex

~ remaining series of reactions of FA synthesis is catalyzed by the multi-functional, dimeric enzyme, FA synthase 1. Adds 2 carbon units from malonyl CoA to the growing chain to form palmitate 2. Palmitate (16:0) is the longest chain humans can make 3. After the addition of 2 carbon units the chain undergoes 2 reductions which require NADPH 4. FA synthase is a large enzyme made up of a homodimer 5. Each subunits has 7 catalytic activities and an acyl carrier protein segment 6. ACP (acyl carrier protein domain) segment contains a phosphopantetheine residue 7. Chain hydrolyzed and palmitate released

Type II Pompe's which enzyme deficient?

α-1,4-glucosidase (lysosomal)

PGH2

• 1st step - oxidative cyclization of free arachidonic acid to form PGH2 - Catalyzed by PG endoperoxide synthase: Microsomal enzyme 2 catalytic activities: 1. Fatty acid cyclooxygenase (COX): Requires 2 O2 2. Peroxidase: Dependent on reduced glutathione •PGH2 → PG's and TX's •PGJ2 - non enzymatic step; powerful effects in vivo

Porphyrin Synthesis which Iron is the most important human and which type porphyrins are important in humans? Forming ALA what are inhibitors and what are the steps involved in porphobilinogen synthesis? There are two forms of ALA where are they and what are they called?

• 2 forms of amino-levulin synthase - Liver (ALAS-1) - Bone marrow (ALAS-2) Fe2+ regulate • Catalyzes rate limiting step for heme synthesis ALA synthase: rate limiting step and is inhibited by glucose(-), and heme (-) aminolevulinic acid dehydrogenase (-) Lead prevents the formation of porphobilinogen 1. Only type III porphyrins are physiologically important in humans 2. Fe2+

List the reactions that encompass the three bypasses of gluconeogenesis and the enzymes that catalyze them.

• 3 irreversible reactions of glycolysis bypassed by unique gluconeogenic enzymes • 7 reversible reactions of glycolysis shared Bypass 1: Phosphoenolpyruvate (pyruvate kinase)--> pyruvate Bypass 2: Frucose-6-P (PFK-1)--> fructose-1,6-BP Bypass 3: Glucose (glucokinase)-->glucose-6P

TPMT & 6-mercaptopurine

• 6-mercaptopurine (Purinethol®) is a chemotherapeutic • Used to treat childhood acute lymphoblastic leukemia (ALL) • 6-MP Incorporates into DNA/ RNA of tumor cells & causes cytotoxicity • 6-MP is inactivated by thiopurine methyltransferase (TPMT) • TPMT activity can be measured in RBCs from patient • SNPs in TPMT affect enzyme activity & ability to inactivate 6-MP • Low/ no TPMT activity => inability to inactivate 6-MP & severe toxicity of 6-MP *** Homozygotes for low activity TPMT alleles are at high risk for drug-induced toxicity!

Glutamine Synthase?

• A second important route in assimilation of ammonia is via glutamine synthetase • It is present in all organisms. • In humans it is most active in the liver. • Glutamine is transported from the liver to other tissues via the blood ~ protect the brain from rising ammonia levels by converting glutamate to glutamine

Cardiolipin Clinical Significance of Cardiolipin what syndrome?

• AKA diphosphatidylglycerol • Considered to be amongst the few glycerophospholipids that is antigenic - Cardiolipin is recognized by antibodies raised against Treponema palladium, the bacterium that causes syphilis • An important part of the inner mitochondrial membrane -Abundant in cardiac cell mitochondria (up to 20% of the phospholipid content) •Its presence is crucial for mitochondrial activity - Interacts with many mitochondrial proteins • Catabolism thought to occur via phospholipase A2 • Barth Syndrome - Infantile cardiomyopathy plus many other symptoms - Linked to X chromosome defect in a cardiolipin processing gene - Abnormal composition of cardiolipin • Abnormal cardiolipins also associated with Alzheimer's disease and Parkinson's disease

Array Comparative Genomic Hybridization

• Adaptation of microarray technique to detect chromosomal abnormalities • Pt. and control DNA labeled with different fluorophores (green or red) • Simultaneously hybridized to array containing probes covering the entire genome • Control DNA labeled with red • Pt. DNA labeled with green • Higher resolution than karyotyping and FISH (can detect even smaller rearrangements) • CANNOT detect abnormalities NOT involving changes in amount of DNA (i.e. inversions & balanced translocations) ** Faster

Purine Catabolism?

• All purine degradation leads to uric acid • Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases in the intestine • Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosides - Direct absorption of nucleosides - Further degradation

Creatine?

• Also called phosphocreatine • High energy compound - Small but rapidly mobilized reserve of high energy phosphates - Helps maintain intracellular levels of ATP

Phosphatidylinositol

• An unusual and important phospholipid • Often contains stearic acid(18:0) at carbon 1 and arachidonic acid (20:4) on carbon 2 of the glycerol backbone • Serves as reservoir of arachidonic acid which is used as a substrate for prostaglandin synthesis • PI has a role in signal transmission across membranes

Antioxidant Vitamins?

