biochem_mod_6

• GGATCC
• CCTAGG

1) RESTRICTION ENDONUCLEASES -- CLEAVE 2X DNA TO CREATE STICKY/BLUNT ENDS

2) VECTORS

3) CONTRIBUTING DNA

1) PLASMID -- SMALL EXTRACHROM, CIRCULAR DNA MOL THAT REPLICATES INDEPENDENTLY. CONFERS Ab RESISTANCE

2) PHAGES -- LINEAR DNA MOLS THAT USE BAC AS HOST

3) VIRUSES -- USE MAMMALIAN CELLS AS HOST

• GENOMIC
• CHROM DNA INCLUDING INTRONS --> RESTRICTION NUCLEASE DIGESTION CREATES DNA FRAGS --> CLONING --> STILL WITH INTRONS

• cDNA
• mRNA --> REVERSE TRANS AND DNA CLONING --> cDNA (NO INTRONS)

1) ANNEALING PROPERTIES OF 2 COMPLEMENTARY STRANDS. H-BONDS

2) DOUBLE HELIX ONLY WHEN STRANDS PERFECT COMPLEMENT. IF NOT, THEN KINK WILL FORM

• SOUTHERN
• 1) DETERMINES IF GENE PRESENT IN TISSUE (DNA)

2) DETERMINE COPY NUMBER OF GENE

3) DETERMINE GROSS ALTERATIONS IN A GENE

• NORTHERN BLOT
• 1) DET ID GENE EXPRESSED AS mRNA

2) DET STRUCTURAL ALTERATION IN mRNA MOL

• WESTERN
• USE Ab TO DETECT PROT OF INTEREST

SIMULTANEOUS DETECTION OF EXPRESSION STATUS OF NUMEROUS GENES USING NUC ACID HYBRIDIZATION

ex -- COMPARISON OF mRNA EXPRESSION OF NORMAL (GREEN) vs CANCEROUS (RED) CELLS AND UNDIFFERENTIATED vs DIFF CELLS

• GREEN -- NORMAL MAKES MORE
• RED -- CANCER MAKES MORE
• DARK/BLACK -- NEITHER PRODUCES

REPEATED COPYING OF x2 DNA --> AMPLIFICATION

1) SEPARATION USING HEAT

2) HYBRIDIZATION OF 2 SPECIFIC PRIMERS MADE OF SHORT COMP STRANDS

3) DNA SYNTH USING HEAT STABLE POL

4) REPEAT 1-3

A TO T TRANSVERSION IN b-GLOBIN GENE DESTROYS RECOG SITE OF RESTRICTION ENDO.

SS HOMO HAS LARGER SINGLE DNA FRAG THAN NORM (2 FRAGS)

DELETION OF b-GLOBIN CHAIN CAUSING SHORTER FRAG THAN NORM

H1N1 AND HIV INFECTION -- AMPLIFY VIRAL DNA

INVASIVE CANDIDIASES

1) PROMOTER BLOCKING -- INTRODUCING FAKE PROMOTER WITH HIGH AFFINITY FOR TRANSCRIPTION FACTORS SO DIVERTING AWAY FROM REAL PROMOTERS

2) ANTISENSE NUCLEIC ACID -- COMP OLIGONUCS TO KNOWN SEQUENCE OF mRNAs OF SELECTED GENES CAN BE USED TO INH TRANSLATION OF SPECIFIC mRNAs.

• 3) GENE REPLACEMENT THERAPY:
• --DNA INTROed INTO CELL BY INTENSE ELECT FIELD

--NON-IMMUNOGENIC LIPID COMPLEXES THAT HELP DNA ENTER CELL

--RECEPTOR MEDIATED ENDO BY COVALENT LINKAGE TO LIGAND

--VIRAL VECTORS INCLUDE LTR, GAG, POL, ENV

ABSENCE OF FUNC T, B, AND NK CELLS. DEFECT IN CYTOKINE RECEPTOR gc GENE

RETROVIRAL VECTOR INSERTED INTO STEM CELLS in vitro THEN RE-INFUSED BACK INTO PATIENT

RANDOM INSERTION OF VECTORS INTO CHROM LEAD TO DEATH

RANDOM VECTOR INSERTION LEAD TO POSITIVE OUTCOME

MITOTIC SPINDLE

INTERPHASE MICROTUBULE NETWORK GUIDES THE MOVEMENT OF VESICLES AND ORGANELLES; INVOLVED IN INTRACELLULAR TRANSPORT/SECRETION

