biochemistry molecular biology high yield

Biochemistry Molecular Biology Part I Megan Murray M.D., Ph.D Candidate University of Buffalo BC01_1- Nucleic Acid Components • DNA and RNA are assembled from nucleotides: Nitrogenous base Pentose Phosphate • • Pentoses: Ribose → RNA; Deoxyribose → DNA Nitrogenous bases: Purines: rings Pyrimidines: ring Pure As Gold •FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1 • ME 3e: 60.3 • ME 4e: 60.3 CUT the pie BC01_1- BC01_1- Nucleic Acid Components • • • Nucleoside: nitrogenous base + ribose Nucleotide: nucleoside + phosphate group(s) Nucleic acids: polymers of nucleoside monophosphates Structural elements of the most common nucleotides Commons.wikimedia.org Used with permission •FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1 • ME 3e: 60.3 • ME 4e: 60.3 BC01_1- BC01_1- Nucleic Acid Composition • Phosphate group links 3’ carbon of deoxyribose to 5’ carbon of another deoxyribose • Each strand has 5’ and 3’ ends • Eukaryotes DNA is double-stranded RNA is single-stranded • Strands are joined by hydrogen bonds between bases • Bases of adjoining strands are always complementary A : T, C : G (DNA) U : T, C : G (RNA) Total purines = pyrimidines • Strands are anti-parallel 5’ to 3’ •FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1 • ME 3e: 60.3 • ME 4e: 60.3 BC01_1- BC01_1- Chromosome Composition • Nuclear DNA is first packaged in a nucleosome Nucleosome • Negatively charged DNA winds around positively charged histone octamer • Histone octamer: copies of H2A, H2B, H3, and H4 • Histone H1: between adjacent nucleosomes • Difference between 10nm and 30nm chromatin is presence or absence of histone H1 •FA 2013: 64.1 • FA 2012: 68.1 • FA 2011: 66.1 • ME 3e: 60.3 • ME 4e: 60.3 BC01_1- BC01_1- Chromosome Composition Euchromatin • Loosely condensed nucleosomes • Accessible for transcription Heterochromatin • Densely condensed nucleosomes • Inaccessible for transcription •FA 2013: 64.1 • FA 2012: 68.2 • FA 2011: 66.1 • ME 3e: 60.3 • ME 4e: 60.3 BC01_1- BC01_1- DNA Replication Definition • Process by which genetic information is transmitted from cell to cell Process • complementary strands of parental DNA are pulled apart • Each is used as template for synthesis of new strand • Bidirectional and semi-conservative •FA 2013: 68.1 • FA 2012: 72.1 • FA 2011: 70.1 • ME 3e: 63.1 • ME 4e: 63.1 BC01_2- BC01_2- DNA Replication Sequence of events 5’ 3’ Base sequence recognized at origin of replication 3’ 5’ Helicase Origin 5’ 3’ 3’ 5’ 3’ 5’ Leading strand 5’ 1 3’ 3’ Lagging strand 5’ 3’ 3’ 3’ 5’ 5’ Single-stranded DNA binding protein (SSB) binds to each strand Primase synthesizes RNA primer (5’→ 3’) at origin of each parental strand to be complementary and antiparallel to DNA template strands DNA polymerase (III in prokaryotes and α/δ in eukaryotes) begins DNA synthesis Leading strand: continuous and toward replication fork Lagging strand: Okasaki fragments, away from replication fork 5’ 5’ Helicase breaks hydrogen bonds holding base pairs together → unwinding begins Site of quinolones, etoposide action! 3’ RNAse H removes primers, DNA polymerase fills gaps DNA polymerase has capacity to proofread by possessing 3’-exonuclease activity DNA ligase seals nicks between Okasaki fragments Topoisomerase II (DNA gyrase) relieves strain of supercoiling Replication is completed when forks meet •FA 2013: 68.1 • FA 2012: 72.1 • FA 2011: 70.1 • ME 3e: 63.1 • ME 4e: 63.1 BC01_2- BC01_2- Telomerases Telomeres • Repetitive sequences at ends of linear DNA molecules • Replication shortens telomere length, contributing to genetic material loss and cell aging Telomerase • Enzyme which maintains telomere length (embryonic, germ, and stem cells) by replacing telomere sequences on parent strand • Inactive in somatic cells (skin, blood, connective tissue) • Prokaryotes have single circular chromosomes, hence no telomerases • Inappropriately present in cancer cells • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC01_2- BC01_2- DNA Repair 3’ Nucleotide excision repair T A 5’ T A 5’ 3’ UV • Excision endonuclease nicks the phosphodiester backbone of damaged strand 3’ • Removes defective oligonucleotide • DNA polymerase fills gap & DNA ligase seals nick 5’ T=T A 5’ A 3’ Excision endonuclease Xeroderma pigmentosum Xeroderma pigmentosum (XP) 3’ 3’ • Enzyme deficiency leads to XP 5’ T=T 5’ • Autosomal recessive 3’ A A 5’ • Extreme sensitivity to sunlight DNA polymerase Nick 3’ • Skin cancer (melanomas) 5’ T A • Diagnosis: excision endonuclease levels 5’ •FA 2013: 69.1 • FA 2012: 73.1 • FA 2011: 71.1 • ME 3e: 64.2 • ME 4e: 64.2 3’ DNA ligase • Skin freckling 5’ T A 3’ BC01_3- BC01_1- DNA Repair Base excision repair • Example: cytosine deamination (loss of amino group leads to conversion of cytosine into uracil) • Glycosylases and AP endonucleases recognize and remove damaged bases Mismatch repair • System for recognizing and repairing DNA replication errors Main genes involved in mismatch repair are hMLH1 and hMSH2 Hereditary nonpolyposis colorectal cancer (Lynch syndrome) • Mutation in genes encoding for mismatch repair enzymes → cancer • ↑↑ risk for colon, endometrial, ovarian, and gastric cancers • Example of incomplete penetrance •FA 2013: 69.1 • FA 2012: 73.1 • FA 2011: 71.1 • ME 3e: 64.2 • ME 4e: 64.2 BC01_3- BC01_1- DNA Repair Mechanisms Summary Repair Mechanism Nucleotide excision Base excision Mismatch Example Key Enzymes Clinical Correlation Phase Xeroderma pigmentosum G1 UV light-induced thymine dimer formation Excinuclease Cytosine deamination Glycosylase AP endonuclease G1 DNA replication error Enzymes encoded HNPCC (Lynch by hMLH1 and syndrome) hMSH2 genes G2 •FA 2013: 69.1 • FA 2012: 73.1 • FA 2011: 71.1 • ME 3e: 64.2 • ME 4e: 64.2 BC01_3- BC01_1- Overview of Transcription • First stage in “expression” of genetic information • Information from double-stranded DNA molecule transcribed into base sequence of single-stranded RNA molecule • Only one strand of DNA molecule (template strand) is read and the other strand (coding strand) is not • Main enzyme is RNA polymerase (reads in the 3’ → 5’ direction, synthesizes RNA molecule in 5’ → 3’ direction) • End product is complementary, antiparallel, single-stranded RNA molecule •FA 2013: 70.3 • FA 2012: 75.1 • FA 2011: 72.3 • ME 3e: 65 • ME 4e: 65 BC01_4- BC01_4- Types of RNA Ribosomal RNA (rRNA) (most abundant type) • Structural component of ribosomes • Most rRNA made in nucleolus Transfer RNA (tRNA) (smallest type) • Carries amino acids to ribosome • Most tRNA made in euchromatin portion of nucleus Messenger RNA (mRNA) (most heterogeneous type) • Carries information specifying amino acid sequence of a protein to ribosomes • Only type that is “translated” into proteins • Different mRNA molecule for each of thousands of proteins • Most mRNA made by euchromatin portion of nucleus Some RNAs of each of these classes are made from mitochondrial DNA, not nuclear DNA •FA 2013: 70.1 • FA 2012: 74.2 • FA 2011: 72.1 • ME 3e: 65 • ME 4e: 65 BC01_4- BC01_4- RNA Polymerases • Prokaryotes: one RNA polymerase Protein factor σ required for transcription initiation Protein factor ῥ required for transcription termination • Eukaryotes: RNA polymerases Actinomycin D inhibits transcription in eukaryotes and prokaryotes Transcription factors help initiate transcription Prokaryotes Eukaryotes RNA polymerase 1: rRNA (nucleolus) Single RNA polymerase RNA polymerase 2: mRNA (nucleoplasm) RNA polymerase 3: tRNA (nucleoplasm) Requires σ to initiate promoter No σ, but Transcription Factors binds before RNA polymerase Sometimes requires ῥ to terminate No ῥ required Inhibited by rifampin RNA polymerase inhibited by α-amanitin •FA 2013: 70.5 • FA 2012: 75.3 • FA 2011: 72.5 • ME 3e: 65 • ME 4e: 65 BC01_4- BC01_4- Transcription Terminology • Promoter region: binding site for RNA polymerase • No primer required • RNA polymerase reads template strand of gene and moves 3’ → 5’ direction • RNA polymerase synthesizes 5’ → 3’ direction • No proofreading • mRNA sequence identical to DNA coding strand (uracil instead of thymine) • First nucleotide transcribed defined as +1 nucleotide • Transcription ends when RNA polymerase reaches termination signal •FA 2013: 69.2 • FA 2012: 74.1 • FA 2011: 71.2 • ME 3e: 65 • ME 4e: 65 BC01_4- BC01_4- Flow of Genetic Information •FA 2013: 69.2 • FA 2012: 74.1 • FA 2011: 71.2 • ME 3e: 65 • ME 4e: 65 BC01_4- BC01_4- Transcription of Eukaryotic mRNA • Genes are composed of exons and introns • Both are transcribed but introns removed during RNA processing • Mature mRNA molecules translated into proteins by ribosomes in the cytoplasm • RNA polymerase II: Recognizes and binds to promoter region (TATA and CAAT box) with help from transcription factors Separates DNA strands to initiate transcription Ends transcription when reaches termination signal The pre-mRNA (primary transcript) undergoes co- and postranscriptional processing •FA 2013: 69.2 • FA 2012: 74.1 • FA 2011: 71.2 • ME 3e: 65 • ME 4e: 65 BC01_4- BC01_4- Co- and Postranscriptional Processing • 7-methylguanosine cap added to 5’ end • Poly-A tail attached to 3’ end • Introns removed by splicing • Excised introns (lariat) degraded in nucleus • Exons joined together • Mutations of 5’ and 3’ splice sites of introns affect accuracy of intron removal • Mutations may lead to introns being left within mature mRNA SLE patients make antibodies against spliceosomal snRNPs •FA 2013: 71.1 • FA 2012: 75.4 • FA 2011: 72.6 • ME 3e: 65 • ME 4e: 65 BC01_4- BC01_4- Alternative Splicing • Production of or more proteins variants from the same gene • Examples: Membrane-bound vs secreted Ig Tropomyosin vs troponin T •FA 2013: 71.3 • FA 2012: 77.1 • FA 2011: 73.2 • ME 3e: 65 • ME 4e: 65 BC01_4- BC01_4- Ribosomes • Ribosomes are complexes of rRNA and proteins • Prokaryotic ribosome: 50S (large) + 30S (small) subunits Combined subunit = 70S • Eukaryotic ribosome: 60S (large) + 40S (small) subunits Combined subunit = 80S • FA 2012: 78.1 • FA 2011: 75.1 • FA 2010: 75.1 • ME 3e: 66.1 • ME 4e: 66.1 BC01_4- BC01_4- tRNA • tRNA carries activated (high energy) amino acid for translation • Each tRNA carries one activated amino acid determined by its anticodon sequence • Amino acid activation catalyzed by aminoacyl-tRNA synthetase Requires high energy bonds from ATP Transfers activated amino acid onto 3’ end of correct tRNA • tRNA has anticodon sequence that allows pairing with mRNA codon • FA 2012: 77.2 • FA 2011: 74.1 • FA 2010: 74.1 • ME 3e: 66.1 • ME 4e: 66.1 BC01_4- 10 BC01_4- Genetic Code Definition • Relationship between sequence of nucleotides in DNA and sequence of amino acids in proteins Characteristics • 64 codons: 61 for AA U Go Away U Are Away U Are Gone stop codons (UGA, UAA, UAG) “AUG” always first codon (methionine) Genetic code • Unambiguous • Degenerate (redundant) • Universal • FA 2012: 70.4 • FA 2011: 72.2 • FA 2010: 72.2 • ME 3e: 66.1 • ME 4e: 66.1 BC01_4- 11 BC01_4- Mutations Definition • Permanent heritable change in DNA base sequence • May be cause of genetic disease, change in enzyme activity, and other properties of cell function Normal Silent mutation Missense mutation Nonsense mutation Frameshift mutation ATG GCA ATT TTA CCT ATA DNA Coding Strand MET ALA ILE LEU PRO ILE Amino Acid ATG GCA ATT TTG CCT ATA MET ALA ILE LEU PRO ILE ATG GCA ATT TCA CCT ATA MET ALA ILE SER PRO ILE ATG GCA ATT TGA CCT ATA MET ALA ILE STOP ATG GCA ATT TAC CTA TAG MET ALA ILE TYR LEU STOP • FA 2012: 71.1 • FA 2011: 69.3 • FA 2010: 69.3 • ME 3e: 64.1 • ME 4e: 64.1 BC01_4- 12 BC01_4- Translation Definition • Decoding of mRNA by ribosome to produce amino acid chain • Location: cytoplasm General characteristics • Separated in time and space from transcription (eukaryotes) • Three stages: Initiation Elongation Termination • Each step requires special protein factors (initiation factor, elongation factor and termination factor or release factor) and energy (GTP) •FA 2013: 73.1 • FA 2012: 78.1 • FA 2011: 75.1 • ME 3e: 66.3 • ME 4e: 66.3 BC01_5- BC01_1- Translation: Initiation Phase • Small ribosomal subunit binds to mRNA 30S small subunit binds to Shine-Dalgarno sequence (prokaryotes) 40S small subunit binds to 5’ cap structure (eukaryotes) • Charged initiator tRNA binds to start codon • Large subunit binds to small subunit forming initiation complex • Ribosomal binding sites Peptidyl site (P site) Aminoacyl site (A site) INITIATION Small ribosomal subunit 5’ cap (Eu) met or ShineDalgarno (Pr) •FA 2013: 73.1 • FA 2012: 78.1 • FA 2011: 75.1 • ME 3e: 66.3 • ME 4e: 66.3 met-tRNA (Eu); or fmet-tRNA (Pr) Large subunit BC01_5- BC01_1- Translation: Elongation Phase • Three-step cycle repeated for each amino acid added to protein after initiator Met • Four high energy bonds used per cycle: Two ATPs for charging tRNA and peptide bond formation Two GTPs for 1st and 3rd steps of the cyclic sequence ELONGATION • Aminoacyl-tRNA binds to A site • Shiga toxins inhibits • (cuts 28S rRNA) • Peptidyl transferase forms peptide bond in large subunit •FA 2013: 73.1 • FA 2012: 78.1 • FA 2011: 75.1 • ME 3e: 66.3 • ME 4e: 66.3 • Translocation of the ribosome nucleotides along the mRNA • Pseudomonas and diphteria toxin inhibit (ADP-ribosylation) eEF-2 BC01_5- BC01_1- Translation: Termination Phase • Occurs when any of the stop codons of RNA moves into A site • Peptidyl transferase + release factor hydrolyzes completed protein from final tRNA in P site • Upon completion, mRNA ribosome, tRNA, assisting factors are reused for further protein synthesis • Multiple ribosomes can bind to single mRNA (polysomes) TERMINATION Stop codon in A site Completed protein released from ribosome Ribosomal subunits separate mRNA Released •FA 2013: 73.1 • FA 2012: 78.1 • FA 2011: 75.1 • ME 3e: 66.3 • ME 4e: 66.3 BC01_5- BC01_1- Antibiotics Aminoglycosides • Gentamicin, neomycin, and tobramycin • Bind to 30S subunit • Inhibit formation of initiation complex Macrolides • Erythromycin, azithromycin • Bind to 50S subunit • Inhibit translocation of ribosome during elongation phase Clindamycin • Binds to 50S subunit • Inhibits translocation of ribosome during elongation phase Chloramphenicol • Inhibits peptidyl transferase → inhibition of protein chain elongation •FA 2013: 73.1 • FA 2012: 78.1 • FA 2011: 75.1 • ME 3e: 66.3 • ME 4e: 66.3 BC01_5- BC01_1- Protein Structure Primary • Sequence of amino acids Secondary • Amino acid chain folded into energetically stable structure (αhelix, β-sheet) Tertiary • Positioning of secondary structures in relation to each other to generate 3-dimensional shape Quaternary • Interaction among multiple subunits in proteins (e.g Hemoglobin) Commons.wikimedia.org Used with permission •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 67.1 • ME 4e: 67.1 BC01_7- BC01_1- Chaperones, Ubiquitin and Proteasomes Chaperones • Specialized proteins • Assist in protein folding Ubiquitin • Peptide which when attached to misfolded proteins targets them for proteolysis Proteasomes • Large cytoplasmic complexes containing multiple proteases for destruction of misfolded proteins • Microbial peptide fragments can be presented in antigen presenting cells •FA 2013: 73.2 • FA 2012: 78.2 • FA 2011: 75.2 • ME 3e: 67.1 • ME 4e: 67.1 BC01_7- BC01_1- Rough ER versus Free Ribosomes Proteins translated on RER • Secreted proteins • Membrane-bound proteins Peroxisome Mitochondrion Lysosome • Lysosomal enzymes Golgi apparatus Proteins translated by free ribosomes Nucleus • Cytoplasmic proteins • Mitochondrial proteins •FA 2013: 74.2 • FA 2012: 79.2 • FA 2011: 76.2 • ME 3e: 67.3 • ME 4e: 67.3 Rough endoplasmic reticulum (RER) RER ribosome RER Free ribosome Smooth endoplasmic reticulum (SER) Microtubule & filament Golgi apparatus BC01_7- BC01_1- Co- and Post-translational Modification of Proteins Definition • Chemical alteration of proteins that will vary depending on ultimate function of resulting product Possible modifications • Disulfide bond formation • N-Glycosylation and/or O-glycosylation • Proteolysis • Phosphorylation • Gamma carboxylation • Prenylation N-terminal hydrophobic signal sequence • Required on proteins destined to be: Secreted outside of cell (e.g., insulin) Embedded in a cell membrane (e.g., Na+/K+-ATPase) Directed to lysosome (e.g., sphingomyelinase) •FA 2013: 73.2 • FA 2012: 78.2 • FA 2011: 75.2 • ME 3e: 67.3 • ME 4e: 67.3 BC01_7- BC01_1- Golgi Apparatus Definition • Organelle that functions as modification and distribution center for proteins and lipids from ER Functions • Modification of N-oligosaccharides • Addition of O-oligosaccharides • Phosphorylation of mannose in lysosomal enzymes Clinical correlates • I-cell disease • Cystic fibrosis •FA 2013: 75.1 • FA 2012: 80.1 • FA 2011: 77.1 • ME 3e: 67.3 • ME 4e: 67.3 BC01_7- BC01_1- Post-translational Modification of Collagen Primary structure composed of tripeptide Gly-X-Y Synthesis of pre pro-α chain with N-terminal hydrophobic signal sequence Rough endoplasmic reticulum Hydroxylation of selected prolines and lysines using vitamin C Removal of signal sequence by signal peptidase Glycosylation of selected hydroxylysines Cytoplasm Triple helix formation (procollagen) Secretion from cell (pro-collagen secreted) Cleavage of pro-peptides Collagen (Tropocollagen) Aggregation to form a collagen fiber •FA 2013: 78.1 • FA 2012: 83.1 • FA 2011: 79.3 • ME 3e: 68.2 • ME 4e: 68.2 Assembly into fibrils Stabilized by lysyl oxidase (Requires O2 and Cu+) BC01_8- BC01_1- Types of Collagen Collagen Type I Characteristics Bundles of fibers High tensile strength II Thin fibrils Structural III Thin fibrils Pliable Tissue Distribution Bone, skin, tendons Cartilage Vitreous humor Blood vessels Granulation tissue Associated Diseases Osteogenesis imperfecta Ehlers-Danlos (various) - - Ehlers-Danlos type IV Keloid formation Goodpasture syndrome IV Amorphous Basement membranes •FA 2013: 77.2 • FA 2012: 82.3 • FA 2011: 79.2 • ME 3e: 68.2 • ME 4e: 68.2 Alport disease Epidermolysis bullosa BC01_8- BC01_1- Disorders of Collagen Disease Scurvy Osteogenesis Imperfecta Ehlers-Danlos Syndrome Menkes Disease Defect Major Symptoms Deficient hydroxylation secondary to ascorbate deficiency Petechiae, ecchymoses, loose teeth, bleeding gums, poor wound healing, poor bone development Mutations in collagen genes Skeletal deformities, fractures, blue sclera Mutagen in collagen genes and lysyl hydroxylase gene Hyperextensible fragile skin, hypermobile joints, dislocations, varicose veins, ecchymoses, arterial and intestinal ruptures Deficient cross-linking secondary to copper deficiency Depigmented (steely) hair, arterial tortuosity and rupture, cerebral degeneration, osteoporosis, anemia •FA 2013: 78.3 • FA 2012: 83.3 • FA 2011: 80.1 • ME 3e: 68.2 • ME 4e: 68.2 BC01_8- BC01_1- Peroxisome General characteristics • Membrane enclosed • Spherical organelle High Yield functions • Synthesis and degradation of H2O2 (contains catalase) • Catabolism of very long chain fatty acids • Bile acid synthesis •FA 2013: 75.2 • FA 2012: 80.2 • FA 2011: NA • ME 3e: 79.3 • ME 4e: 79.3 BC01_9- BC01_1- Smooth Endoplasmic Reticulum High yield functions • Detoxification of drugs and poisons • Steroid synthesis • Sequestration and release of Ca++ (sarcoplasmic reticulum) •FA 2013: 74.3 • FA 2012: 79.3 • FA 2011: 76.3 • ME 3e: 79.1 • ME 4e: 79.1 BC01_9- BC01_1- Microtubules • Hollow tubes composed of polymerized dimers of α and β tubulin • Tracks for intracellular transport • Specific ATPase motor molecules Dynein: retrograde transport Kinesin: anterograde transport Location • True cilia and flagella • Mitotic spindle (during mitosis/meiosis) Drugs acting on microtubules • Mebendazole/ thiabendazole • Griseofulvin • Vincristine/vinblastine, paclitaxel • Colchicine •FA 2013: 76.1 • FA 2012: 81.2 • FA 2011: 78.1 • ME 3e: 81.1 • ME 4e: 81.1 BC01_9- BC01_1- Chédiak–Higashi Syndrome • Autosomal recessive • Microtubule polymerization defect → impaired fusion of phagosomes and lysosomes Symptoms • Neutropenia • Recurrent pyogenic infections • Partial albinism • Peripheral neuropathy CreativeCommons Used with permission •FA 2013: 76.1 • FA 2012: 81.2 • FA 2011: 78.1 • ME 3e: 81.1 • ME 4e: 81.1 BC01_9- BC01_1- Cilia Structure • Nine peripheral pairs of microtubules • Two central microtubules Function • Mucociliary clearance • Motility of sperm cells Commons.wikimedia.org Used with permission Kartagener syndrome • Absence of dynein • Immotile spermatozoa → infertility • Chronic respiratory infections •FA 2013: 76.2 • FA 2012: 81.3 • FA 2011: 78.2 • ME 3e: 81.1 • ME 4e: 81.1 BC01_9- BC01_1- Cytoskeletal Structures Actin and myosin • Components of muscle sarcomeres, microvilli, adherence junctions • Role in cytokinesis Microtubules Intermediate filaments • Type I: keratins (all epithelial cells) • Type II: Desmin (skeletal, cardiac, and GI tract smooth muscle cells) Vimentin (fibroblasts, fibrocytes, endothelial cells, vascular smooth muscle) Glial fibrillary acidic protein (astrocytes, Schwann cells) Peripherin (peripheral nerve axons) • Type III: neurofilaments (neurons) • Type IV: lamins (form meshwork inside nuclear envelopes of all cells) •FA 2013: 76.3 • FA 2012: 81.4 • FA 2011: 78.3 • ME 3e: 81.1 • ME 4e: 81.1 BC01_9- BC01_1- Biochemistry Molecular Biology Part II Megan Murray M.D., Ph.D Candidate University of Buffalo Chromatin Remodeling • DNA packaged in chromatin structures • Gene expression requires chromatin remodeling Transcription factor (proteins) bind to specific DNA sequences and recruit other coactivators Histone acetylase, acetylates lysyl residues on histones to neutralize their positive charges, weakening interactions between histones and DNA Remodeled chromatin has more exposed DNA for additional transcription factors and other proteins to bind and initiate transcription •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC02_1- Enhancers Characteristics • DNA sequences that to which specific transcription factor proteins bind • May be located upstream, downstream, or within an intron of the gene • Can act in a tissue-specific manner • Multiple DNA sequences called response elements may be grouped within the enhancer region •FA 2013: 70.3 • FA 2012: 75.1 • FA 2011: 72.3 • ME 3e: NA • ME 4e: NA BC02_1- Transcription Factors Definition • Activator proteins that bind to response elements within enhancer regions Domains: within a transcription factor • DNA binding domain Zinc fingers ( e.g steroid hormone receptors) Leucine zippers (e.g., cAMP dependent transcription factors) Helix-turn-helix (e.g., embryonic gene expression transcription factors) • Activation domain, which allows Binding to other transcription factors Interaction with RNA polymerase Recruitment of chromatin modifying proteins •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 65 • ME 4e: 65 BC02_1- Stimulation of Transcription Types of Transcription Factors • General: bind to DNA sequences within the promoter, allowing RNA polymerase to form initiation complex Found in most genes