• Antioxidant vitamins terminate free radical chain reactions initiated by superoxide anion & hydroxyl radicals 1. Vitamin E (tocopherols): - Lipid soluble - Protect against lipid peroxidation in membranes 2. Vitamin C (ascorbate): - Water soluble->circulates in blood & ECF - Regenerates reduced (functional) form of vitamin E 3. Beta-carotene (precursor of vitamin A): - Lipid soluble - Similar in function to vitamin E - Protects against damage from sunlight in retina & skin

The pathology associated with cystic fibrosis as well as the methods of diagnosis and screening, as well as:

• Autosomal recessive • Chronic pulmonary disease • Exocrine pancreatic insufficiency • Failure to thrive, meconium ileus in infants • Sinusitis, nasal polyps • Infertility in males • Elevated sweat electrolyte levels

Fetal Cell Sampling

• Based on detection & analysis of circulating cell-free fetal (ccff) nucleic acids in blood sample from pregnant woman • Can detect ccff at 12th week of pregnancy • No risk of interference of ccff DNA from previous pregnancies (ccff undetectable 2 hrs postpartum) but twins/multiples are an issue • Non-invasive & no risk for miscarriage • Utilizes next generation sequencing methods & determines ratio of sequences from each chromosome (if ratio >< than expected then fetus has trisomy, monosomy or deletion) • Detects trisomies 13, 18, 21, sex aneuploidies & some microdeletions (cris-du- chat, DiGeorge & others)

Biochemical Testing for Inborn Errors of Metabolism? Enzyme assay?

• Biochemical testing : ~measure enzyme activity in patient cells (from biopsy or blood draw) • Enzyme assay : mix substrate analog & enzyme in extract of patient cells => reaction generates color/fluorescence=>quantifiable & signal is proportional to amount of enzyme activity

CYP2D6 & CYP2C19 and different levels of enzyme activity 4?

• CYP2D6 & CYP2C19 are members of P450 family of cytochromes • Involved in drug metabolism in liver =>affect how well drug reaches therapeutic level & how well drug is cleared • Genetic variation in CYP2D6 & CYP2C19 gives rise to alleles with different levels of enzyme activity & different metabolic phenotypes for drugs 1) Extensive metabolizers (normal enzyme activity & metabolism) 2) Intermediate metabolizers (reduced activity & slower than normal metabolism) 3) Poor metabolizers (no activity & almost no metabolism) 4) Ultrarapid metabolizers (higher than normal activity & faster than normal metabolism)

multiplex FISH

• Can simultaneously use chromosome-specific probes for ALL chromosomes • Probes color-coded by chromosome according to convention • Method called M-FISH (multiplex FISH) or SKY (spectral karyotyping) • Allows for simultaneous visualization of all chromosomes

Regulation of Pyrimidine synthesis?

• Carbamoyl synthetase II is inhibited by UTP and activated by PRPP

Types of screening: when is screening performed?

• Carrier (asymptomatic) screening using cells obtained from parents preconception for reproductive counseling (blood sample) • Pre-implantation screening using cells from in vitro fertilized embryo (Pre- implantation Genetic Diagnosis/PGD) • Prenatal screening using fetal cells obtained during pregnancy (via amniocentesis, chorionic villus sampling, cordocentesis) • Neonatal (newborn) screening using cells obtained from newborn (dried blood spot for MS/MS)

Stem Cell Requirements?

• Cells that Persist for the Lifetime of the Organism • Continues to Reproduce Itself • Generates Differentiated Progeny (Involves Transit Amplifying Cells)

Metabolic change in Type II diabetes?

• Characterized by hyperglycemia and dyslipidemia 1. Hyperglycemia - decreased uptake by GLUT4 in muscle, adipose - increase in gluconeogenesis via loss of PEPCK repression 2. Hypertriacylglycerolemia -Due to elevated chylomicrons &VLDLs because of decrease in lipoprotein lipase activity

CoA and Pantothenic Acid

• CoA (coenzyme-A) synthesized from pantothenate/pantothenic acid (vitamin B5) • Dietary sources=> most foods but eggs & yeast are good sources • AI is 5 mg/day Animals require pantothenic acid to synthesize coenzyme-A (CoA) • Deficiency very very rare; Causes burning foot syndrome (observed in POWs in Asia during WWII) - painful burning feet - irritability, restlesness - fatigue, apathy, sleep disturbances - GI distress, nausea, vomiting, abdominal cramps

Copper

• Cofactor for enzymes - lysyl oxidase (crosslinks collagen & elastin) - tyrosinase (melanin production) - dopamine β-hydroxylase (catecholamine production) - cytochrome c oxidase (ETC) - superoxide dismutase (scavenges superoxide radicals & prevents membrane damage) - ceruloplasmin (promotes absorption of iron) Deficiency: • Effects - weak-walled blood vessels - failure of pigmentation - neurological effects - decreased ATP formation - tissue damage - iron deficiency anemia

Important facts about the CF gene

• Complete gene locus spans 250 kb • 27 exons • Mature mRNA of 6500 bases • Encodes an ion channel of 1480 amino acids (CFTR) • Three-nucleotide deletion of codon 508 (phe) in 70%

Glycerophospholipids

• Constitute the major class of phospholipids • Phosphatidic acid is the simplest phosphoglyceride and serves as the precursor for other species of this group • Other phospholipids are formed from phosphatidic acid and an alcohol - Serine - Ethanolamine - Choline - Inositol

Thiamine Deficiency

• Decreased ATP production which affects NS activity • Accumulation of lactate & pyruvate (lactic acidosis) • Decreased NADPH formation via HMP & affects fatty acid synthesis (myelin synthesis affected & peripheral neuropathy) • 1. Beri-beri - dry skin - irritability - progressive paralysis 2. Wernicke's encephalopathy - confusion, ataxia - nystagmus & eye paralysis - peripheral neuropathy • If untreated, may progress into Korsakoff 's psychosis - irreversible syndrome - amnesia & deficits in learning & memory Causes of Deficiency: • Alcoholism • Systemic disease (cancer , AIDS) • Insufficient dietary intake • Severe liver disease