FORM AXONEMES OF CILIA AND FLAGELLA

CONSTANTLY GROW AND SHRINK

ONLY PLUS ENDS

ALLOWS MICROTUBES TO EXPLORE ENV

DYN BEHAVIOR STOPS WHEN BIND TO TARGET STRUCTURES

SIGNAL GIVEN THAT ALLOWS CHROM TO SEPATATE AND ANAPHASE BEHINS

WITHOUT DYN, THOUSANDS OF MICROTUBES WOULD HAVE TO FORM TO MAKE CERTAIN EACH CHROM IS CONNECTED

DIMER OF POLYPEPTIDES CALLED a AND b

ARRANGED END TO END

3rd FORM CALLED g-TUBULIN FOUND IN CENTROSOMES AND ACTS AS MICROTUBULE "ROOT" & NUCLEATES MICROTUBES

1) BIND SITE FOR LARGE VARIETY OF LIGANDS

-a & b BIND GTP FOR MICROTUBE ASSEMBLY. ONCE ADDED, GTP HYDROLYSED TO GDP, THEN REPEAT

-OTHER BINDINGS SITES FOR COLCHICINE, VINCA, AND TAXANE

2) C-TERM REGIONS OF a AND b HIGHLY NEGATIVELY CHARGED

-CONSIST LARGELY OF GLUT AND ASP RESIDUES

-STICK STRAIGHT OUT; MAY PLAY ROLE IN SIGNALING

3) NUMBER AND NATURE OF POST-TRANSLATIONAL MODS

-LYS40 OF a-TUBE ACETYLATED FOR STABILITY (AXON)

-a-TUBE SYNTHED WITH C-TERM TYROSINE RES (REMOVED BY TUB CARB-PEP AND REPLACED BY TUB TYR LIGASE) FORM PEPTIDE BOND NON-RIBOSOMALLY. TYR SIGNAL TO REG STABILITY (NO TYR = MORE STABLE)