within most cells Involved in basal transcription of genes • Specific: localized to specific cells and tissues Bind to enhancer/silencer regions, modulating formation of initiation complex DNA binding domain Specific transcription factor (example) Activation domain General transcription factor (example) •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 65 • ME 4e: 65 BC02_1- Key Specific Transcription Factors Transcription Factor (DNA-binding protein) Response Element (Binding site) Function Protein class Steroid receptors HRE Steroid response Zinc finger cAMP response element binding (CREB) protein CRE Response to cAMP Leucine zipper Peroxisome proliferatoractivated receptors (PPARs) PPREs Regulate multiple aspects of lipid metabolism Zinc finger NFkB (nuclear factor kappa-B) kB elements Regulates expression of many genes in immune system Rel domains Homeodomains proteins - Regulate gene expression during development Helix-turn-helix • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC02_1- Recombinant DNA Definition • Splicing together of DNA from sources (usually a human gene and a bacterial vector) • The recombinant DNA can replicate and possibly be expressed into protein autonomously in microorganisms Uses • Provides a means of analyzing and altering genes and proteins • Provides reagents for genetic testing • Provides a source of specific proteins (e.g., insulin) Commons.wikimedia.org Used with permission •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 69 • ME 4e: 69 BC02_2- Recombinant Genomic DNA Major points • Restriction endonucleases cut DNA at palindrome sequences → restriction fragments (usually containing a gene of interest) • Restriction fragments are cloned in vectors • Cloned fragments are re-isolated from cloned recombinant vectors and sequenced Genomic libraries used to identify • Protein-coding genes • Restriction endonuclease sites • Genetic markers (short tandem repeats, single nucleotide polymorphisms) • Non-expressed DNA (enhancers, promoters, introns) •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 70 • ME 4e: 70 BC02_2- Recombinant cDNA Characteristics • cDNA (expression library) is made when the goal of cloning is to have a complete cloned gene expressed in the cell Entire coding sequence must be cloned intact Sequence of events • Cytoplasmic mRNA is isolated • Reverse transcriptase produces ds cDNA • cDNA recombined with vector to produce recombinant DNA for cloning Results • All genes expressed in a given cell will be cloned along with desired gene • None of the nonexpressed will be cloned • Each cDNA represents complete coding sequence • cDNA has no introns, as cytoplasmic DNA was the source • Expression library produced at the end of procedure •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 70 • ME 4e: 70 BC02_2- DNA Libraries Genomic Libraries cDNA (expression) Library Source of DNA Chromosomal DNA mRNA (cDNA) Enzymes to make library Restriction endonuclease DNA ligase Reverse transcriptase DNA ligase Contains nonexpressed sequences of chromosomes Yes No Cloned genes are complete sequences Not necessarily Yes Cloned genes contained introns Yes No Promoter and enhancer sequences present Yes, but not necessarily in same clone No Gene can be expressed in cloning host (recombinant proteins) No Yes Can be used for gene therapy or constructing transgenic animals No Yes •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 70 • ME 4e: 70 BC02_3- Applications of Gene Cloning Recombinant proteins • Human insulin • Hepatitis B surface antigen (vaccination) • Clotting factor VIII Gene therapy • Promising treatment for: Cystic fibrosis Thalassemia Severe combined immunodeficiency Transgenic mice Commons.wikimedia.org Used with permission • Knock-in mice: introducing a disease-producing allele to study the process from embryonic development through adulthood • Knock-out mice: deletion of gene to study effect of gene absence •FA 2013: 81.2 • FA 2012: 86.1 • FA 2011: 82.2 • ME 3e: 70 • ME 4e: 70 BC02_3- Blotting Techniques Purpose Detection and visualization of specific DNA, RNA and proteins among complex mixtures of contaminating molecules General method •FA 2013: 80.1 • FA 2012: 85.1 • FA 2011: 81.2 • ME 3e: 71.1 • ME 4e: 71.1 BC02_4- Types of Blots Blot type Southern Northern Material analyzed DNA RNA Electrophoresis Probe used required Yes Yes 32P- 32P- Purpose DNA Determines which restriction fragments of DNA are associated with a particular gene DNA Measures size and amounts of specific mRNA molecules to answer questions about gene expression 125I- Western Protein Dot (slot) RNA, DNA, or protein Yes or enzyme linked antibody Measures amount of antigen (proteins) or antibody No Same as blots above Detects specific DNA, RNA, protein, or antibody •FA 2013: 80.1 • FA 2012: 85.1 • FA 2011: 81.2 • ME 3e: 71.1 • ME 4e: 71.1 BC02_4- Polymerase Chain Reaction Definition • Amplification of selected DNA region of chromosome Uses • Comparison of DNA samples in forensic cases • Paternity testing • Direct mutation testing • Diagnosis of bacterial and viral infections • HIV testing •FA 2013: 79.3 • FA 2012: 84.3 • FA 2011: 81.1 • ME 3e: 69.2 • ME 4e: 69.2 BC02_4- Polymerase Chain Reaction Sequence Region of DNA to be amplified Ingredients • DNA sample • Primers • Heat-stable DNA polymerase • Deoxyribonucleotides Denaturation: heat DNA →ssDNA Strand Strand Cycle Strand Strand Strand Annealing: cool sample to allow primers to anneal Strand Cycle Strand Strand Strand Strand Extension/elongation: heat to temp required for DNA polymerase → DNA fragment amplification Cycle Strand Strand Cycles to 20 Repeat ~20x Present in about 106 copies •FA 2013: 79.3 • FA 2012: 84.3 • FA 2011: 81.1 • ME 3e: 69.2 • ME 4e: 69.2 BC02_4- Enzyme-Linked Immunosorbent Assay (ELISA) Definition • Immunologic technique which tests for antigen–antibody reactivity Technique • Patients’ serum is probed with either test antibody or test antigen • Antibody/antigen coupled to colorgenerating enzyme • If antigen/antibody present → color reaction seen Uses Commons.wikimedia.org Used with permission • HIV, West Nile antibodies, E coli and rotavirus in feces, hepatitis B, and food allergens •FA 2013: 80.3 • FA 2012: 85.3 • FA 2011: 81.4 • ME 3e: 71.4 • ME 4e: 71.4 BC02_4- Biochemistry Medical Genetics Megan Murray M.D., Ph.D Candidate University of Buffalo Definitions Gametes • • Ova and sperm cells Haploid: one copy of each type of chromosomes (1-22 and either X or Y) Somatic cells • • • All other cells Diploid: two copies of each type of chromosomes (1-22 and either XX or XY) Two chromosomes in each pair are homologous Contain same genes May have different alleles at some loci Allele • Different form of a particular gene Gene • Sequence of DNA encoding specific proteins or non-translated RNA Locus • Physical location of a gene on a chromosome •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC03_1- Definitions Polymorphism • DNA sequence variations of a gene occupying a specific site on chromosome Genotype • Specific DNA sequence at locus • Homozygous: same allele of both homologs at particular locus • Heterozygous: two different homologs at particular locus Phenotype • Expression of a genotype in terms of observable characteristics Mutations • Loss of function: missing protein product or decreased activity • Gain of function: producing protein with new function or increased activity Recurrence risk • Probability that offspring of a couple will express genetic disease •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC03_1- Pedigree Definition • Diagram plotting the genetic history of a disease trait within a family • First individual identified with a genetic disease within a pedigree known as a proband •FA 2013: 84.1 • FA 2012: 89.1 • FA 2011: 85.1 • ME 3e: 71.1 • ME 4e: 72.1 BC03_1- Autosomal Dominant Inheritance Characteristics • Disease observed in every generation • Skipped generations are not seen • Males and females affected equally • Affected genes often structural genes • Typical mating pattern is heterozygous affected individual with homozygous normal individual Recurrence risk • 50% (heterozygous affected with homozygous normal) • 75% (both heterozygous affected) •FA 2013: 84.1 • FA 2012: 89.1 • FA 2011: 85.1 • ME 3e: 71.1 • ME 4e: 71.1 • • • • Familial hypercholesterolemia Huntington’s disease MEN syndromes Hereditary spherocytosis BC03_2- Autosomal Recessive Inheritance Characteristics • Clinically expressed only when both homozygous alleles are defective • Offspring must inherit one copy of disease causing allele from each parent • Typically skips generations, but disease seen in multiple people in one generation of pedigree • Males and females equally affected • Typical mating pattern: heterozygous parents (carriers) Recurrence risk • 25% (2 heterozygous parents) •FA 2013: 84.1 • FA 2012: 89.1 • FA 2011: 85.1 • ME 3e: 71.1 • ME 4e: 71.1 • • • • • Glycogen storage diseases PKU Thalassemias Sickle cell anemia Cystic fibrosis BC03_2- X-Linked Recessive Inheritance Characteristics • Males > females • Males are hemizygous for X chromosome • Skipped generations commonly seen • Male-to-male transmission never seen •FA 2013: 84.1 • FA 2012: 89.1 • FA 2011: 85.1 • ME 3e: 71.1 • ME 4e: 71.1 • • • • G6PD deficiency Duchenne muscular dystrophy Lesch-Nyhan syndrome Hemophilia A and B BC03_2- Recurrence Risk in X-Linked Recessive Inheritance Recurrence risk • Carrier status of parents • Sex of offspring Affected male-homozygous normal female (X chromosome with mutation in lower case) x Y X Xx XY X Xx XY Possible patterns • Affected male with homozygous normal female All daughters heterozygous carriers All sons homozygous normal • Normal male-carrier female Normal male with carrier female Half of sons will be affected Half of daughters will be carriers X Y If fetal sex known, recurrence rate in daughter = 0; in son = 50% X XX XY If fetal sex not known, recurrence rate 25% x Xx xY •FA 2013: 84.1 • FA 2012: 89.1 • FA 2011: 85.1 • ME 3e: 72.2 • ME 4e: 72.2 BC03_2- X-Linked Dominant Inheritance Characteristics • • • • • Relatively rare • Male-to-male transmission never seen • Heterozygous females are affected • Seen twice as often in females • Disease phenotype seen in multiple generations • Skipped generations unusual •FA 2013: 84.1 • FA 2012: 89.1 • FA 2011: 85.1 • ME 3e: 72.2 • ME 4e: 72.2 Fragile X syndrome Rett syndrome Hypophosphatemic ricketts Charcot-Marie-Tooth syndrome BC03_2- Recurrence Risk in X-Linked Dominant Inheritance Affected male with homozygous normal female • None of sons affected • All daughters affected Normal male with heterozygous affected female • 50% sons and 50% of daughters affected Affected male-homozygous normal female (the mutation-carrying chromosome is upper case) X Y x Xx xY x Xx xY •FA 2013: 84.1 • FA 2012: 89.1 • FA 2011: 85.1 • ME 3e: 72.2 • ME 4e: 72.2 Normal male-heterozygous affected female x Y X Xx XY x xx xY BC03_2- Mitochondrial Inheritance Characteristics • Mitochondria have own DNA • DNA encodes for 22 tRNAs, RNAs, and 13 sub-units of complexes in ETC • Mitochondria inherited exclusively through females • Disease transmitted only from affected females • Both males and females affected • No offspring of affected male affected • Diseases are typically neuropathies and/or myopathies •FA 2013: 84.1 • FA 2012: 89.1 • FA 2011: 85.1 • ME 3e: 72.2 • ME 4e: 72.2 BC03_2- Algorithm for Determining Mode of Inheritance in Pedigree •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 72.3 • ME 4e: 72.3 BC03_2- Factors Influencing Phenotypic Expression in Single Gene Diseases Environmental factors • Degree of exposure to UV light Xeroderma pigmentosum XP Commons.wikimedia.org Used with permission Allelic heterogeneity • Nonsense vs missence mutation in factor VIII gene Hemophilia A Bleeding Commons.wikimedia.org Used with permission •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC03_3- Factors Influencing Phenotypic Expression in Single Gene Diseases Incomplete penetrance • Individuals with disease genotype but not display disease phenotype Retinoblastoma Retinoblastoma Commons.wikimedia.org Used with permission Variable expression • Nature and severity of disease vary from patient to patient, but there is some pathology seen Phlebotomy/ hemochromatosis Commons.wikimedia.org Used with permission •FA 2013: 82.1 • FA 2012: 87.1 • FA 2011: 83.1 • ME 3e: 73 • ME 4e: 73 Hemochromatosis BC03_3- Factors Influencing Phenotypic Expression in Single Gene Diseases Pleiotropy • Single disease causing mutation affects multiple organ systems Marfan syndrome Cystic fibrosis Hypermobile joints/Marfan syndrome Commons.wikimedia.org Used with permission Locus heterogeneity • Same disease phenotype can be caused by mutations in different loci Osteogenesis imperfecta Osteogenesis imperfecta Commons.wikimedia.org Used with permission •FA 2013: 82.1 • FA 2012: 87.1 • FA 2011: 83.1 • ME 3e: 73 • ME 4e: 73 BC03_3- Factors Influencing Phenotypic Expression in Single Gene Diseases New mutation • Disease caused by new mutation transmitted from unaffected parent to affected offspring • Pedigree shows no family history • Recurrence risk for siblings of affected individual remains low • Recurrence for offspring of affected individual will be same as of any individual who inherited the disease causing mutation •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC03_3- Factors Influencing Phenotypic Expression in Single Gene Diseases Delayed age of onset • Mutation manifests as disease later in life Dr George Huntington-1872 Anticipation • Affected individuals in the most recent generation of a pedigree developed disease at earlier age or with greater severity than in earlier generations •FA 2013: 82.1 • FA 2012: 87.1 • FA 2011: 83.1 • ME 3e: 73 • ME 4e: 73 BC03_3- Factors Influencing Phenotypic Expression in Single Gene Diseases Imprinting • Small number of genes are transcriptionally active only when transmitted by one of sexes • Homologous locus in other parent is transcriptionally inactive • Some loci only have the maternal or paternal loci active • Occurs in specific loci on several chromosomes • Occurs during gametogenesis • Maintained in all somatic cells of offspring • Erased and re-established according to individuals sex during gametogenesis • Imprinting involves cytosine methylation of an allele to imprint or inactivate the appropriate loci •FA 2013: 82.1 • FA 2012: 87.1 • FA 2011: 83.1 • ME 3e: 73 • ME 4e: 73 BC03_3- Factors Influencing Phenotypic Expression in Single Gene Diseases 15q • Transcriptionally active gene may be deleted from chromosome during gametogenesis → offspring has no active gene at locus Normally imprinted in paternal 15 Prader–Willi syndrome • Loss from paternal chromosome Angelman syndrome 15q 15q Normally imprinted in paternal 15 •FA 2013: 83.2 • FA 2012: 88.2 • FA 2011: 84.2 • ME 3e: 73 • ME 4e: 73 Deletion in paternal chromosome 15 Prader-Willi Normally imprinted in maternal 15 Normally imprinted in paternal 15 • Loss from maternal chromosome Normally imprinted in maternal 15 Normally imprinted in maternal 15 Deletion in maternal chromosome 15 Angelman BC03_3- Prader-Willi Syndrome General characteristics • Deletion of an allele in the paternal chromosome at an imprinted locus mapping to 15q11-13 • Affects both males and females Signs and symptoms • Neonatal hypotonia • Hypogonadism • Mental retardation • Poor feeding in neonatal period • Hyperphagia and obesity by 2-4 years • Small hands and feet •FA 2013: 83.2 • FA 2012: 88.2 • FA 2011: 84.2 • ME 3e: 77.1 • ME 4e: 77.1 Prader-Willi syndrome phenotype at age15 Note presence of typical PWS facial features and mild truncal obesity Commons.wikimedia.org Used with permission BC03_3- Angelman Syndrome General characteristics • Deletion of an allele in the maternal chromosome 15 at an imprinted locus • Affects males and females Signs and symptoms • Mental retardation • Seizures • Ataxia • Puppet-like posture of limbs • Happy disposition • Inappropriate laughter •FA 2013: 83.2 • FA 2012: 88.2 • FA 2011: 84.2 • ME 3e: 77.1 • ME 4e: 77.1 "Boy with a Puppet" or “Child with a drawing" by Giovanni Francesco Caroto Commons.wikimedia.org Used with permission BC03_3- Hardy-Weinberg Equilibrium Definition • Predicts the genotype and allele frequencies in a non-evolving population Characteristics • Constant and predictable relationship between genotype frequencies and allele frequencies p2 + 2pq + q2 = p: frequency of allele (most common normal allele) q: frequency of allele (minor, disease producing allele) p2: frequency of genotype 1-1 (homozygous normal individuals) 2pq: frequency of genotype 1-2 (heterozygous individuals; carriers) q2: frequency of genotype 2-2 (homozygous affected individuals) • For test purposes: since p is very close to (most of the alleles of a particular gene are normal), the equation can be simplified to + 2q + q2 ≈ •FA 2013: 83.1 • FA 2012: 88.1 • FA 2011: 84.1 • ME 3e: 77.2 • ME 4e: 77.2 BC03_4- Hardy-Weinberg Equilibrium A 20-year-old college student is taking a course in human genetics She is aware that she has PKU, an autosomal recessive genetic disease with a known prevalence in the population of 1/10,000 live births She asks her genetics professor, what is the chance that she would marry a man with the diseaseproducing allele? 1.Carrier frequency is 2q 2.Disease prevalence is q2 (1/10.000) 3.√1/10.000 = 1/100 (frequency of disease-producing allele) 4.Therefore 2q = 1/50 Answer: The woman has a 1/50 chance of marrying a carrier of this allele What is the probability that she will have a child with PKU? 1.Probability that she will marry a carrier is 1/50 2.Probability that he will pass his PKU allele is ½ (50%) 3.Multiplication of these probabilities = 1/100 Answer: The probability that the woman will have a child with PKU is 1/100 •FA 2013: 83.1 • FA 2012: 88.1 • FA 2011: 84.1 • ME 3e: 77.2 • ME 4e: 77.2 BC03_4- Hardy-Weinberg Equilibrium Dominant diseases • Most affected individuals will be heterozygous • Prevalence is 2q • p≈1 • q2 prevalence of homozygous affected individuals X-linked recessive conditions • Most cases occur in hemizygous males • q = disease-producing allele frequency, prevalence of affected males • q2 is prevalence of disease in females • 2q prevalence of female carriers •FA 2013: 83.1 • FA 2012: 88.1 • FA 2011: 84.1 • ME 3e: 77.2 • ME 4e: 77.2 BC03_4- Factors Responsible for Genetic Variations in Populations • New mutations • Introduction of new mutation from outside • Non-random mating • Natural selection • Genetic drift • Gene flow Map of the migration of modern humans out of Africa, based on mitochondrial DNA Colored rings indicate thousand years before present Commons.wikimedia.com Used with permission •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 77.2 • ME 4e: 77.2 BC03_4- Natural Selection Characteristics • Acts upon genetic variation • Increases frequency of alleles which promote survival or fertility (fitness) • Decreases frequency of alleles which reduce fitness • Example: sickle cell disease & malaria Modern distribution of malaria Commons wikimedia.org Used with permission •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 77.2 • ME 4e: 77.2 Distribution of the sickle-cell trait shown in pink and purple Commons.wikimedia.org Used with permission Green shows historical distribution of malaria (no longer endemic in Europe) Commons Wikimedia.org Used with permission BC03_4- Genetic Drift Definition • Change in genetic composition of population due to chance or random events rather than natural selection • Results in changes in allele frequencies over time Results • When new mutation or founder effect occurs in small populations, genetic drift can make allele prevalence +/- than statistically predicted • If population size dramatically decreases, genetic drift may change allele frequencies •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 77.2 • ME 4e: 77.2 BC03_4- Gene Flow Definition • Exchange of genes among populations • Populations located close to one another often have similar gene frequencies • Gene flow can cause gene frequencies to change through time • Example: sickle cell in African Americans Gene flow in and out of Beringia, according to human mitochondrial DNA haplogroups Commons Wikimedia.org Used with permission •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 77.3 • ME 4e: 77.3 BC03_4- Consanguinity Definition • Mating of individuals related to one another (second cousin or closer) Coefficients of relationship • Siblings share ½ of genes • First cousins share⅛ of genes • Second cousins share 1/32 of genes Bernard Gui’s Arbor genealogiae regum Francorum, showing consanguinity of the kings of France Commons.wikimedia.org Used with permission •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC03_4- Cytogenetics General characteristics • Chromosome abnormalities are seen in ≈ 1/150 live births • Leading known cause of mental retardation & pregnancy loss • Fetuses with chromosome abnormalities lost prenatally 50% spontaneous fetal loss during first trimester 20% during second trimester • Fetuses carried to term with chromosome abnormalities demonstrate: Presence of extra chromosomes Loss of chromosomes or chromosomal material •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA Karyotype for trisomy Down syndrome Notice the three copies of chromosome 21 Commons Wikimedia.org Used with permission BC03_5- Karyotype Definition • Display of 23 pairs of human chromosomes in typical somatic cells Characteristics • Chromosomes are most easily visualized during metaphase of mitosis • 1-22 are autosomes; other two are sex chromosomes • In each chromosome, the p arm is the shorter and q arm is the longer regions • Acrocentric chromosomes 13-15; 21-22 have very tiny p arms Commons.Wikimedia.org Used with permission •FA 2013: 81.4 • FA 2012: 86.3 • FA 2011: 82.4 • ME 3e: NA • ME 4e: NA BC03_5- Numerical Chromosome Abnormalities Euploidy • Normal 46, XX (female) or normal 46, XY (male) Triploidy • Three copies of each chromosome, 69 total • Usually resulting from fertilization of an ovum by sperm cells • Vast majority lost prenatally Tetraploidy • Four copies of each chromosome, 92 total Aneuploidy • Deviation from euploid number (gain or loss) • Monosomy (loss of chromosomes) All autosomal monosomies are inconsistent with life • Trisomy (gain of chromosomes) Only trisomies of chromosomes 13,18, and 21 are consistent with life • Aneuploidy involving sex chromosomes Relatively common and less severe At least X chromosome required for survival If Y chromosome is present, phenotype is male (with few exceptions, e.g testicular feminization syndrome) If >1 X chromosome present all but one will become a Barr body •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC03_5- Normal Meiosis Disjunction During Meiosis Homologues chromosomes pair with each other at the Metaphase plate During Anaphase 1, homologues