Phospholipid Metabolism

• Degradation of phospholipids is carried out by specific phospholipases found in all tissues • Phospholipases hydrolyze the phosphodiester bonds of phosphoglycerides • Each phospholipase cleaves bonds at a specific site • Phospholipases not only degrade but can remodel phospholipids

Celecoxib/Rofecoxib

• Develop blood clots due to -decreased production anti-thrombotic (clot blocking) prostaglandin (PGI2) by endothelial cells lining small blood vessels -Lack of inhibition of COX-1-mediated formation of pro-thrombotic thromboxanes in platelets

Advantages of Patient Specific iPS Cell Lines

• Differentiated cells will be a perfect immunological match to the donor, permitting grafting back without the use of immunosuppressive drugs. • Permits treatment of genetic diseases or cancers (e.g., repopulation of bone marrow). • Tumor formation represent major concern due to mechanisms of viral gene insertion (introduction of pluripotency genes). Host genes may be interrupted. In addition, silenced genes (which occur during passage) may be reactivated.

PDH deficiency what is it due to?

• Due to mutation (usually in E1)=> lactic acidosis & neurological symptoms • X-linked mode of inheritance • Most severe: - Mental retardation, Spasticity, Seizures, Structural abnormalities in CNS • Treatment: management of symptoms via nutritional & pharmacological intervention: - ketogenic diet (low carbohydrate/high fat) - thiamine administration - muscle relaxants & anti-convulsants

Cofactor Requirements for PDH which ones are permanently attached to the enzyme and which are diffusible?

• E1: pyruvate carboxylase => Thiamine Pyrophosphate (TPP) cofactor • E2: dihydrolipoyl transacetylase => Lipoic Acid cofactor (target of arsenic poisoning) • E3: dihydrolipoyl dehydrogenase=>FAD cofactor • Also requires NAD+ and CoA as cofactors • TPP, lipoic acid & FAD => prosthetic groups (permanently attached to enzyme) • NAD+ and CoA => diffusible

Type II due to mutations in dihydropteridine reductase treatment ? What levels are affected?

• Elevated plasma Phe levels but patients not responsive to low Phe (still developed neurological impairment & mental retardation) suggests defects in tetrahydrobiopterin biosynthesis or regeneration • BH4 levels tested in all newborns as part of NBS (via tandem MS) • Identification of these individuals is key because treatment is very different than for classic PKU • Treat by low Phe diet (not to extent as with classic PKU), supplementation with L- dopa, carbidopa, 5-OH-Trp & pharmacologic doses of BH4

Approaches for Classifying Novel Missense Variants

• Examine genetic code, nature of AA substitution • Position of affected residue in 3-D protein structure • Study of additional family members • General population studies • Evolutionary conservation • Correlation with phenotype: family history, histology, molecular/biochemical defects (e.g., MSI, ER/PR) • Functional studies

Important facts about the dystrophy gene. How is multiplex PCR used in diagnosis of mutated genes?

• Example of disease for which the gene is known but the mutation is not • Gowers sign - primarily males, can only stand up by climbing up • One of the Largest genes known: >2.5 million bp • 79 exons • Mature mRNA transcript of 14 kb • Deletions found in 2/3 of patients, and 1/3 have single nucleotide changes • Multiplex amplification primer o PCR primers spaced equally across gene → PCR product diff size → run thru gel electrophoresis → if one was not hybridized that's where mutation is

How do we identify a gene responsible for a genetic disorder? How does one find chromosomal location?

• First, the pattern of inheritance (autosomal/ sex-linked; dominant/recessive) is determined by pedigree analysis. • The linkage of the gene to a specific chromosome is established. • Gene is then cloned by applying a "positional cloning" approach. - -> Identification or cloning of a disease gene based on its chromosomal location. --> Some early examples of genes identified by positional cloning: • DMD, CF, Huntington disease, adult polycystic kidney disease, colorectal cancer - By linkage mapping: • Link the disease to a region of genome by using polymorphic markers. • Analyze the region for the presence of most likely candidate. • Confirm that the candidate gene is mutated in affected individuals.

Thyroid Hormones where are they made? What are some products and what do they ultimately become?

• Follicular cells of thyroid gland produce thyroglobulin • Iodination of tyrosine residues in thyroglobulin produces - Monoiodotyrosine (MIT) - Diiodotyrosine (DIT) • MIT and DIT undergo coupling reactions to produce - 3,5,3-triiodothyronine (T3) - 3,5,3,5-tetraiodothyronine (T4) : AKA thyroxine

Plasmalogens

• Formed when an unsaturated alkyl group replaces the fatty acid at carbon 1 and is attached to the glycerol backbone via an ether bond (instead of usual ester bond) • Ex: - Phosphatidalethanolamine (found in nerve tissue) - Phosphatidalcholine (found in heart muscle) • Also termed ether lipids • Function unclear

Vitamin C

• Functions as coenzyme in hydroxylation reactions (collagen & adrenaline/ noradrenaline synthesis) = critical for collagen synthesis • Required for maintainance of normal connective tissue & wound healing • Reducing agent (maintains active site metals for catalysis) • Facilitates absorption of dietary iron • Antioxidant & free radical scavenger (oxygen radicals; maintains Vit. D in reduced state)

Describe how molecular profiling tests use tumor gene expression signatures to determine survival, risk of recurrence, risk of metastasis & treatment outcome.