- BOTH a & b POLYGLYCYLATED. MAY RESULT IN UNUSUAL DOUBLET MICROTUBES

BLOCK DYNAMIC INSTABILITY

MOST SUCCESSFUL CANCER THERAPY AVAILABLE

TAXANES USEFUL IN BREAST, OVARIAN, AND LUNG CANCER

VINCA USEFUL IN HODGKIN AND ACUTE LYMPHOCYTIC LEUKEMIA

EXCESS URIC ACID PRODUCTION AND DEPOSITION AS CRYSTALS IN JOINTS

WHITE BLOOD CELLS RELEASE INF FACTORS

COLCHICINE FOR ACUTE GOUT BINDS TO TUBULIN MOLS AT PLUS ENDS AND FREEZES DYNAMICS

TARGETS WBCs TO LOSE PSEUDOPODIAL MOTILITY SO CANNOT MIGRATE TO JOINTS

IF IN JOINTS, PREVENTS SECRETION OF ORGANELLES CONTAINING INF FACTORS

LONG FILAMENTOUS PROTS LACKING ENZ ACT THAT BIND TO C-TERM REGIONS OF MICROTUBES

PROMOTER DOMAIN THAT BINDS TO MICROTUBE AND PROJECTION DOMAIN THAT STICKS OUT

• PROMOTERS INDUCE GROWTH, INH DYN
• INSTABILLITY

PROJ INDUCE SPACING AND REG VELOCITY OF MOTOR PROTS THAT TRAVEL ON MICROTUBULES

PHOS OF MAPS MAKES THEM MORE NEG --> MICROTUBES ALSO NEG, SO DIMINISHES INTERACTION

SENILE PLAQUES MADE OF AMYLOID PROT; PAIRED HELICAL FILAMENTS MADE OF TAU WHICH IS HYPERPHOSED

TAU TOO NEG TO BIND AND STABILIZE NEURONAL MICROTUBES

INSTEAD SELF-ASSEMBLES TO FORM PAIRED HELICAL FILS

NEURONAL TUBES BEGIN TO SHRINK AND ARE UNABLE TO TRANSPORT SYN VESICLES --> DEMENTIA

KINESIN, CYTOPLASMIC DYNEIN, AND AXONEMAL DYNEIN

KINESIN CARRIES CARGO IN ANTEROGRADE DIRECTION

DYNEIN - RETROGRADE

AXONEMAL DYNEIN POWERS CILIARY AND FLAGELLAR MOTILITY

GENETIC

LESIONS IN AXONEMAL DYNEIN

CILIA AND FLAG CANNOT MOVE. SPERM AND MUCUS ELEVATOR IMMOBILE

MALE INFERT, FREQ BRONCH INF, IN 50% SITUS INVERSUS TOTALIS

INC SPASTICITY AND NEUROMUSCULAR WEAKNESS, ESP IN LOWER EXT

MUTATED KINESINS

ONE TYPE ATPase ACTIVITY INH SO KINESIN CANNOT WALK ON THE MICROTUBE

MOSTLY IN NEURONS --> INH ANTEROGRADE TRANS

MUTATION IN INTRAFLAGELLAR RAFT

G-PROT REC DO NOT GET INTO THE PRIMARY CILIA AND SIGNALING COMPROMISED --> DEV PROBS

RETINOPATHY, CARDIOMYOPATHY, POLYDACTYLY, HYPOGONADISM, M&PR, AND OBESITY

1 IN 500 LIVE BIRTHS

ACCUMULATION OF CSF

ONE FACTOR IS LACK OF HYDIN PROT --> PART OF CILIARY AXONEMES --> AXON MOTILITY

BEATING OF CILIA IRREGULAR AND UNABLE TO PUMP CSF OUT OF VENTRICLES

INFECT WIDE RANGE OF CELL TYPES

UNLIKE RETRO, CAN TRANSFER INTO NON-DIVIDING CELLS

GENERALLY NON-INTEGRATING (EPISOMAL)

DISADVANTAGES

EPISOMAL VECTORS OFTEN LOST AFTER CELL DIVISION

IMMUNOGENIC

INSERT INTO WIDE RANGE, THUS HARD TO TARGET SPECIFIC CELL TYPES

MOST ABUNDANT PROT; HIGH TENSILE STRENGTH

SUBUNITS CALLED TROPOCOLLAGEN. FORM POLYMERS --> FIBERS OR NETWORKS. FIBERS ARE 3 POLYPEP CHAINS FORMING TRIPLE HELIX

FIBERS COVALENTLY CROSS-LINKED TO EACH OTHER AND WITHIN x3 HELIX

INDIVIDUAL T. COLL IN x3 HELIX HAS HELICAL SHAPE CALLED TYPE II TRANS HELIX. STABILIZED BY STERIC REPULSION USING PROLINE, MANY OF WHICH ARE HYDROXYLATED FORMING 4-HYD.PROLINE THAT H-BOND

LYSINE IN COLLAGEN HYDROXYLATED TO MAKE HYD.LYSINE --> ATTACHMENT POINT FOR GALACTOSE AND GLUCOSE --> H-BOND AND CROSS-LINK FOR STABILIZATION

GLYCINE = 1/3 OF COLLAGEN; EVERY 3rd POSITION. POINT TOWARD CENTER OF HELIX. IF OTHER AA, SIDE CHAIN WOULD CREATE BULGE AND DESTABILIZE

MANY POST TRANSLATIONAL MODS

1) PREPROCOLLAGEN SYNTHED ON RIBOS OF RER. NEEDS O2 AND ASCORBIC ACID (VIT C) AS CO-FACTOR FOR SERIES OF HYDROXYLATIONS

2) PREPROCOLLAGEN ENTERS LUMEN OF ER. SUGARS ARE ADDED (GLUC & GAL) --> OLIGOSACC ADDED AND RELEASED INTO LUMEN OF ER --> 3 GLUC REMOVED AND DISULFIDE BRIDGES FORM AND x3 HELIX MADE SPONT. --> NOW PROCOLLAGEN

3) SECRETION OF PROCOLLAGEN FROM CELL --> ENTERS GOLGI WHERE SUGARS ADDED/REMOVED

4) PROCESSED IN EXTRACELLULAR MATRIX --> CLEAVAGE OF N & C-TERM REGIONS AND WHAT REMAINS IS TROPOCOLLAGEN --> IF DENATURED, x3 HELIC WILL NOT REFORM (JOB OF C-TERM)

5) FORMATION OF COLLAGEN FIBER BY SPONT. POLYMERIZATION, N & C-TERM HELPED PREVENT THIS

6) CROSS-LINKING OF FIBERS. LYSYL OXIDASE REQUIRES COPPER AND DOES NOT DIRECTLY MAKE CROSS-LINKS --> CLEAVES a-NH2 GROUP MAKING ALDEHYDE DERIVATIVE OF LYSINE OR HYDROXYLYSINE (VERY REACTIVE (O2))

VARIOUS FORMS OF COLLAGEN MUTATION (a1(I) OR a2(I) COLLAGENS)

ONE LETHAL FORM, MUTATION REPLACES GLYCINE WITH ALANINE --> SMALL CHARGE DISRUPTS x3 HELIX --> PROTEOLYTIC DIGESTION --> INFANTS MINIMAL BONE MINERALIZATION AND DIE SOON AFTER BIRTH

MUTATION IN COLLAGEN SYNTH PATHWAY

HYPER-EXTENSIBILITY

TYPE IV: DEFECT IN a1(III) COLLAGEN

TYPE VI: DEFECT IN LYSYL HYDROXYLASE

NUTRITIONAL DISORDER CAUSED BY LACK OF ASCORBIC ACID (VIT C)