disjoin (separate) At the end of Meiosis 1, each daughter cell has one homolog •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA Disjunction During Meiosis During Metaphase 2, each chromosome aligns individually at the metaphase plate In Anaphase 2, sister chromatids migrate to opposite poles and each daughter cell gets one chromatid BC03_5- Nondisjunction during Meiosis I •FA 2013: 88.1 • FA 2012: 93.1 • FA 2011: 88.3 • ME 3e: 74 • ME 4e: 74 BC03_5- Nondisjunction During Meiosis II Disjunction During Meiosis Non- Disjunction During Meiosis •FA 2013: 88.1 • FA 2012: 93.1 • FA 2011: 88.3 • ME 3e: 74 • ME 4e: 74 BC03_5- Structural Chromosomal Abnormalities Characteristics • Occur when chromosomes are broken by clastogens Radiation Viruses Chemicals Unbalanced alterations • Loss or gain of genetic material Balanced alterations • Without loss or gain of genetic material Structural alterations • Germ cells (can be transmitted to offspring) • Somatic cells (can alter genetic material → cancer) •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC03_5- Reciprocal Translocation Definition/characteristics • Genetic material exchanged between non-homologous chromosomes • Translocation during gametogenesis → offspring will carry translocation in all cells (translocation carrier) Often no clinical consequences other than during reproduction Reproduction by translocation carrier can transmit genetic material to offspring causing partial trisomies and monosomies Typically pregnancy loss • Translocation in somatic cells Often no clinical consequences When oncogene or tumor suppressor gene alteration occurs → abnormal growth (CML, AML, follicular lymphomas, Burkitt’s lymphoma) •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 75.2 • ME 4e: 75.2 BC03_5- Robertsonian Translocation Characteristics • More common than reciprocal translocations • Occurs only in acrocentric chromosomes (centromeres toward the end) • Involves loss of short p arms and subsequent fusion of long q arms of chromosomes • No clinical consequences for carrier Short p arms of acrocentric chromosomes contain no essential genetic material Clinical correlate: ≈ 4% of Down syndrome cases result from Robertsonian translocation affecting chromosomes 14 and 21 •FA 2013: 88.2 • FA 2012: 93.2 • FA 2011: 89.1 • ME 3e: 76 • ME 4e: 76 BC03_5- Deletion Definition • Loss of some genetic information within a chromosome Causes • Agents causing chromosome breaks • Unequal crossover during meiosis Types • Interstitial Material within the chromosome is lost • Terminal End of chromosome lost Clinical correlate: Cri-du-chat syndrome due to microdeletion in p arm of chromosome •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 75.1 • ME 4e: 75.1 BC03_5- 10 Other Types of Structural Abnormalities Inversions • Pericentric (include centromere) • Paracentric (without centromere) • Carriers almost always unaffected but their offspring may have small partial trisomy Ring chromosomes • Often lost with resulting monosomy • Turner syndrome Loss of ring X chromosome •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 75.1 • ME 4e: 75.1 BC03_5- 11 Other Types of Structural Abnormalities Isochromosomes • Two copies of one arm, no copy of the other • Turner syndrome Isochromosome of long arm of X chromosome Uniparental disomy • Both copies of particular chromosome contributed by one parent Clinical Correlates Prader-Willi: maternal uniparental disomy of chromosome 15 Angelman: paternal uniparental disomy of chromosome 15 •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC03_5- 12 Flourescence In Situ Hybridization (FISH) Used for detection of • Aneuploidies • Translocations • Deletions • Microdeletions Method • Chromosome specific DNA segment labeled with fluorescent tag → probe • Probe is hybridized with patient’s chromosomes → visualization under fluorescence microscope • Probe will mark presence of tested chromosome segment •FA 2013: 81.1 • FA 2012: 85.4 • FA 2011: 82.1 • ME 3e: NA • ME 4e: NA Metaphase cell positive for the bcr/abl rearrangement (associated with chronic myelogenous leukemia) using FISH The chromosomes can be seen in blue In chromosome with green and red spots (upper left), the rearrangement is present Commons.wikimedia.org Used with permission BC03_5- 13 Biochemistry Genetic Diseases Megan Murray M.D., Ph.D Candidate University of Buffalo Achondroplasia • Autosomal dominant disorder • Most common form of inherited dwarfism • Missense mutation in fibroblast growth factor receptor (FGFR3) on chromosome → inhibition of cartilage synthesis at epiphysial growth plate • Decreased endochondral bone formation and premature ossification of growth plates • Long bones of extremities are abnormally short and thick • Cranial and vertebral bones are spared → large head and trunk Lavinia Warren, an actress with dwarfism Commons.wikimedia.org Used with permission •FA 2013: 85.1 • FA 2012: 90.1 • FA 2011: 86.1 • ME 3e: 442.1 • ME 4e: 442.1 BC04_1-2 Autosomal Dominant Polycystic Kidney Disease • Mutation of the PKD-1 gene on chromosome 16 • Enlarged kidneys with cysts filled with serous, turbid, or hemorrhagic fluid, bilaterally • Functioning nephrons found between cysts • Cysts arise from tubular structures of nephrons • Patients are asymptomatic with normal renal function until middle age → renal insufficiency, hematuria, HTN • Extra-renal manifestations • Liver cysts, berry aneurisms, mitral valve prolapse, colonic diverticula •FA 2013: 85.1 • FA 2012: 90.1 • FA 2011: 86.1 • ME 3e: 327.1 • ME 4e: 327.1 BC04_1-3 Familial Adenomatous Polyposis • Autosomal dominant disorder • Mutation of APC gene on chromosome 5q21 • Thousands of colonic polyps after puberty • After age 40, ≈100% of patients develop adenocarcinoma • Increased risk for development of adenocarcinoma of duodenum and ampulla of Vater Endoscopic image of sigmoid colon with familial adenomatous polyposis Commons.wikimedia.org Used with permission •FA 2013: 85.1 • FA 2012: 90.1 • FA 2011: 86.1 • ME 3e: 358.4 • ME 4e: 358.4 BC04_1-4 Familial Hypercholesterolemia • Also known as type II hyperlipidemia • Most common inherited disorder • Autosomal dominant • Mutation of LDL receptor gene on chromosome 19 • Elevation of serum LDL levels Xanthoma tendinosum Commons.wikimedia.org Used with permission Heterozygotes: 2-3x above normal Homozygotes: 5-6x above normal • Xanthomas: collections of lipid-laden macrophages within skin • Xanthelasmas around eyes • Premature atherosclerosis • Homozygotes can develop MI early in life •FA 2013: 85.1 • FA 2012: 90.1 • FA 2011: 86.1 • ME 3e: 53.1 • ME 4e: 53.1 Xanthelasma palpebrarum, yellowish patches consisting of cholesterol deposits above eyelids Commons.wikimedia.org Used with permission BC04_1-5 Hereditary Hemorrhagic Telangiectasia • Also known as Osler-Weber-Rendu syndrome • Autosomal dominant disorder • Telangiectasias of skin and mucous membranes • Lips, tongue, and fingertips commonly affected • Recurrent epistaxis • Skin discoloration • Arterio-venous malformations • Possible iron deficiency anemia Lips and tongue with characteristic telangiectasias Commons wikimedia.org Used with permission •FA 2013: 85.1 • FA 2012: 90.1 • FA 2011: 86.1 • ME 3e: NA • ME 4e: NA BC04_1-6 Hereditary Spherocytosis • Autosomal dominant disorder • Defect in proteins ankyrin and spectrin (components of erythrocyte cell membrane) • RBCs contract to a sphere shape • Spherocytes removed in spleen by macrophages Signs and symptoms • Splenomegaly • Hemolytic anemia • Jaundice • Pigmented gallstones • Increased MCHC • Treatment: splenectomy •FA 2013: 85.1 • FA 2012: 90.1 • FA 2011: 86.1 • ME 3e: 467.5 • ME 4e: 467.5 Peripheral blood smear of hereditary spherocytosis Commons.wikimedia.org Used with permission BC04_1-7 Huntington’s Disease • Autosomal dominant disorder • HD gene on chromosome • Codes for Huntingtin protein • Expansion of trinucleotide repeat CAG • Degeneration of GABAergic neurons of caudate nucleus • Removal of inhibitory input on extrapyramidal circuit → chorea • Manifests between age 20–40 • Personality changes • Dementia •FA 2013: 85.1 • FA 2012: 90.1 • FA 2011: 86.1 • ME 3e: 216.1 • ME 4e: 216.1 Human brain Commons.wikimedia.org Used with permission BC04_1-8 Marfan Syndrome • Autosomal dominant disorder • Mutation of fibrillin gene on chromosome 15q21 • Glycoprotein that functions as scaffold for elastic fiber alignment • Marfan disease illustrates the concept of pleotrophy • Tall, thin body habitus • Bilateral subluxation of lenses • Cystic degeneration of media of elastic arteries → aneurysms • Aortic and mitral valve insufficiency •FA 2013: 85.1 • FA 2012: 90.1 • FA 2011: 86.1 • ME 3e: 73.1 • ME 4e: 73.1 Antoine Marfan (1858-1942); portrait by Henry Bataille Commons.wikimedia.org Used with permission BC04_1-9 Multiple Endocrine Neoplasia Syndromes • Autosomal dominant disorders • Hyperplasia and tumors of endocrine glands MEN I: mutation of MEN I tumor supressor gene MEN IIA: mutation of RET proto-oncogene MEN IIB (III): mutation of RET proto-oncogene • Parathyroid (most common presentation) • Parathyroid hyperplasia or adenoma • Pheochromocytoma • Pituitary (prolactinomas) • Pheochromocytoma • Medullary carcinoma of thyroid • Pancreas (insulinomas, Zollinger-Ellison) • Medullary carcinoma of thyroid • Muco-cutaneous neuromas • Marfanoid habitus • FA 2012: 90.1 • FA 2011: 86.1 • FA 2010: 86.1 • ME 3e: 381.7 • ME 4e: 381.7 BC04_1-10 Neurofibromatosis • Autosomal dominant disease Neurofibromatosis type I (Von Recklinghausen disease) (90% of cases) • Mutation of tumor suppressor gene NF-1 on chromosome 17 • Multiple neurofibromas (may undergo malignant transformation) • Café-au-lait spots • Lisch nodules • Increased risk for meningiomas and pheochromocytomas Neurofibromatosis type II • Mutation of tumor suppressor gene NF-2 on chromosome 22 • Bilateral acoustic neuromas • Neurofibromas • Café-au-lait spots • Increased risk for meningiomas and ependymomas • FA 2012: 90.1 • FA 2011: 86.1 • FA 2010: 86.1 • ME 3e: 73.1 • ME 4e: 73.1 Back of an elderly woman with neurofibromatosis Commons.wikimedia.org Used with permission BC04_1-11 Tuberous Sclerosis • Autosomal dominant disease • Multiple cortical, retinal, and pulmonary hamartomas Benign focal malformations; resemble neoplasias • Ash leaf spots • Renal cysts • Renal angiomyolipomas • Cardiac rhabdomyomas • Cortical tubers • Increased incidence of astrocytomas • Seizures • Mental retardation • FA 2012: 90.1 • FA 2011: 86.1 • FA 2010: 86.1 • ME 3e: 261.3 • ME 4e: 261.3 Tuberous sclerosis showing facial angiofibromas in characteristic butterfly pattern Commons.wikimedia.org Used with permission BC04_1-12 von Hippel-Lindau Disease • Autosomal dominant disease • Mutation of tumor suppressor gene VHL on chromosome 3p • Main action of normal gene product is to tag proteins with ubiquitin for degradation (e.g., hypoxia-inducible factor-1A) • Expression of HIF → activation of angiogenic growth factors • Hemangioblastomas • Retina : von Hippel tumor • Cerebellum • Brainstem Lindau tumors • Spinal cord • Cysts of liver, pancreas, and kidneys • Multiple, bilateral renal cell carcinomas • FA 2012: 90.1 • FA 2011: 86.1 • FA 2010: 86.1 • ME 3e: 73.1 • ME 4e: 73.1 Typical distribution of hemangioblastomas in Von Hippel-Lindau disease Commons.wikimedia.org Used with permission BC04_1-13 Autosomal Recessive Diseases • Albinism • Autosomal recessive polycystic kidney disease • Glycogen storage diseases • Hemochromatosis • Mucopolysaccharidoses (except Hunter’s, X-linked recessive) • Phenylketonuria • Sickle cell anemia • Sphingolipidoses (except Fabry, X-linked recessive) • Thalassemias • Cystic fibrosis •FA 2013: 86.1 • FA 2012: 91.1 • FA 2011: 87.1 • ME 3e: 72 • ME 4e: 72 BC04_2-1 Cystic Fibrosis • Most common lethal genetic disorder in Caucasians • Mutation of cystic fibrosis transmembrane regulator (CFTR) chloride channel protein (chromosome 7) • Most common mutation: 3-nucleotide deletion that encodes phenylalanine at 508 position • Abnormal protein folding → degradation of chloride channel • Diagnosis: sweat test • Treatment is multimodal: N-acetylcysteine Treatment of accompanying infections Clubbing in the fingers of a person with cystic fibrosis Commons.wikimedia.org Used with permission •FA 2013: 86.2 • FA 2012: 91.2 • FA 2011: 87.2 • ME 3e: 73 • ME 4e: 73 BC04_2-2 X-Linked Recessive Disorders • Bruton’s agammaglobulinemia • Wiskott-Aldrich syndrome • Fabry’s disease • G6PD deficiency • Ocular albinism • Lesch-Nyhan syndrome • Duchenne’s and Becker’s muscular dystrophy • Hunter’s syndrome • Hemophilia A and B •FA 2013: 86.3 • FA 2012: 91.3 • FA 2011: 87.3 • ME 3e: 72 • ME 4e: 72 BC04_3-1 Duchenne’s Muscular Dystrophy • Frame-shift mutation of dystrophin gene on X chromosome → ineffective dystrophin protein • Onset of symptoms by age • Calf pseudo-hypertrophy • Proximal muscle weakness • Gower’s maneuver • Cardiac myopathy • Respiratory insufficiency and pulmonary infections • Elevated CPK • Muscle biopsy: necrosis, degeneration and regeneration of fibers, fatty infiltration, decreased dystrophin • Diagnosis: DNA analysis by PCR •FA 2013: 86.4 • FA 2012: 91.4 • FA 2011: 87.4 • ME 3e: 450 • ME 4e: 450 BC04_3-2 Fragile X Syndrome • Second most common genetic cause of mental retardation (after Down syndrome) • Triplet nucleotide repeat mutation in familial mental retardation -1 gene (FMR-1) on X chromosome • CGG repeats hundreds to thousands of times • X-linked dominant inheritance • 100% males – 50% females • Autism/ADHD • Elongated face with large jaw • Large everted ears • Macroorchidism • Diagnosis: PCR or DNA probe analysis •FA 2013: 87.1 • FA 2012: 92.1 • FA 2011: 87.5 • ME 3e: 72 • ME 4e: 72 USA.gov Used with permission BC04_4-1 Friedreich’s Ataxia • GAA sequence expansion in frataxin gene on chromosome • Frataxin protein essential for mitochondrial iron regulation; in its absence, mitochondrial iron builds up, leading to free radical damage and mitochondrial dysfunction • Autosomal recessive inheritance • Onset in early childhood • Degeneration of nerve tissue in spinal cord • Gait ataxia (pes cavus, hammer toes) • Dysarthria • Hand clumsiness • Loss of sense of position • Impaired vibratory sensation • Areflexia in all limbs •FA 2013: 87.2 • FA 2012: 92.2 • FA 2011: 88.1 • ME 3e: NA • ME 4e: NA Pes cavus Commons.Wikimedia.org.Used with permission BC04_4-2 Myotonic Dystrophy • CTG repeat sequence expansion • Autosomal dominant disorder • Muscle loss • Myotonia • Cardiac conduction defects • Testicular atrophy • Frontal baldness • Cataracts A 40-year-old patient with myotonic dystrophy presenting with bilateral cataracts and complete heart block Commons.wikimedia.org Used with permission •FA 2013: 87.2 • FA 2012: 92.2 • FA 2011: 88.1 • ME 3e: 450 • ME 4e: 450 BC04_4-3 Down Syndrome (Trisomy 21) • Most common of the chromosomal disorders • Incidence: Maternal age dependent • Most common cause of congenital mental retardation Causes • Meiotic nondisjunction of homologous chromosomes (95% of cases) Advanced maternal age • Robertsonian translocation (4% of cases) • Mosaicism from mitotic nondisjunction (1% of cases) Tommy Jessop, actor with Down syndrome Commons wikimedia.org Used with permission • FA 2012: 93.1 • FA 2011: 88.2 • FA 2010: 88.2 • ME 3e: 74 • ME 4e: 74 •FA 2013: 88.1 • FA 2012: 93.1 • FA 2011: 88.2 • ME 3e: 74 • ME 4e: 74 BC04_5-1 Down Syndrome (Trisomy 21) • Mongoloid facial features: flat face, low-bridged nose, epicanthal folds • Muscular hypotonia • Broad, short neck • Simian creases • Congenital endocardial cushion defects • ≈100% develop Alzheimer disease by age 40; ↑ risk for duodenal atresia, Hirschsprung disease, ALL • Pre-natal screening: Brushfield spots, visible in irises of baby with Down syndrome Commons.wikimedia.org Used with permission ↓ α-fetoprotein ↑ β-HCG ↓ unconjugated estriol ↑ inhibin A •FA 2013: 88.1 • FA 2012: 93.1 • FA 2011: 88.2 • ME 3e: 74 • ME 4e: 74 Single transverse palmar crease Commons.wikimedia.org Used with permission BC04_5-2 Edwards Syndrome (Trisomy 18) • Second most common trisomy resulting in live birth • Increased risk with advanced maternal age • Caused by nondisjunction • Mental retardation • Low set ears, micrognathia • Overlapping flexed fingers • Rocker-bottom feet • Congenital heart defects • Renal malformations • Screening ↓ α-fetoprotein ↓ β-hCG ↓ estriol Clenched hand and overlapping fingers: index finger overlaps third finger and fifth finger overlaps fourth finger, characteristically seen in Trisomy 18 Commons.wikimedia.org Used with permission Normal inhibin A •FA 2013: 88.1 • FA 2012: 93.1 • FA 2011: 88.2 • ME 3e: 74 • ME 4e: 74 BC04_5-3 Patau Syndrome (Trisomy 13) • Increased risk with advanced maternal age • Caused by nondisjunction • Mental retardation • Cleft lip/palate • Cardiac defects • Renal abnormalities • Microcephaly/holoprosencephaly • Polydactyly • Screening: Normal markers •FA 2013: 88.1 • FA 2012: 93.1 • FA 2011: 88.2 • ME 3e: 74 • ME 4e: 74 A 37 2/7 week gestational age boy with Patau syndrome demonstrating polydactyly Commons.wikimedia.org Used with permission BC04_5-4 Cri-Du-Chat Syndrome • Deletion of short arm of chromosome • High pitch cat-like cry • Mental retardation • Congenital heart disease (ventricular septal defect) • Microcephaly Facial features of a Cri du Chat patient Age months (A), years (B), years (C), years (D) Commons.wikimedia.org Used with permission •FA 2013: 89.1 • FA 2012: 94.1 • FA 2011: 89.2 • ME 3e: 77 • ME 4e: 77 BC04_6-1 Williams Syndrome • Microdeletion of long arm of chromosome (including elastin gene) • Mental retardation • “Elfin” face • Hypercalcemia (increased sensitivity to vitamin D) • Cheerful demeanor and ease with strangers • Developmental delay coupled with strong language skills • Cardiovascular problems (supravalvular aortic stenosis) •FA 2013: 89.2 • FA 2012: 94.2 • FA 2011: 89.3 • ME 3e: NA • ME 4e: NA BC04_6-2 Microdeletion at Chromosome 22q11 General characteristics • Abnormal facies • Cleft palate • Thymic aplasia→ T-cell deficiency • Parathyroid aplasia→ hypocalcemia • Cardiac defects DiGeorge syndrome • Cardiac defects • Thymic aplasia • Hypocalcemia/hypoparathyroidism CATCH-22 Cardiac anomalies Abnormal facies Thymic aplasia Cleft palate Hypocalcemia/ hypoparathyroidsm 22 chromosome Velocardiofacial syndrome • Cardiac defects • Abnormal facies • Cleft palate •FA 2013: 89.3 • FA 2012: 94.3 • FA 2011: 89.4 • ME 3e: 77 • ME 4e: 77 BC04_6-3 Biochemistry Signal Transduction Systems, Vitamins Megan Murray , M.D., Ph.D Candidate University of Buffalo Hormones: General Characteristics • Any compound produced by a cell, which, by binding to its cognate receptor, alters the metabolism of a cell bearing a hormone-receptor complex Classification • Autocrine hormones: bind to receptors on cell which produces them Example: prostaglandins • Paracrine hormones: act on cells close to cell which produces them Example: neurotransmitters Secreted in interstitial space Very short half-life • Endocrine hormones: have target cell at a distant site Secreted into blood stream Longer half-life •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC05_1-2 Hormones Classification Water-Soluble Lipid-Soluble Receptor in cell membrane Receptor inside cell Second messenger often involved Protein kinase activated Hormone-receptor complex binds hormone response elements (HRE, of enhancer regions) in DNA Protein phosphorylation to modify activity of enzymes (requires minutes) Control of gene expression through proteins such as cAMP response element binding (CREB) protein (requires hours) Examples: Insulin Glucagon Catecholamines •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 370 • ME 4e: 370 -Control of gene expression (requires hours) Examples: Steroids Calcitriol Thyroxines Retinoic acid BC05_1-3 Signal Transduction by Water Soluble Hormones Pathway cAMP PIP2 cGMP Insulin, growth factors G Protein Gs (Gi) Gq None Monomeric p21ras Enzyme Second Messenger(s) Adenyl cyclase Phospholipase C cAMP DAG, IP3, Guanyl cyclase •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 84-85 • ME 4e: 84-85 Ca2+ cGMP Protein Kinase Examples Protein kinase A Glucagon Epinephrine (β, α2) Vasopressin (V2, ADH) kidney Protein kinase C Vasopressin (V1, V3) Vascular smooth muscle Epinephrin (α1) Protein kinase G Atrial natriuretic peptide (ANP) Nitric oxide (NO) Tyrosine kinase activity of receptor Insulin Insulin-like growth factor (IGF) Platelet-derived growth factor (PDGF) Epidermal growth factor (EGF) BC05_1-4 Trimeric G Protein GDP GTP αs: stimulates αi: inhibits GDP •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 84 • ME 4e: 84 BC05_1-5 cAMP and PIP2 Systems helix receptors •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 84 • ME 4e: 84 BC05_1-6 Atrial Natriuretic Peptide and Nitric Oxide Systems • ANP secreted by atrium of heart • Location of ANP receptor Vascular smooth muscle and kidney •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 85 • ME 4e: 85 BC05_1-7 Insulin Receptor •FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 84 • ME 4e: 84 Growth factors stimulating tyrosin kinase receptors • Platelet-derived growth factor • Epidermal growth factor BC05_1-8 Vitamin Classification Water-Soluble Lipid-Soluble Thiamine (B1) Riboflavin (B2) Retinol (A) Retinoic acid (A) Niacin (B3) Pantothenic acid (B5) Pyridoxine (B6) Biotin (B7) Folic acid (B9) Cobalamin (B12) Ascorbate (C) • FA 2013: 90.1 • FA 2012: 94.4 • FA 2011: 90.1 • ME 3e: 56-57 • ME 4e: 56-57 Calcitriol (D) K E BC05_2-1 Thiamine (B1) Characteristics • Coenzyme form: thiamine pyrophosphate Pyruvate dehydrogenase (glycolysis–TCA cycle) α-ketoglutarate dehydrogenase (TCA cycle) Branched chain ketoacid dehydrogenase (metabolism of valine, leucine, and isoleucine)→ maple syrup urine disease Transketolase (HMP shunt) Deficiency • Alcoholism → interferes with absorption Clinical manifestations • Wernicke’s (ataxia, nystagmus, ophthalmoplegia) • Korsakoff’s (psychosis, confabulation) • Dry beriberi (muscle wasting, partial paralysis) • Wet beriberi (high output cardiac failure, peripheral edema) • FA 2013: 90.4 • FA 2012: 95.2 • FA 2011: 91.1 • ME 3e: 56 • ME 4e: 56 Patient with Beriberi, turn of 20th century in southeast Asia Commons.wikimedia.org Used with permission BC05_2-2 Riboflavin (B2) Characteristics • Coenzyme forms: FAD/FMN • Coenzyme for several dehydrogenases • Involved in oxidation/reduction reactions Deficiency • Poor dietary intake (usually accompanied by deficiency of other vitamins) • Malabsorption syndromes Clinical manifestations • Corneal neovascularization • Cheilosis/stomatitis Stomatitis • Magenta-colored tongue • FA 2013: 91.1 • FA 2012: 95.3 • FA 2011: 91.2 • ME 3e: 56 • ME 4e: 56 Commons.wikimedia.org Used with permission BC05_2-3 Niacin (B3) Characteristics • Coenzyme forms: NAD(H) and NADP(H) • Coenzyme for many dehydrogenases • Synthesized from tryptophan Deficiency • Hartnup disease: autosomal recessive disorder→ impaired AA absorption from intestines and reabsorption in kidneys→ tryptophan deficiency → niacin deficiency • Malnutrition → pellagra Clinical manifestations • Diarrhea • Dementia • Dermatitis • Death • FA 2013: 91.2 • FA 2012: 96.1 • FA 2011: 91.3 • ME 3e: 56 • ME 4e: 56 Dermatologic features of this disorder, including desquamation, erythema, scaling, and keratosis of sun-exposed areas Commons.wikimedia.org Used with permission BC05_2-4 Pantothenic Acid (B5) Characteristics • Found in nearly every food product • Synthesis of coenzyme A, important for Pyruvate → TCA cycle α-ketoglutarate → succinyl-CoA Fatty acid metabolism Cholesterol synthesis • Required for acylation and acetylation Involved in signal transduction and enzyme activation/deactivation Deficiency • Extreme starvation (rare) Clinical manifestations • Dermatitis, enteritis, alopecia, adrenal insufficiency • FA 2013: 91.3 • FA 2012: 96.2 • FA 2011: 91.4 • ME 3e: 56 • ME 4e: 56 BC05_2-5 Pyridoxine (B6) Characteristics • Converted to pyridoxal phosphate in the body Coenzyme for • Aminotransferase reactions (protein metabolism) Alanine aminotransferase (ALT) Aspartate aminotransferase (AST) • ↑ in liver & muscle disease Delta-aminolevulinate synthase Rate-limiting enzyme in heme synthesis Deficiency • Isoniazid therapy (most common cause) Clinical manifestations Ring sideroblast, visualized by Prussian blue stain • Sideroblastic anemia • Cheilosis/stomatitis • Convulsions • FA 2013: 91.4 • FA 2012: 96.3 • FA 2011: 91.5 • ME 3e: 56 • ME 4e: 56 Commons.wikimedia.org Used with permission BC05_2-6 Biotin (B7) Characteristics • Cofactor for carboxylation enzymes Pyruvate carboxylase Gluconeogenesis: Pyruvate Oxaloacetate Acetyl-CoA carboxylase Fatty acid synthesis: Odd carbon fatty acid synthesis: Acetyl-CoA Malonyl-CoA Propionyl-CoA Propionyl-CoA carboxylase Methylmalonyl-CoA Deficiency • Excessive consumption of raw eggs → contain avidin (biotin-binding protein) Clinical manifestations • Alopecia • Bowel inflammation • Muscle pain Commons.wikimedia.org Used with permission • FA 2013: 92.1 • FA 2012: 98.2 • FA 2011: 93.1 • ME 3e: 56 • ME 4e: 56 BC05_2-7 Folic Acid (B9) Characteristics • Found in green leafy vegetables • Stored in liver • Tetrahydrofolate is a coenzyme for Enzymes for 1-carbon transfer/methylation reactions, e.g., thymidylate synthase (de novo pyrimidine synthesis) Deficiency • Body stores folate for up to 3-4 months • Most common vitamin deficiency in the U.S • Folate pool quickly depleted in alcoholics and pregnancy Deficiency in pregnancy → neural tube defect • Side effect of several drugs Peripheral blood smear shows hypersegmented neutrophils, characteristic of megaloblastic anemia Commons.wikimedia.org Used with permission Phenytoin, sulfonamides, methotrexate Clinical manifestations • Macrocytic megaloblastic anemia • Homocysteinemia (cardiovascular disease, DVT, thromboembolism, stroke) • FA 2013: 92.2 • FA 2012: 97.2 • FA 2011: 92.2 • ME 3e: 56 • ME 4e: 56 BC05_2-8 Cobalamin (B12) Characteristics • Converted to coenzymes methyl- or deoxyadenosyl cobalamin for: Homocysteine methyltransferase (homocysteine → methionine) Methymalonyl-CoA mutase (methylmalonyl-CoA → succinyl CoA) Deficiency (develops in years) • • • • • • Pernicious anemia (most common cause) Aging Chronic pancreatitis Long-term total vegetarian diet Infection with Diphyllobothrium latum Resection of terminal ileum Clinical manifestations • • • • Immunofluorescence staining pattern of gastric May be associated with secondary deficiency of parietal cell antibodies on a stomach section active tetrahydrofolate Commons.wikimedia.org Used with permission Megaloblastic anemia Homocysteinemia (CV disease, DVT, thromboembolism, stroke) Neuropathies • FA 2013: 93.1 • FA 2012: 97.1 • FA 2011: 92.1 • ME 3e: 56 • ME 4e: 56 BC05_2-9 Ascorbate (Vitamin C) The only water-soluble vitamin not converted to a coenzyme before use Characteristics • Cofactor for: Prolyl and lysyl hydroxylases (collagen synthesis) Dopamine β-hydroxylase (dopamine→ norepinephrine) • Enhances Fe absorption in GI tract • Antioxidant Deficiency • Dietary deficiency (devoid of citrus fruits and green vegetables) Clinical manifestations • Scurvy Poor wound healing, easy bruising Scorbutic gums, a symptom of scurvy Note gingival redness in triangle-shaped interdental papillae between teeth Perifollicular hemorrhaging, bleeding gums, anemia Commons.wikimedia.org Used with permission Increased bleeding time Painful glossitis • FA 2013: 94.1 • FA 2012: 98.3 • FA 2011: 93.2 • ME 3e: 56 • ME 4e: 56 BC05_2-10 Vitamin A Functions • Maintenance of healthy epithelium • Vision Structures (differ on basis of functional group on C1) • Retinol (hydroxyl) • Retinoic acid (carboxyl) • Retinal (aldehyde) Maintenance of epithelium Retinol and retinoic acid Bind to intracellular receptors (zinc fingers) Regulate transcription through specific response elements Normal differentiation of epithelial cells into specialized tissue Prevention of squamous metaplasia • FA 2013: 90.3 • FA 2012: 95.1 • FA 2011: 90.4 • ME 3e: 57 • ME 4e: 57 BC05_2-11 Vitamin A and Vision • Trans-retinal → cis-retinal • Transfer to opsin in rod cells→ light receptor rhodopsin (also called transducin) • Rod cell membrane partially depolarized at rest and hyperpolarized on stimulation • Darkness (rest)→ glutamate release → inhibition of bipolar cells Light Activation of cGMP phosphodiesterase (↓cGMP) Closure of cGMP-gated Na+ channels (hyperpolarization) Cessation of glutamate release ↓ inhibition of bipolar cells Signal initiation • FA 2013: 90.3 • FA 2012: 95.1 • FA 2011: 90.4 • ME 3e: 57 • ME 4e: 57 BC05_2-12 Vitamin A Deficiency and Clinical Uses • Extreme malnutrition • Fat malabsorption • Liver cirrhosis Most common causes in the U.S Clinical manifestations • Night blindness Most common cause of blindness in developing countries • Metaplasia of corneal epithelium • Xerophthalmia, follicular hyperkeratosis • Frequent infections (bronchitis, pneumonia, measles) Clinical uses • Isotretinoin: wrinkles and acne (topically) • ATRA: acute myelogenous leukemia (M3 subtype) Toxicity: Teratogenic in pregnancy • FA 2013: 90.3 • FA 2012: 95.1 • FA 2011: 90.4 • ME 3e: 57 • ME 4e: 57 Keratosis pilaris rubra on a right upper arm Commons.wikimedia.org Used with permission BC05_2-13 Vitamin D Forms • Vitamin D2 (ergocalciferol): derived from plants • Vitamin D3 (cholecalciferol): sun exposure or vitamin D fortified milk • Calcifediol (25-hydroxy vitamin D): storage form in liver • Calcitriol: active vitamin D (1,25-dihydroxy vitamin D); formed in kidney in response to PTH Function (↑ Ca 2+ and phosphate) • GI tract: induces synthesis of Ca2+ binding proteins, ↑ phosphate absorption • Kidneys: stimulates reabsorption of Ca2+ and secretion of phosphate • Bone: acts upon osteoblast receptors → osteoclast activation → bone resorption • FA 2013: 94.2 • FA 2012: 99.1 • FA 2011: 93.3 • ME 3e: 57 • ME 4e: 57 BC05_2-14 Vitamin D Deficiency Childhood rickets (skeletal abnormalities) Adults osteomalacia (fewer deformities) • Mild deficiency: Chvostek’s/Trousseau’s signs • Severe deficiency → hypocalcemic tetany • Breast-fed only infants→ vitamin D deficiency Chest x-ray shows changes consistent with rickets, often called Rosary beads of rickets Commons.wikimedia.org Used with permission Radiograph of 2-year old rickets sufferer, with genu Wrist x -ray shows changes in rickets, mainly varum (bowing of femurs) and decreased bone opacity, cupping is seen here suggesting poor bone mineralization Commons.wikimedia.org Used with permission • FA 2013: 94.2 • FA 2012: 99.1 • FA 2011: 93.3 • ME 3e: 57 • ME 4e: 57 Commons.wikimedia.org Used with permission BC05_2-15 Vitamin D Toxicity Causes • Excessive consumption • Sarcoidosis Pathogenesis • Bone resorption → hypercalcemia → impairment of renal function → Ca2+ deposition → irreversible kidney damage • FA 2013: 94.2 • FA 2012: 99.1 • FA 2011: 93.3 • ME 3e: NA • ME 4e: NA BC05_2-16 Vitamin K General characteristics • • Required for γ-carboxylation of glutamyl residues on several Ca2+-dependent proteins (co-translational modification on RER) Example: coagulation factors II, VII, IX, and X Anticoagulation proteins C and S Deficiency • • • • Fat malabsorption Prolonged treatment with broad spectrum antibiotics Newborns Infants born to mothers who received anticonvulsants (phenytoin) Clinical manifestations • • • Prolonged bleeding (↑ PT, ↑ PTT, normal bleeding time) Easy bruising Potentially fatal hemorrhage Clinical use • Warfarin antidote in the non-acute setting • FA 2013: 95.1 • FA 2012: 99.3 • FA 2011: 94.1 • ME 3e: 57 • ME 4e: 57 BC05_2-17 Vitamin E (Tocopherol) Characteristics • Antioxidant • Protects other lipids from oxidative damage by preventing peroxidation of fatty acids in cell membranes • Maintains normal cellular membrane fluidity Deficiency • Fat malabsorption • Premature, low-weight newborns Clinical manifestations • Hemolysis • Posterior column and spinocerebellar tract demyelination • Retinitis pigmentosa • FA 2013: 94.3 • FA 2012: 99.2 • FA 2011: 93.4 • ME 3e: 57 • ME 4e: 57 BC05_2-18 Biochemistry Overview of Metabolism: Carbohydrates Megan Murray, MD, PhD Candidate University of Buffalo Energy from Different Sources Hydrolyzation (GI tract) Hydrolyzation (GI tract) NAD → NADH FAD → FADH2 Oxidative phosphorylation • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_1-2 Energy Storage ATP • Formed in catabolic pathways by phosphorylation of ADP • Provides energy for muscle contraction, biosynthesis pathways, active transport systems Storage Forms of Energy • Creatine phosphate: its energy can rapidly be transferred to form ATP • Triglycerides: fatty acids + glycerol • Glycogen: polymer of glucose • Protein: when degraded in excess amounts for fuel indicates a pathological situation • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_1-3 Levels of Metabolic Pathways Control • Regulation of metabolic pathways depends on nutritional status Well fed state Fasting Starvation • Shifts between metabolic patterns regulated by anabolic/catabolic hormonal ratios Anabolic hormone: insulin Catabolic hormones: glucagon, cortisol, GH, epinephrine • Levels of control of metabolic pathways Allosteric inhibitors and activators of rate limiting enzymes (rapid) Control of gene expression by insulin/glucagon (slow) Phosphorylation/dephosphorylation of rate limiting enzymes (rapid) • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_1-4 Metabolic Profile of the Well-Fed (Absorptive) State ↑INSULIN • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_1-5 Metabolic Profile of the Post-Absorptive State ↑Glucagon ↑Epinephrine • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_1-6 Well-Fed Vs Fasting States • Fats: kcal/g • Carbohydrates, Protein: kcal/g Organ Well-Fed Fasting Liver Glucose and amino acids Fatty acids Resting skeletal muscle Glucose Fatty acids, ketones Cardiac muscle Fatty acids Fatty acids, ketones Adipose tissue Glucose Fatty acids Brain Glucose Glucose (ketones prolonged fast) RBC Glucose Glucose • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_1-7 Exercise Vs Resting Skeletal Muscle Fast twitch fibers Slow twitch fibers • High capacity for anaerobic glycolysis • High capacity for aerobic glycolysis • Quick to fatigue • Resist fatigue • Short term, high intensity exercise • Prolonged low/moderate exercise *Slow twitch fibers and number of mitochondria ↑↑↑ in trained athletes • Short high