• Gene expression analysis by microarrays & real time RT-PCR • Molecular profile (signature) of tumor (disease sub-type) • Gene expression profile of tumor used to predict likelihood of metastasis, recurrence 1. Looking at RNA of the tumor->Oncotype DX Breast Cancer Assay • Gene expression profile of 21 gene panel used to calculate a recurrence score within 10 years of initial diagnosis & assesses whether women will benefit from certain types of chemotherapy • Test performed on a small amount of tissue removed during tumor resection using real time RT-PCR 2. Mammaprint ® Breast Cancer Assay-> microarray • Gene expression profile of 70 gene panel used to predict risk of metastasis over 10 years • Test performed on tumor biopsy removed during tumor resection by microarray hybridization

Genome Wide association studies?

• Genome-wide association study: Case-control study to identify specific SNPs with higher frequency in cases vs controls (i.e. associate with disease in statistically significant fashion) • SNPs identified are in linkage with (close to/flanking) disease gene(s) • Allows to narrow down region where disease gene(s) is(are) located • Can then identify disease gene(s) (susceptibility locus/loci) with fine mapping methods

Glucagon and Fasting/Starving in relation to glycolysis and gluconeogenesis?

• Glucagon => repression of transcription of PFK-1& Pyruvate Kinase => glycolysis OFF • Glucagon => induction of transcription of PEPCK => gluconeogenesis ON

Metabolic effects of glucagon?

• Glucagon increases blood glucose levels • Stimulates access of stores • Stimulates hepatic glucose production • mRNA transcribed & protein translated in α cells of pancreas • Preproglucagon processed to glucagon via proteolytic events • Mature protein is 29 aa & single polypeptide chain 1. Glucagon bind to the GPCR (glucagon receptor) allows for the G protein coupled activation of adenylate cyclase --> cAMP active--> Increase in intracellular levels of second messenger cAMP: • Activation of cAMP-dependent Protein Kinase A; PKA phosphorylates: Metabolic enzymes & mediates downstream effects 1. Gene expression Induction/ repression Change in enzyme levels 2. Enzyme Phosphorylation Change in enzyme activity (Reversal of insulin effect)

Glycogen properties

• Glucose polymer (10,000-40,000X) • Branching increases solubility & provides multiple sites for adding or removing glucose residues (synthesis & degradation) • Branches every 8-12 residues • α (1,4) bonds between glucose in linear chain • α (1,6) bonds between glucose at branchpoints Glycogen synthesis & degradation occur in cytoplasm • Glycosidic bonds form between OH group of anomeric C (C-1) of free glucose & a free OH group of glucose in glycogen *** Each glycogen molecule has 1 reducing end and many non-reducing ends: free OH at C-1 => free oxygen can be oxidized => Reducing end

How does elongation occur in glycogenesis? What does glycogen synthase need? What is glycogenin?

• Glycogen synthase creates α(1,4) between C-4 of non-reducing end of glycogen & C-1 of UDP-glucose; cycle repeats 11X before new branch formation • Glycogen synthase needs primer for elongation: 1. Existing glycogen molecule (has to be at least 4 glucose residues long) 2. glucosyl chain attached to glycogenin • De novo chain synthesis performed by glycogenin • Glycogenin autoglycosylates at a tyrosine residue and then autoglycosylates again until chain is long enough to be a substrate for glycogen synthase • Glycogenin remains associated with & forms core of glycogen molecule

Energy use of Glycogenesis?

• Glycogenesis is an anabolic process • 1ATP & 1UTP utilized per new glucose added • Glycogenesis occurs in fed state => ATP is from oxidation of dietary glucose

Glycogenolysis & Homeostasis of Blood Glucose Levels

• Glycogenolysis in liver & muscle provides glucose • Muscle glycogen is a source of glucose during exercise • Liver glycogen is an easily accessible store of glucose to be used during fasting • Occurs in fasting state in liver and during exercise in muscle • Helps prevent hypoglycemia because it provides a source of blood glucose Glycogenliver =>=> Glcblood

Branching enzyme how does it work in glycogenesis?

• Hydrolyzes α(1,4) between glucose residues in linear chain • Transfer of terminal 6-8 glucose segment from non- reducing end to internal position • Creates α(1,6) bond between glucose in internal position & terminal glucose of fragment • New non-reducing end at branch is substrate for glycogen synthase

Hyperphenylalanemias and Biopterin Defects

• Hyperphenylalanemias : Increase in Phe serum levels due to defects in Phe metabolism • Can be due to defects in biosynthesis or regeneration of tetrahydrobiopterin cofactor (BH4) for phd hydroxylase=>Type II & III hyperphenylalanemias (less common; 1-3% of cases) • Type II due to mutations in dihydropteridine reductase; Type III due to mutations in dihydrobiopterin synthetase

Hyperphenylalanemias

• Hyperphenylalanemias caused by defects in metabolism of Phe (essential aa) elevated serum Phe • Elevated serum Phe disruption of important cellular processes in brain (myelination & protein synthesis)=>severe mental retardation • Early identification of affected individuals & nutritional & therapeutic management is KEY • Can be due to mutations in phenylalanine hydroxyls = Classic PKU (type I; more common) manifests as lethargy feeding difficulty, eczema, mousey odor • Treatment is dietary restriction (low Phe diet; complete lack of Phe is fatal) => must start latest 4 weeks after birth & should be life-long

The significance of ΔF508 (what does this abbreviation mean?).