LOSS OF TEETH, BLEEDING, FATALITY

• NEEDED FOR:
• 4-PROLYL HYDROXYLASE
• 3-PROLYL HYDROXYLASE
• LYSYL HYDROXYLASE

SHORT REPEATING SEGS OF SMALL HYDROPHOBIC AAs

HIGHLY CROSS-LINKED USING LYSYL OXIDASE

OILED COIL: UPON STRETCHING, SIDE CHAINS EXPOSED TO AQUEOUS ENV AND DESTABILIZED. WHEN PRESSURE RELEASED, RETURN TO ORIGINAL SHAPE

COLLAGEN STEP 6) LYSYL OXIDASE NEEDS Cu TO REMOVE a-NH2 GROUP AND INDUCE REACTIVITY OF LYSINE/HYDROXYLYSINE

LYSYL OXIDASE USED SIMILARLY IN ELASTIN

ELASTIN MONOMERS POLYMERIZE TO FIBERS MADE OF PROT CALLED FIBRILLIN

MUTATIONS IN FIBRILLIN CAUSE MARFAN

CONNECTING ELASTIN MONOMERS???

PROT THAT HAS CARBS COVALENTLY ATTACHED

CAN BE SINGLE SUGAR OR OLIGOs

SMALL GENERALLY WELL-DEFINED POLYMER OF DIFF SUGARS

ARRANGEMENT USUALLY NON-RANDOM

LINEAR POLYMER OF REPEATING DISACCHARIDE UNITS OFTEN BEGINNING WITH CORE NON-REPEATING STRUCTURE

DIFFERENCE BETWEEN GLYCOSAMINOGLYCANS AND OLIGOs IS THAT GAGs HAVE UNDEFINED AND RELATIVELY LARGE NUMBER OF SUGARS

PROTS WHICH A GAG IS COVALENTLY ATTACHED

N-LINKED: OLIGOs ATTACH TO AMIDE NITROGEN OF ASPARAGINES

O-LINKED: OLIGOs ATTACH TO HYDROXYL OXYGEN OF SERINE, THREONINE, OR HYDROXYLYSINE

NUCLEOTIDES

EXTREMELY HYDROPHOBIC ISOPRENOID

MADE AS A DETOUR FROM THE CHOL SYTH PATHWAY

ANCHORED IN MEM OF ER

SUGARS ATTACHED TO LIPID (DOLICHOL) NOT PROTEIN! ON CYTO SIDE OF ER --> FLIPS TO ER SIDE AND OLIGOs TRANSFERED FROM DOLICHOL TO ASPARAGINE RESIDUE ON PROT

DOLICHOL THEN DE-PHOSED AND FLIPS BACK TO CYTO SIDE FOR ANOTHER ROUND

1) BIOSYNTH OF SUGER COMPONENTS: SUGARS SYNTHED AND ATTACHED TO NUCLEOTIDE.

• UDP - GLU, FAL, GlcNAc, GalNAc
• GDP - Man, Fuc
• CMP - SA

2) SYNTH OF LIPID-LINKED OLIGOs: SUGARS ATTACHED ONE-BY-ONE TO DOLICHOL CORE ON CYTO SIDE OF ER MEM --> FLIPS TO LUMEN-SIDE OF ER

3) TRANSFER OF OLIGOs TO PROT: FROM DOLICHOL TO ASPARAGINE RESIDUE ON PROT

4) PROCESSING OF PROT-BOUND OLIGOs IN GOLGI: MANY SUGARS REMOVED LEAVING OLIGO CORE --> SUGARS ADDED BACK TO FORM EITHER THE COMPLEX OR THE HIGH MANNOSE STRUCTs OR SOME HYBRID OF THEM

IF GOING TO LYSOSOME, 2 MANNOSE-6-PHOSs ADDED WHICH ARE RECOGNIZED BY M-6-P RECEPTOR

GLYCOPROTEINS

SUGARS ADDED TO OLIGO CALLED H ANTIGEN

DOES NOT INVOLVE DOLICHOL

• O: BARE
• A: GalNAc
• B: Gal

1) PROTECT PROTS FROM DEGRADATION

2) HELP TARGET PROTEINS

3) CELL-CELL RECOGNITION

4) MAY HELP DETERMINE CONFORMATION OF PROT

HEMA: BINDS TO SIALIC ACID CAUSING RECEPTOR MEDIATED ENDOCYTOSIS

NEURA: CLEAVES SIALIC ACID TO FREE NEW VIRUSES FROM CELL

DEFICIENCY OF N-ACETYLGLUCOSAMINE-1-PHOSPHOTRANSFERASE WHICH CREATES M6P

LYSOSOME UNABLE TO DEGRADE GAGs WHICH ACCUMULATE IN CELLS AS INCLUSION BODIES

DEFECTS IN DEGRADATION OLIGOs IN LYSOSOME

MENTAL RETARDATION CAUSED BY ACCUMULATION AND EXCRETION OF INCOMPLETELY DIGESTED OLIGOs

HYALURONIC ACID: NOT ATTACHED TO PROTS AND CONSISTS OF REPEATING DISACC MANDE OF GlcNAc AND GLUCURONIC ACID. HUGE MOLECULE FUNCTIONS AS LUBEF AND SHOCK ABSORBER IN JOINTS AN MAJOR PART OF VITREOUS HUMOR OF EYE

CHONDROITIN SULFATE: TENSILE STRENGTH OF CARTILAGE, TENDONS, LIGS, AND WALL OF AORTA

HEPARIN SULFATE: SPONGY QUALITY TO BASEMENT MEMBRANE

KNOW THIS

SUGARS ATTACHED SEQUENTIALLY TO SERINE AND ASP OF PROT CORE

SUGARS ALWAYS BOUND TO UDP PRIOR TO ATTACHMENT

SULFATES ADDED

BOTH GAGs AND OLIGOs METABOLIZED IN LYSOSOMES

HURLER

HUNTER

SANFILIPPO

MORQUIO

MAROTEAUX-LAMY

SLY

DEFECTIVE GLYCOSYLATION OF DYSTROGLYCAN

DG IS A PROT THAT BRIGDES CELL MEM. EXTERNALLY, DG BINDS TO COMPS OF EXTRACELLULAR MATRIX -- INTERNALLY BINDS TO DYSTROPHIN WHICH BINDS TO ACTIN

MUSCLE WEAKNESS AND SEVERE NEUROPATHY

ADHESIVE PROT

DIMER OF 2 CHAINS CONNECTED BY DISULFIDE BRIDGES

BINDING SITES FOR HEPARIN, GANGLIOSIDES, FIBRIN, COLLAGEN AND INTEGRINS.

INTEGRINS IMPLANTED IN CELL MEMs, FIBRONECTIN AND LINK THEM W/ COLLAGEN AND HELP SHAPE CELLS OR GROW IN CERTAIN DIRECTIONS

IMPORTANT IN BLOOD COAGULATION. LACK CAN LEAD TO HEMOPHILIA

TUMOR CELLS PRODUCE LESS FIBRIN --> BREAK FREE (METASTASIZE)

STAPH AND STREP CONTAIN FIBRONECTIN RECEPTORS --> INC INFECTIVITY AND PENETRATE EXT MATRIX

ADHESIVE PROT FOUND IN BASAL LAMINA

FLEXIBLE THIN MATS THAT SURROUND EPI SHEETS & TUBES, MUSCLE FAT & SCHWANN CELLS.

3 CHAINS (a, b, g) ARRANGED TO FORM A CROSS

BIND TO TYPE IV COLLAGEN, ENTACIN, HEPARIN, HEPARAN SULFATE, AND INTEGRINS

STIMS NERVE AXON GROWTH IN EMBRYOS

ENACTIN WITH LAMININ, T-IV-C AND OTHER PROTEOGLYCs FORM BL

ADHESIVE PROT

BLOOD BONE OTHER TISSUES

LIKE FIBRONECTIN TO MEDIATE CELL ADHESION TO EXTRACELLULAR MATRIX AND PROMOTE BLOOD COAG

ADHESION PROT

ENCODED BY 3 DIFF GENES AND SUBJECT TO ALT mRNA SPLICING

PRODUCED BY PLATELETS AND OTHER CELLS

STIM CELL PROLIFERATION IMPORT PART OF WOUND HEALING AND NERVE GROWTH

INHIBIT DEV OF NEW CAPILARIES

PROD BY MACROPHAGES THAT ARE DESTROYING LEUKOCYTES THAT DIED BY APOP -- BIND THE DYING CELL TO MACROs

ADHESION PROT

BINDS CHONDROCYTES TO T-II-C TO PROTEOGLYCs OF CARTILAGE

ADHESION PROT

PROD LARGELY IN EMBRYO TISSUE

BIND TO FIBRONECTIN AND SYNDECAN, A PROTEOGLYC BOUND TO CELL MEM

PROMOTE OR INH CELL ADH

REPELS GROWING NEURONS AND PLAYS ROLE IN REG OF CELL MIGRATION THAT IS IMP IN EMBRYO DEV

MEM PROTS THAT ACT AS RECEPTORS FOR EXTRACELL MATRIX PROTS.