• Important for post translation process; high freq delta - deletion F - single letter symbol for phenylalanine 508 - codon/gene

Sources of Human Embryonic Stem Cells

• In vitro fertilization for reproductive purposes→ eggs from donor fertilized & allowed to develop in vitro (in culture) • Fertilized eggs implanted into mother in fertility clinics • Extra embryos frozen →excess embryos that can be used to harvest ES cells • Inner cell mass harvested from blastocyst (30 cells) → cultured with feeder cells • Feeder cells provide matrix for attachment & nutrients • Feeder cells can contaminate ES cells with viruses or other molecules (safety issues in patient transplantation)

Insulin and Fed state in relation to glycolysis and glyconeogenesis?

• Insulin => induction of transcription of PFK-1& Pyruvate Kinase => glycolysis ON • Insulin => repression of transcription PEPCK => gluconeogenesis OFF

Secretion of Insulin

• Insulin mRNA transcribed & protein synthesized in β-cells of pancreas • Proteolytic processing in ER of β cells • Mature insulin stored in cyto. for exocytosis • Mature protein is 51 aa; A & B chain linked by disulfide bonds • Exocytosis stimulated by glucose, aminoacids & GI hormones (cholecystokinin & gastric inhibitory peptide)

Replenishing TCA Cycle Intermediates: Anaplerotic Reactions

• Intermediates replenished by anaplerotic reactions • 2 types of anaplerotic reactions: - 4 & 5C acids replenished via aminoacid degradation - oxaloacetate regenerated from pyruvate by pyruvate carboxylase

Duplications & Inversions what are they due to?

• Inversions & duplications occur due to errors in replication & repair • Inversions involve reversing positions of chromosomal segments (no loss or gain of DNA) • Inversions can be: - Pericentric => include centromere - Paracentric => do not include centromere • Inversion carriers are asymptomatic • Pairing & crossing over of sister chromatids during meiosis affected • Offspring of carrier can have clinical consequences

What molecules CANNOT enter gluconeogenesis?

• Ketone bodies, fatty acids & AcCoA cannot be used to generate glucose • No direct route from acetyl CoA to pyruvate because the PDH reaction is irreversible (and no bypass exists) ** need to be converted to pyruvate then OAA and rest of the steps can continue

Diseases Associated with ETC Components

• Leber's Hereditary Optic Neuropathy (LHON): - caused by mutations in any of several mt genes encoding Complex I subunits - progressive loss of central vision & blindness (degeneration of optic nerve) • Mitochondrial encephalopathy, lactic acidosis & stroke-like episodes (MELAS): - caused by mutations in any of several mt genes (genes encoding mt tRNAs ex. leucine, glutamine or genes encoding Complex I subunits) - progressive neurodegenerative disease & stroke like episodes • Myoclonus, Epilepsy and Ragged-Red Fiber disease (MERRF): - caused by mutations in any of several mt genes encoding mt tRNAs ex. phenylalanine, serine - progressive myoclonic epilepsy, slow progressive dementia • Leigh syndrome (subacute necrotizing encephalopathy): - caused by mutations in mt genes encoding Complex IV subunits - atrophy of optic nerve - respiratory abnormalities - hypotonia & spasticity ~ Leigh syndrome also caused by PDH mutations (X-linked)

Nitric oxide?

• Liberated in the conversion of arginine to citrulline • Important in macrophages for creating NO for the generation of free radicals which are bacteriocidal • Stimulates the influx of calcium ions into vascular endothelial cells with the activation of cGMP - Results in relaxation of vascular smooth muscle

What is source of ATP/ GTP & NADH during fasting & starvation?

• Lipolysis generates fatty acids & glycerol • Fatty acids used as fuel via β-oxidation • Glycerol used as a substrate for gluconeogenesis *** Fatty acid oxidation provides ATP & NADH for gluconeogenesis • ATP CANNOT be provided from glycolysis because glycolysis is OFF in 36 liver

Biochemical Testing for Inborn Errors of Metabolism: Tandem Mass Spectrometry (MS/MS)

• MS/MS identification of multiple compounds in complex biological mixture (blood sample) • Increased levels of certain metabolites result from specific enzyme deficiencies • Heel-prick of newborn (by 6th day of life ->dried blood spot ->complete aminoacid, organic acid & fatty acid panel=>can simultaneously test for several enzyme deficiencies & metabolic disorders **Can test several metabolites (i.e. enzyme deficiencies) at the same time

Phosphatidic Acid Synthesis

• Main precursor - phospholipids and triacylglycerols • All except mature RBC make phosphatidic acid • Triacylglycerols only made by liver, adipose tissue, lactating mammary glands and intestinal mucosal cells

Lung Surfactant

• Major lipid component of lung surfactant = dipalmitoyl- phosphatidylcholine (DPPC, or dipalmitoylecithin) - Positions 1 and 2 on the glycerol backbone is occupied by palmitate (16:0) - Secreted by granular pneumocytes - Serves to decrease the surface tension of the extracellular fluid lining the alveoli • This reduces the pressure needed to reinflate the alveoli and so preventing alveolar collapse (atelectasis) • Insufficient production is associated with respiratory distress syndrome in pre term infants • Considered a significant cause of neonatal deaths in developed countries

Risks for Mendelian and non-Mendelian Conditions: Counseling

• Mendelian- theory dictates risks (such as 1⁄2 or 1⁄4, depending on the pattern of inheritance: dominant or recessive) - Assume that a couple is CF carrier - What would be the risk for their next child affected? =1/4 (regardless of # of children) • Non-Mendelian conditions: risks are predicted from population survey (not from theory) - Neural tube defects: 1/25 in UK - If a couple has affected child- = Risk of having the next child affected: 1/12