BIND TO ARG-GLY-ASP SEQUENCE IN ADHESIVE PROTS

CONSIST OF 2 POLYPEP CHAINS (a, b) BOTH OF WHICH TRANSVERSE THE CELL MEM

LINK EXTRACELLULAR MATRIX TO CYTOSKEL (LIKE A BRIDGE THROUGH CELL MEM, WITH OUTER PART BINDING TO ADHESIVE PROTS OF EXTRACELL MAT AND INNER PART TO CYTOSKEL PROT TALIN, VINCULIN OR a-ACTININ, WHICH BINDS TO ACTIN

HIGHLY REGULATED -- EXTRACELL MAT SIGNALS CYTOSKEL & VICE VERSA

1) PROMOTE BINDING OF CELL TO EXT MATRIX

2) PLATELETS -- HELP THEM BIND TO FIBRONECTIN --> COAG

3) MITOSIS -- IN CYTO DOMAIN OF FIBRONECT BINDING INTEGRINS IS PHOSED --> LOSE ABILITY TO BIND FIBRONECT. ALLOWS CELL TO TAKE SPHERICAL SHAPE REQUIRED FOR MITOSIS. "SIGNAL" TO OUTSIDE

4) ON LEUKOCYTES -- DONT BIND EXT MATRIX. BIND ENDO CELL RECEPTORS AND ALLOWS LEUKOs TO CROSS THE ENDO LINING OF CAPILLARIES

5) MECHANOCHEMICAL TRANSDUCERS -- ALLOW CHANGES IN EXT MAT STRUCT TO ALTER SHAPE OF CELL. "SIGNAL" FROM OUTSIDE

6) BIND TO LAMININ -- ESP a6b4, STABILIZE SCHWANN CELLS SO THEY GEN MYELIN

RARE MUTATION IN b2-INTEGRIN (CD18) EXPRESSED IN LEUKOCYTES.

TROUBLE EXITING CAPILLARIES AND BINDING BACTERIA

WOUND HEALING

FAILURE TO PRODUCE PUS

MYCOBACTERIUM LEPRAE

BINDS TO LAMININ-2 (a2b1y1) IN NERVOUS SYSTEM, ALLOWING ACCESS

ALSO BINDS INTEGRIN a6b4 ON SCHWANN CELLS --> ENTERS CELL WHICH LOSES MYELIN --> PERIPHERAL NERVES DIE

SUPEROXIDE ANION

HYDROXYL RADICAL

HYDROGEN PEROXIDE

WHEN O2 REDUCED, TOXIC INTERMEDIATES PRODUCED AND REACT/DAMAGE

REACTIONS WITH CARBS LEAST SERIOUS EXCEPT IN UNTREATED DIABETES SINCE THESE HAVE INC GLUC LEVELS --> CATARACTS

MORE SERIOUS ARE LIPIDS, NUC ACIDS, PROTS

• SOD CONVERTS SUPEROXIDE ANION INTO HYDROGEN PEROXIDE:
• O2- + O2- + 2H+ --> H2O2 + O2

3 FORMS: EXTRACELLULAR, CYTOSOLIC Cu-Zn, MITOCHONDRIAL MANGANESE (ONLY PROT KNOWN TO HAVE MANG, PROTECTS MITO)

• CAT IS A HEME-CONTAINING ENZ THAT DESTROYS HYDROGEN PEROXIDE:
• H2O2 + H2O2 --> 2 H2O + O2

ONE OF MOST UNUSUAL ENZ IN BODY, ACTIVE SITE CONTAINS AN ATOM OF SELENIUM IN THE FORM OF THE AA SELENOCYSTEINE

BREAKS DOWN H2O2

ONE ISOFORM PHGPX

CAN ALSO REACT WITH PEROXIDES OF LIPIDS AND OTHER ORGANIC COMPOUNDS

MALE INFERTILITY: GLUTATHIONE PEROXIDASE ISOFORM PHGPX ACTS TOGETHER WITH VIT E TO REVERSE PEROXIDE INDUCED DAMAGE TO MEMBRANES. CONCENTRATED IN TESTES AND SPERM CELLS. PHGPX LOSES ACTIVITY AND POLYMERIZES INTO CAPSULE TO PROTECT MITO (STRUCT INTEGRITY). WITHOUT, SPERM HEAD & TAIL FALL APART.

KESHAN: RURAL CHINA WHERE SOIL IS POOR IN SELENIUM. CARDIAC FAILURE AT EARLY AGE DUE TO H2O2 DAMAGE. ALSO MUST HAVE DIETARY VIT C, E, AND Cu (OR INFECTION) FOR DISEASE. MACROs PRODUCE THESE INTERMEDIATES TO KILL BAC, SO INFECTION WILL EXACERBATE OXIDATIVE STRESS.