Regulation of Blood Glucose Levels by Catecholamines (fight or flight)

• Metabolic effects of epinephrine => mobilization of fat & glycogen stores • Released during physical exertion, stress, cold exposure & hypoglycemia

Function & Classification of Minerals

• Minerals needed through diet • Major minerals are needed in larger amounts & are used mainly in bone (calcium, phosphorus, magnesium), are electrolytes (sodium, potassium, chloride), and are components of aminoacids (sulfur) • Trace elements are needed at very low levels for protein function (12 trace elements) • Trace elements can cause toxicity at high doses

Phosphatidylcholine Synthesis

• Most abundant phospholipid (along with phosphotidylethanolamine) • Primary route of synthesis is from choline (and ethanolamine) from the diet or from phospholipid turnover • Choline is considered an essential nutrient - Cells can make choline but the amount is not sufficient so choline is heavily reutilized

Why geneticists study the Ashkenazi Jewish population.

• Most of them have deltaF508 and W1282X

Cofactor and enzyme deficiency?

• Mutations that indirectly affect enzyme activity by affecting biosynthesis of cofactor on a DIFFERENT gene (gene involved in biosynthesis, regeneration, absorption or transport of cofactor) • All enzymes utilizing same cofactor affected ! multiple pathways affected ~Disease due to mutation in any of the genes involved in biosynthesis/ availability of cofactor(s) ~ Can affect several enzymes due to the cofactor deficiency

NADH and NADPH

• NAD+/NADH&NADP+/NADPH=>2e- donor/acceptor • Participate in redox reactions (dehydrogenase enzymes) • NAD+/NADH in catabolic pathways • NADP+/NADPH in reductive biosynthesis • Diffusible & not permanently attached to enzymes

Naming conventions for nucleosides and nucleotides?

• Nucleosides: 1. Purine nucleosides end in "-sine" • Adenosine, Guanosine 2. Pyrimidine nucleosides end in "-dine" • Thymidine, Cytidine, Uridine • Nucleotides: ~ Start with the nucleoside name from above and add "mono-", "di-", or "triphosphate" • Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate

Why genetic testing of children is not usually done for some of the adult onset diseases (e.g. Huntington's disease). Under what circumstances genetic testing should be done?

• Onset is during middle age, needless stigmatism, especially for disease with no treatment o Cause psychological distress • Genetic testing should be done if teenager (considered a child) is pregnant • the importance of "40 or above" repeats (mistakenly placed under fragile X...belongs under Huntington's disease instead) 40 or above - have Huntingtin disease (100% penetrance)

The role of pre-implantation genetic diagnosis.

• Option for Pro-Life clients (don't want to abort) • In-vitro fertilization -grow to 8-cell stage → test for disease → microdissect fibroblast → do PCR if neg for disease → only implant "normal" embryo • Risky because very challenging to do PCR on single cell, liability issues

Function and classification of Vitamins

• Organic molecules • Cannot be synthesized by humans and are therefore required through diet • Absence of vitamin causes deficiency =>=> disease

PCR amplification

• PCR is a way to rapidly amplify desired segment of patient DNA • After PCR amplification additional method is needed to screen for mutation in patient (sequencing, RFLP)

How Dr. Grody's study of CF screening was considered to be one of the best studies of the incidence of CF mutation frequency and why did his group screen for 6 different mutations.

• PCR- based screening for CF carrier mutations in ethnically diverse pregnant population • 6 mutations found in first ~3200 subjects; the extreme heterogeneity of CF mutations limits the sensitivity of avail DNA screening tests; also greater pick up rate bc it covers more of general population • Negative test results means: Increased - 0% Reduced, but not zero - 92% Zero - 8%

Regulation of PDH Complex

• PDH activity is highly regulated via: 1) allosteric effectors on PDH catalytic subunits 2) regulation of catalytic subunits by PDH kinase & PDH phosphatase (regulatory subunits of PDH) • Allosteric effectors of PDH => inhibition by AcCoA & NADH (feed-back inhibition) • PDH kinase => phosphorylates PDH => PDH INACTIVE • PDH phosphatase => dephosphorylates PDH => PDH ACTIVE Regulation: • PDH kinase (activated : NADH, acetyl coA, ATP) ; inhibited: ADP, pyruvate, Ca2+ • PDH phosphatase also regulated insulin + stimulation

Glucagon: Reciprocal Regulation of Glycolysis & Gluconeogenesis via F-2,6-BP

• PFK-2 & fructose 2,6-Biphosphatase are regulated by glucagon & insulin signaling • Glucagon => activates f-2,6-Bpase => loss of fructose-2,6-BP => 1) removal of PFK-1 allosteric activator => glycolysis inhibited 2) removal of F-1,6-Bpase allosteric inhibitor => gluconeogenesis activated

Insulin: Reciprocal Regulation of Glycolysis & Gluconeogenesis via F-2,6-BP

• PFK-2 & fructose 2,6-Biphosphatase are regulated by glucagon & insulin signaling • Insulin => activates PFK-2 => gain of fructose-2,6-BP => 1) presence of PFK-1 allosteric activator => glycolysis activated 2) presence of F-1,6-Bpase allosteric inhibitor => gluconeogenesis inhibited

Purine nucleotide degradation?

• Phosphate and ribose are removed first • Then nitrogenous base is oxidized

Melanin?

• Pigment occuring in -Eyes, Hair, Skin • Synthesized by melanocytes from Tyrosine • Provides protection from harmful effects of sunlight

Glycerol entry into Gluconeogenesis and Bypass 2?

• Point of entry of glycerol: - glycerol =>glycerol-3P by glycerol kinase (ATP hydrolysis) - glycerol-3P => DHAP by glycerol-3P dehydrogenase (NADH used)

"polygenic disorders" and why these polygenic diseases are difficult to identify using molecular techniques.

• Polygenic disorder - multiple genes in combination w/ lifestyle and environmental factors (ex. heart disease, diabetes, cancer) • Hard to identify w/molecular techniques bc of other variables involved

Pyrimidine Synthesis de novo?

• Pyrimidine ring synthesis completed first; then attached to ribose-5-phosphate • UMP synthesized first -CTP synthesized from UMP Pyrimidine base production. Requires aspartate. 1. Start with 2 ATP + CO2 + Glutamine(Carbamoyl phosphate synthetase II) --> carbamoyl phosphate 2. Aspartate used -> carbamoyl aspartate 3. Orotate --> OMP 4. ---> UMP (uridine 5' monophosphate) 5. UMP (ATP and Glutamine)--> CTP Pyrimidines: 1. Make temporary base (orotic acid) 2. Add sugar + phosphate (PRPP) 3. Modify base

From pyruvate to AcCoA and PDH reaction

• Pyruvate (cytoplasm) => pyruvate (mitochondrial matrix) via pyruvate transporter • Pyruvate => AcCoA by Pyruvate Dehydrogenase Complex (PDH) • Oxidative decarboxylation • Irreversible & highly regulated • PDH is a multi-subunit enzyme - 3 catalytic subunits (E1-E3; - Require 5 cofactors for activity) - 2 regulatory subunits (PDH kinase & phosphatase)

Pyruvate Carboxylase & Biotin

• Pyruvate carboxylase catalyzes pyruvate=> OAA • Occurs in mitochondria of liver & kidney • Functions to: 1) replenish OAA 2) in gluconeogenesis • Pyruvate carboxylase requires biotin as a cofactor • Biotin deficiency caused by eating raw eggs (avidin) • Deficiency symptoms: - Dermatitis - Glossitis - Loss of appetite

Reciprocal versus Robertsonian translocation

• Reciprocal translocations: - breaking & exchange between chromosomes - formation of 2 new derivative chromosomes - affects pairing & segregation during meiosis - can be balanced or unbalanced - incidence is 1:500 in general population • Robertsonian translocation: - involves 2 acrocentric chromosomes (13, 14, 15, 21, 22) - acrocentric chromosomes fuse at centromere - formation of new derivative chromosome - loss of satellite material from arms of acrocentric chromosomes - affects pairing & segregation during meiosis (familial Down syndrome (14q;21q)) Translocation=>break & exchange of chromosome frag. 1. Unbalanced translocations: loss /gain of genetic mat. 2. Balanced translocations: NO loss/gain of genetic mat. • Translocations can be identified by banding or FISH Carriers of translocations (parents) may be asymptomatic but may experience reproductive problems (infertility, miscarriage & having a child with anunbalanced chromosome complement)

The "economy class syndrome" and the relationship to Factor V - Leiden.

• Refers to long plane flights sitting in "Economy class" • Develop pulmonary embolism, Factor V - Leiden - clotting factor • If heterozygous for Gln506 mutation → 35 fold increase of embolism

Regulation TCA cycle

• Regulated = allosteric effectors • Citrate synthase: (-) citrate, ATP • Isocitrate dehydrogenase: (-) NADH; (+)ADP & NAD+ • α-ketoglutarate dehydrogenase: (-) succinyl-CoA, NADH & ATP • Net effect => Low energy charge ON ==>High energy charge OFF • OFF when intermediates accumulate

Iodine

• Required for synthesis of thyroid hormones (thyroxine & triiodothyronine) Deficiency: • Causes goiter =>enlargement of thyroid gland due to overproduction of Thyroid Stimulating Hormone (TSH) • Deficiency during pregnancy may result in hypothyroidism in newborn => growth & mental development may be impaired => cretinism 1) Lack of dietary iodine decreases T3 & T4 synthesis 2) Negative feedback on hypothalamus & anterior pituitary is released 3) Increase in release of TRH & TSH causes hyperplasia

Nucleoside

• Result from linking one of the sugars with a purine or pyrimidine base through an N- glycosidic linkage 1. Purines bond to the C1 carbon of the sugar at their N9 atoms 2. Pyrimidines bond to the C1 carbon of the sugar at their N1 atoms

Recommendation for genetic testing of children and how MEN and FAP are used to argue for/against genetic testing of children.

• Scenario: husband does not want to know if he has Huntingtin disease, mother does not want Huntingtin child so she wants to Preconception Dx • If prenatal diagnosis is performed and the fetus is positive for a CAG expansion, then the father has been diagnosed, too • Recommendation: Preimplantation Dx (uses in-vitro) - if disease gene is found in embryo, can notify parents that they've implanted so many "normal embryos" w/out revealing that there was a disease gene found (bc then father has been diagnosed, too) • MEN: Multiple Endocrin Neoplasi -Early detection will prevent cancer • FAP - Polyps in colon have 100% chance of becoming cancerous - Early testing able to start early aggressive removal of polps and eventual colonoscopy

Vitamin C Deficiency

• Scurvy (symptoms due to decrease in collagen synthesis) - swollen, sore & spongy gums with bleeding & loose teeth - anemia - spontaneous bruising - poor wound healing - swollen joints & muscle pain Causes of Deficiency: • Insufficient dietary intake of fresh fruits & vegetables • Seen in elderly people & alcoholics ~Vitamin C is critical for collagen maturation

Describe Glycogenolysis? What are the two products that are made by two different enzymes needed to degrade the linear chain and the branched chain? Which product gives the most glucose in the glycogen?