IDENTICAL TO SERINE AND CYSTEINE AA EXCEPT WITH SELENIUM ATOM

COTRANSLATIONAL MOD USING SPECIAL tRNA: AA-tRNA SYNTH --> SER-tRNA

SERINE + tRNAsec + ATP --> SER-tRNAsec

THEN SELENOCYSEINE (SEC) SYNTH REPLACES OH IN SER WITH SELENIUM --> SEC-tRNAsec

mRNA FOR SEC HAS SPECIAL INSERTION SEQUENCE WHICH BINDS TO ITS PROTEIN AND CONVERTS UGA STOP CODON TO SEC CODON

TRIPEPTIDE (g-GLU-CYS-GLY)

• DOES NOT USE RIBOSOMES.
• GLUT + CYS + ATP --> g-GLU-CYS

g-GLU-CYS + GLY +ATP --> GLUTATHIONE

ASIDE FROM GLUTATHIONE PEROXIDASE, GLUTA FIXES DAMAGED HEMOGLOBIN BY REDUCTION AND REDUCES DISULFIDE BRIDGES

EACH CELL DAMAGED 10,000 TIMES EVERY DAY BY OX TOX. DAMAGE --> MUTATION --> TUMOR. ALSO, INC H2O2 INCs TRANSCRIPTION OF MATRIX METALLOPROTEINASES WHICH DEGRADE COLLAGEN --> METASTASIS.

• RETROLENTAL FIBROPLASIA:
• PREMATURE INFANTS IN HYPERBARIC OXYGENINC O2 --> RETINAL DAMAGE & BLINDNESS

IIFRD: DEC O2 --> INC XANTHINE OXIDASE --> THEN O2 RESTORED AND SUPEROXIDE ANIONS PRODUCED AND CAUSE DAMAGE. MAY DAMAGE PROTEOSOME WHICH CAUSES INC IN DAMAGED PROTS

PHOTOAGING: UV LIGHT INC FREE RADICALS eg SUPEROXIDES IN SKIN --> SUPER OX DISMUTASE CONVERTS TO H2O2 --> INC MATRIX METALLOPROTINASES DEGRADE COLLAGEN --> WRINKLES

AGE SPOTS: ACCUM OF PRODUCTS OF LIPID PEROXIDATION BY TOX OX

LEUKOs USE TO TARGET PATHOGENIC ORGANISMS

APOPTOSIS USING CYTOCHROME C FROM MITO

THIOREDOXIN USES SELENOCYSTEINE RESIDUE TO ACT AS A SENSOR TO DETECT REACTIVE OX SPECIES AND THEY INC ITS ACTIVITY (REDUCING aka BREAKING DISULFIDE BRIDGES)

HYDROXYLATION OF MANY SUBSTRATES BY REACTIONS CALLED MONOOXYGENATIONS (PUTS 1 ATOM OF O2 INTO SUBSTRATE AND MAKES H2O WITH OTHER)

2e- REQUIRED FOR EACH REATION, ACCEPTED FROM NADH OR NADPH

TYPE OF REACTION DEPENDS ON WHERE e- INSERTED INTO ELEC TRANSPORT CHAIN

MICROSOMAL: IN SMOOTH ER, USES SINGLE NADPH-SPECIFIC P450 REDUCTASE ON MEM CONTAINING FAD AND FMN AS COFACTORS. MAKE COMPONENTS MORE HYDROPHILIC. USED IN DRUG AND XENOBIOTIC, STERIOD, FA, AND EICOSANOID METABOLISM

NADPH --> FAD --> FMN --> P450

MITOCHONDRIAL: INNER MEM OF MITO AND USED FOR STEROID METABOLISM. USES ADRENODOXIN REDUCTASE AND ADRENODOXIN CONTAINING FAD AND IRON-SULFATE PROT RESPECTIVELY TO HAND e- TO P450. MANY ISOFORMS (CYPs)

CYP11A1 CONVERTS CHOL TO PREGNONALONE

SUBSEQUENT REACTIONS MAKE TESTOSTERONE, ALDOSTERONE, ESTRADIOL etc

• TWO PHASES:
• I -- FUNCTIONAL GROUP eg OH ADDED TO DRUG BY CYP450

II -- CONNECT BY FUNC GROUP TO ENDOGENOUS SUBSTRATE eg GLUCURONIC ACID. MAKES DRUG MORE WATER SOLUBLE AND EASIER TO EXCRETE

INACTIVATION: DRUG CONVERTED TO INACTIVE FORM eg VALIUM. CYP3A4 MAJOR PLAYER, LOC IN GI AND LIVER AND IS WHY ORAL DRUGS BROKEN DOWN SO FAST.

ACTIVATION: eg TERFENADINE (SELDANE) ACTIVATED TO FEXOFENADINE (ALLEGRA) BY CYP3A4. TERFEN HAS SEVERE CARDIAC SIDE EFFECTS. GRAPEFRUIT JUICE INHIBITS CYP3A4 AND INC TERF []

LEVELS AND ACTIVITY OF P450 VARY IN PEOPLE AND GREATLY INFLUENCE EFFECT OF DRUGS.

POLY = DIFF IN DNA SEQUENCE IN 1% OR MORE OF POPULATION. ex CYP2D6 DIVIDED AMONG POP INTO EXTENSIVE AND POOR METABOLIZERS. POOR AT RISK FOR ADVERSE DRUG REACTIONS WHILE EXTENSIVE MAY FIND DRUGS INEFFECTIVE.

ex ANTIARRHYTHMICS, ANTIDEPRESSANTS, ANTIPSYCHOTICS, BETA-BLOCKERS, ANALGESICS

UNINTENDED EFFECTS OCCUR WHEN LEVELS OF CYP450 ARE INC/INH BY OTHER DRUGS

ex CYP2E1 IS INDUCED BY ALCOHOL OR ISONIAZID --> METABOLIZE SOME DRUGS INTO PROTS HARMFUL TO LIVER --> IMMUNE SYSTEM ATTACKS CAUSING FORM OF HEP. PEEPS WITH INC CYP231 AT HIGHER RISK.

NO IS HIGHLY REACTIVE FREE RADICAL AND INVOLVED IN NEUROTRANSMISSION, VASODILATION, IMMUNE RESPONSE, PLATELET AGG, ERECTION, SEPTIC SHOCK, HYPOTENSION, VASC LEAK, AUTO IMMUNE -- RA ULCERATIVE COLLITIS DIABETES MS ASTHMA, GvsH DISEASE, LIVER CIRRHOSIS

PRODUCED BY NO SYNTHASES

ACTIVATES: SOLUBLE GUANYLYL CYCLASE

INHIBITS: CYP450 HEMOGLOBIN, ACONITASE, MITO COMPLEXES I,II

S-NITROSATION, OXIDATION

NOx FORMATION

SUPER OXIDE ANION PEROXYNITRITE FORMATION

DNA STRAND CLEAVAGE, DEAMINATION

L-ARG + 2 MONOOXYGENATIONS SIMILAR TO CYTP450 OXIDOREDUCTASE SYSTEM

e- PASSED FROM NADPH TO FAD TO FMN THEN TO HEME WHERE THEY ACTIVATE O2 FOR MONOOXYGENASE REACTION. BETWEEN FMN AND HEME IS CALMODULIN (CaM) BINDING SITE, WHICH MUST BE BOUND WITH CaM FOR e- TRANSFER.

CaM BINDS WHEN INTRACELLULAR Ca INCed ABOVE BASAL.

• ENDOTHELIAL NOS:
• VASCULAR ENDO CELLS --> VASODILATION INH OF VASOCONSTRICTORS, INH OF PLATELET ADHESION AND AGG. INC Ca --> CaM BINDING --> MUSCLE LIGHT CHAIN KINASES (MLCK) --> CONTRACTION. DIALATE BY NO DIFFUSE ACROSS MEM TO SMOOTH MUSCLE CELL --> SOL GUAN CYCLASE --> cGMP INC PKG --> INH Ca INFLUX --> THEN REVERSED BY PDE (esp PDE5)

• NEURONAL:
• CNS (NEUROTRANS), PNS (SPHINC RELAX), SKEL MUSCLE (MOD CONTRAC FORCE). PRODUCED IN POST-SYNAP NEURON AND DIFFUSES BACK. SOL GUAN CYCLASE ACTIVATED BY NO --> cGMP --> GLUT & NOR SYNTH. PNS MYENTERIC, CARIAC, URO NO ACTIVATES SOL GUAN CYC WHICH RELAXES SMOOTH MUSCLE. SKEL RELAXED SIMILARLY.

• INDUCIBLE NOS:
• MACROs, NEUTROPHILS, LIVER. (NANOMOLAR). TRANSCRIPTIONAL VIA CYTOKINE OR ENDOTOXIN INDUCTION. CaM BOUND TO iNOS IN BASAL CONDITIONS SO IS ALWAYS IN ACTIVATED STATE. CYTOTOXICITY BY PEROXYNITRITE FORMATION, DNA CLEAVAGE AND DEAMINATION, NITROSATION OF PROTS, AND INH OF HEME PROTS.

OVERPRODUCTION BY iNOS

ERECTION PROD BY NO-MEDIATED GENERATION OF cGMP --> VASODILATION VIA PKG. cGMP REMAIN ELEVATED

cGMP DESTROYED BY PDE5 --> VIAGRA BINDS COMPETITIVELY TO cGMP THUS BLOCKING PDE5.