• Sequential removal of glucose residues from non-reducing ends • Mechanisms for degradation of linear chain phosphorolysis of α(1,4) bonds & for branch removal α(1,6) bonds • Glycogen phosphorylase degrades α(1,4) bonds & debranching enzyme degrades α(1,6) bonds 1. Glycogen phosphorylase produces glucose-1-phosphate (92% of total glucose in glycogen) then is converted to G-6-P via phosphoglucomutase 2. Debranching enzyme produces glucose (8% of total glucose in glycogen)

COX-3

• Some evidence suggests the existence of a third isoform of PGH2 Synthase, designated COX-3 - with roles in mediating pain and fever - subject to inhibition by acetaminophen (Tylenol) • Acetaminophen has little effect on COX-1 or COX-2, and thus lacks anti-inflammatory activity

Phospholipid Synthesis

• Synthesized SER • Transported to the Golgi and then to membranes of organelles/ plasma membrane • Are also secreted from the cell -> exocytosis 1. Donation of phosphatidic acid from CDP-diacylglycerol → alcohol OR 2. Donation of phosphomonoester of alcohol fr CDP-alcohol → 1,2-diacylglycerol • Activation of diacylglycerol or alcohol to be added by linkage to CDP (NT cytidine diphosphate)

PDH & Thiamine Deficiency What syndrome? What is the vitamin?

• Thiamine deficiency (vitamin B1) => Beriberi & Wernicke-Korsakoff syndrome • Thiamine deficiency in areas where polished rice is major component of diet= Causes Beriberi • Beriberi can be wet or dry: - wet: edema & cardiovascular symptoms (cardiac failure) - dry: muscle wasting & peripheral neurologic dysfunction • In industrialized countries thiamine deficiency observed in chronic alcoholics & gives rise to neuropsychiatric syndromes (EtOH interferes with thiamine absorption) - Wernicke encephalopathy: reversible (ataxia, confusion, nystagmus) - Korsakoff syndrome: irreversible (deficits in learning & memory) - Wernicke-Korsakoff syndrome Note: Thiamine also required for transketolase (HMP) and α-ketoglutarate dehydrogenase (TCA cycle)

Glutathione describe?

• Tripeptide synthesized from glutamate, cysteine and glycine • Contains unusual linkage between glutamate side chain carboxylate group and the nitrogen of cysteine - Antioxidant role - Transports amino acids across cell membranes: Glutamyl cycle

Ultrasonography : Prenatal Screening for Congenital Disorders

• Ultrasonography can reveal structural abnormalities associated with chromosomal defects & with certain single gene & multifactorial disorders • Performed at 18 weeks gestation Defects associated with single gene disorders: 1. Holoprosencephaly 2. Infantile polycystic kidney disease 3. Merkel-Gruber syndrome (encephalocele, polydactyly & polycystic kidneys) 4. Fyrns syndrome (abnormalities in face, diaphragm, limbs, GU tract & CNS) Defects associated with multifactorial disorders: 1. Cleft lip 2. Club foot 3. Congenital heart defects 4. Neural tube defects

Henderson-Hasselbach equation Buffers

• consist of a weak acid and its conjugate base or a weak base and its conjugate acid • Resist changes in pH when [H+] or [OH-] are added • Working range: 1 pH unit above and below the pKa

Neurofibromatosis 1: Linkage Mapping. How was linkage of NF1 determined?

• von Reclinghausen disease • Autosomal dominant - Brownish spots on skin (café au lait) - Benign nodules on iris - Neurofibromas (fleshy benign tumors along the course of nerves) - Serious growth abnormalities and deformities • Linked to chromosome 17 • Linkage was determined: Restriction fragment length polymorphism (RFLP) markers were used -RFLP marker sequences are either susceptible or resistant to digestion with specific restriction enzymes mutations -By using a probe can distinguish between 2 alleles with 1 EcoRI site is intact (restriction site) and another where the EcoRI site has been mutated get different fragment this way ** determined there is recombination and there is a marker to a certain allele, marker is NOT the gene use the marker to find the linkage

COX-1 and COX-2

•2 isozymes of PG endoperoxide synthase •COX-1 -Always ON made constantly; in most tissues -Required for normal gastric tissue, platelet aggregation and renal homeostasis •COX-2 -Inducible in response to products of immune and inflammatory cells -PG's produced mediate pain, heat, redness, and inflammation and fever -Inflammation assoc w/ up-regulation of COX-2 and increased prostaglandins -Increased in arthritis, seen in some cancer cells -Angiogenesis (blood vessel development) → tumor growth -Overexpression → inc VEGF (vascular endothelial growth factor) -NSAIDs dec colorectal cancer (due to inhibition of COX2) ** potential anti-cancer drugs -may have role in memory COX-2

Platelet Activating Factor

•Ether glycerophospholipid w/ saturated alkyl group, link to carbon 1 (16-18 Carbons long) • Carbon 2 - acetyl group instead of FA • Functions: Activate inflammatory cells, mediate hypersensitivity, acute inflammatory and anaphylactic rxns • Platelet aggregation and degranulation • Promotes superoxide radicals formation from neutrophils and alveolar macrophages • Potent bioactive cmpd -low amount can illicit a biological response