intensity exercise → anaerobic glycolysis from muscle glycogen • High continuous exercise → oxidation of glucose and fatty acids • Prolonged exercise → ↓intensity of exercise to a rate that can be supported by fatty acid oxidation • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_1-8 Overview of Carbohydrate Metabolism Characteristics • All cells carry out glycolysis • Erythrocytes depend exclusively on glycolysis to generate ATP • Glucose is the major monosaccharide entering the pathway • Fructose and galactose can also be used Initial steps in glucose metabolism • Transport of glucose into the cell (because glucose is polar it cant enter by itself) • Phosphorylation using ATP by kinase enzymes to prevent glucose from leaving the cell (e.g hexokinase and glucokinase) • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_2-1 Major Glucose Transporters Name Tissues GLUT Most tissues (brain, RBC) GLUT Liver Pancreatic β-cells GLUT Most tissues GLUT Skeletal muscle Adipose tissue Km Glucose ~1mM ~15 mM Functions Basal uptake of glucose Uptake and release of glucose by the liver β-cell glucose sensor ~1mM Basal uptake ~5 mM Insulin-stimulated glucose uptake; also stimulated by exercise in skeletal muscle Note: Glut has high Km for glucose uptake • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 38 • ME 4e: 38 BC06_2-2 Insulin Regulation of Glucose Basal transport of glucose occurs in all cells independently of insulin Insulin: ↑rate of glucose transport into adipose tissue and resting muscle via GLUT • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC06_2-3 Introduction to Glycolysis Characteristics • Cytoplasmic pathway in all cells • Converts a molecule of glucose into two pyruvates (no carbon loss) • Can be aerobic or anaerobic • Provides intermediates for other pathways • Important enzymes: Hexokinase Glucokinase Phosphofructokinase (PFK-1 and PFK-2) Pyruvate kinase Lactate dehydrogenase (LDH) • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 38 • ME 4e: 38 BC06_3-1 Glycolysis Insulin ATP ADP Glucose Glucose Transport NAD Mg2+ Glucose- 6P Fructose- 6P ETC/O2 Mitochondria Pi PFK-1 + (phosphofructokinase) ADP Glyceraldehyde-3P Fructose- 2,6 bis -P ATP ADP ATP Hexokinase Glucokinase (liver) PFK-2 Fructose- 1,6 bis -P Glyceraldheyde 3P dehydrogenase Isomerase Dihydrooxyacetone-P NADH 1,3-Bisphosphoglycerate ADP ATP Glycerol 3P dehydrogenase (RBC) Phosphoglycerate kinase 3-Phosphoglycerate 2,3 Bisphosphoglycerate Pyruvate Glycerol-3P TGL system Electron shuttle Mutase Acetyl CoA 2-Phosphoglycerate Enolase NADH NAD Lactate Phosphoenolpyruvate ADP Pyruvate kinase ATP Lactate dehydrogenase Pyruvate • FA 2013: 98.1 • FA 2012: 102.1 • FA 2011: 96.1 • ME 3e: 38 • ME 4e: 38 TCA Fatty acid synthesis CO2 ↑ATP BC06_3-2 ATP Production from Glycolysis 2NAD 2NADH 4-6 ATP 2ATP 2ADP Glucose Pyruvate 4ADP 4ATP *The Malate shuttle and Glycerol phosphate shuttle are necessary to transport cytoplasmic NADH to the mitochondria Anaerobic glycolysis: 2ATP 2NAD 2NADH Pyruvate *6-8ATP ATP 2Acetyl- CoA 2GDP 2Acetyl- CoA 2GTP TCA Cycle 6NAD 6NADH 2FAD • FA 2013: 99.1 • FA 2012: 103.1 • FA 2011: 97.1 • ME 3e: 38 • ME 4e: 38 4CO2 24 ATP 18 ATPs 2FADH ATP BC06_3-3 2,3- Biphosphoglycerate and Pyruvate Kinase Deficiency Pyruvate kinase deficiency • Second most common genetic deficiency causing hemolytic anemia Characteristics • Chronic hemolysis • ↑ 2,3-BPG → ↓Oxygen affinity to Hb • No Heinz bodies (this differentiates PK deficiency from G6PD deficiency) • ↓ATP production → ↓Na/K- ATPase function → Loss of ion balance → → Swelling and lysis • FA 2013: 101.1 • FA 2012: 105.1 • FA 2011: 99.2 • ME 3e: 38 • ME 4e: 38 BC06_3-4 Galactose Metabolism Lactose Glucose • Symptoms of lactase deficiency after ingestion of lactose (milk products) include: • Diarrhea • Bloating • Cramps Lactase Galactose Blood Lens Galactose Galactose If galactose accumulates Liver, Brain, and Other Tissues ATP ADP Galactokinase Galactose1-P UDP-Gal epimerase UDP-Glc UDP-Gal GAL 1-P uridyl transferase Glucose 1-P Glycogen Glucose 6-P Glycolysis Glucose • FA 2013: 105.1 • FA 2012: 109.1 • FA 2011: 103.2 • ME 3e: 38 • ME 4e: 38 Galactitol Galactitol trapped in the lens causes swelling and cataracts • Galactokinase deficiency: • Cataracts in early life • • • • • • Gal 1-P uridyltransferase deficiency Cataracts early in life Vomiting, diarrhea (post-lactose ingestion) Lethargy Liver damage (hyperbilirubinemia) Mental retardation • In the well-fed state, galactose can enter glycolysis or contribute to glycogen storage • Administration of galactose during hypoglycemia induces an increase in blood glucose BC06_4-1 Fructose Metabolism Fructokinase Aldolase B • FA 2013: 104.2 • FA 2012: 108.2 • FA 2011: 103.1 • ME 3e: 38 • ME 4e: 38 BC06_4-2 Pyruvate Dehydrogenase • PDH is an irreversible enzyme → cannot be used to convert Acetyl-CoA to pyruvate or glucose Cofactors • Thyamine pyrophosphate • Lipoic acid Irreversible reaction • Coenzyme A • FAD (H2) from riboflavin • NAD (H2) from niacin Tender Loving Care For Nancy • FA 2013: 100.3 • FA 2012: 104.3 • FA 2011: 99.1 • ME 3e: 41 • ME 4e: 41 + Insulin (liver) ↑ADP ↑ Ca+ levels ↑NAD+/NADH ratio BC06_5-1 Pyruvate Dehydrogenase Deficiency • Accumulation of substrates: pyruvate and alanine → Lactic acidosis Clinical manifestations • Neurological defects (e.g microcephaly, mental retardation) Treatment • Diet limited to ketogenic nutrients • Diet high in fat content • Diet rich in lysine, leucine Microcephaly Commons.wikimedia.org Used with permission • FA 2013: 101.1 • FA 2012: 105.1 • FA 2011: 99.2 • ME 3e: 41 • ME 4e: 41 BC06_5-2 Citric Acid Cycle Acetyl- CoA NAD + FAD +GDP + Pi 2CO2 NADH + FADH2 +GTP • All enzymes located in the mitochondrial matrix except succinate dehydrogenase No hormonal control • FA 2013: 101.3 • FA 2012: 105.3 • FA 2011: 100.1 • ME 3e: 39 • ME 4e: 39 BC06_6-1 Electron Transport Chain NADH + O2 FADH2 + O2 NAD + H2O FAD + H2O ∆G = -56 kcal/mol ∆G = -42 kcal/mol NADH • ATP / mitochondrial NADH • or ATP / cytoplasmic NADH (depends on shuttle) FADH2 • ATP • FA 2013: 102.2 • FA 2012: 106.1 • FA 2011: 100.2 • ME 3e: 40 • ME 4e: 40 BC06_6-2 Electron Transport Chain Mitochondrial Matrix Inner Membrane H+ Pathways NADH NADH Dehydrogenase Complex I NAD Succinate Dehydrogenase e FADH2 Complex II e e - e - - Fatty Acyl Coenzyme Q CoA (ubiquinone) Dehydrogenase e FADH2 Cyto b/c1 Complex III H+ e - H+ H+ Cyto a/a3(Cu+) Complex IV Cytochrome Oxidase H20 F1 ADP H+ ATP + 2,4-DNP • FA 2013: 102.2 • FA 2012: 106.1 • FA 2011: 100.2 • ME 3e: 40 • ME 4e: 40 Glycerol-P Shuttle FADH2 H+ H+ H+ H+ + H H+ + H+ + H H+ + H+ H+ H H H+ H+ H+ Cyt C - Cyanide CO - Olygomicin F0 ATP Synthase Pi - - e O2 Cytoplasm Side Barbiturates Intermembrane Space Rotenone (an insecticide) NADH Dehydrogenase Complex I Energy lost as heat without ATP synthesis ATP ADP Pi Uncouplers 2,4-DNP + H+ Aspirin (high dose) Thermogenin (brown adipose) BC06_6-3 Pathologies Affecting the Electron Transport Chain Mitochondrial Matrix Inner Membrane H+ Pathways NADH NADH Dehydrogenase Complex I NAD Sources of CO poisoning • Propane heaters • Gas grills • Vehicle exhaust • Tobacco smoke • House fires • Methylene based paint H+ strippers Treatment • Hyperbaric O2 Succinate Dehydrogenase e FADH2 Complex II e e - - Fatty Acyl Coenzyme Q CoA (ubiquinone) Dehydrogenase e FADH2 Cyto b/c1 Complex III e - H+ H+ Cyto a/a3(Cu+) Complex IV Cytochrome Oxidase H20 F1 ADP ATP + 2,4-DNP • FA 2013: 102.2 • FA 2012: 106.1 • FA 2011: 100.2 • ME 3e: 40 • ME 4e: 40 Glycerol-P Shuttle FADH2 H+ H+ H+ H+ + H H+ + H+ + H H+ + H+ H+ H H H+ H+ H+ Sources of Cyanide poisoning • Polyurethane • Nitroprusside Treatment • Nitrites Cyt C - Cyanide CO - Olygomicin F0 ATP Synthase Pi - - e O2 H+ e - Cytoplasm Side Barbiturates Intermembrane Space Rotenone (an insecticide) NADH Dehydrogenase Complex I Energy lost as heat without ATP synthesis ATP ADP Pi Uncouplers 2,4-DNP + H+ Aspirin (high dose) Thermogenin (brown adipose) BC06_6-4 Uncoupling Mitochondrial Matrix Inner Membrane H+ Pathways NADH NADH Dehydrogenase Complex I NAD Succinate Dehydrogenase e FADH2 Complex II e e - e - - Fatty Acyl Coenzyme Q CoA (ubiquinone) Dehydrogenase e FADH2 Cyto b/c1 Complex III H+ e - H+ H+ Cyto a/a3(Cu+) Complex IV Cytochrome Oxidase H20 F1 ADP H+ ATP + 2,4-DNP • FA 2013: 102.2 • FA 2012: 106.1 • FA 2011: 100.2 • ME 3e: 40 • ME 4e: 40 Glycerol-P Shuttle FADH2 H+ H+ H+ H+ + H H+ + H+ + H H+ + H+ H+ H H H+ H+ H+ Cyt C - Cyanide CO - Olygomicin F0 ATP Synthase Pi - - e O2 Cytoplasm Side Barbiturates Intermembrane Space Rotenone (an insecticide) NADH Dehydrogenase Complex I Energy lost as heat without ATP synthesis ATP ADP Pi Uncouplers 2,4-DNP + H+ Aspirin (high dose) Thermogenin (brown adipose) BC06_6-5 Glycogen Characteristics • Branched polymer of glucose (storage form) • Synthesis and degradation occurs primarily in liver and skeletal muscle • Stored in cytoplasm • Liver glycogen is mobilized during hypoglycemia to supply other tissues • Muscle glycogen is energy reserve for muscle contraction • FA 2013: 110.2 • FA 2012: 114.2 • FA 2011: 109.2 • ME 3e: 43 • ME 4e: 43 BC06_7-1 Glycogen Synthesis and Breakdown • FA 2013: 111.1 • FA 2012: 115.1 • FA 2011: 110.1 • ME 3e: 43 • ME 4e: 43 BC06_7-2 Glycogen Storage Diseases Type Deficient Enzyme Clinical Features Glycogen Structure Normal I: von Gierke Glucose-6-phosphatase Severe hypoglycemia, lactic acidosis, hepatomegaly, hyperlipidemia, hyperuricemia, short stature, doll-like facies, protruding abdomen, emaciated extremities II: Pompe Lysosomal α1,4glucosidase Cardiomegaly, muscle weakness, death by years Glycogen-like material in inclusion bodies III: Cori Glycogen debranching enzyme Mild hypoglycemia, hepatomegaly Short outer branches Single glucose residue at outer branch IV: Andersen (amylopectinosis) Branching enzyme Infantile hypotonia, cirrhosis, death by years Very few branches, especially toward periphery V: McArdle Muscle glycogen phosphorylase Muscle cramps and weakness on exercise, myoglobinuria Normal VI: Hers Hepatic glycogen phosphorylase Mild fasting hypoglycemia, hepatomegaly, cirrhosis Normal • FA 2013: 111.2 • FA 2012: 115.2 • FA 2011: 110.2 • ME 3e: 43 • ME 4e: 43 BC06_7-3 Glycogen Storage Disease Type I: Von Gierke’s Disease Glucose 6-phosphatase Glucose 6-P Glucose Enzyme deficiency: glucose-6-phosphatase in liver Signs and symptoms • Severe fasting hypoglycemia • Accumulation of glycogen in the liver → hepatomegaly • Lactic acidosis • Hyperlipidemia (secondary to hypoglycemia) • Hyperuricemia • FA 2013: 111.2 • FA 2012: 115.2 • FA 2011: 110.2 • ME 3e: 43 • ME 4e: 43 BC06_7-4 Glycogen Storage Disease Type II: Pompe’s Disease Enzyme deficiency: lysosomal α-1,4- glucosidase- enzyme found in lysosomes which participates in digestion of glycogen-like material Signs and symptoms • Hepatomegaly • Cardiomegaly • Cardiac failure • Death before years of age Computerized tomography of hepatomegaly Commons.wikimedia.org Used with permission • FA 2013: 111.2 • FA 2012: 115.2 • FA 2011: 110.2 • ME 3e: 43 • ME 4e: 43 BC06_7-5 Glycogen Storage Disease Type III: Cori’s Disease Enzyme deficiency: glycogen debranching enzyme Signs and symptoms • Accumulation of excessive amounts of glycogen with altered structure • Branched a1,6 branched glucoses are not removed • Hypoglycemia, hyperlipidemia and failure to thrive • Myopathy, hypotonia • Cardiomyopathy • FA 2013: 111.2 • FA 2012: 115.2 • FA 2011: 110.2 • ME 3e: 43 • ME 4e: 43 BC06_7-6 Glycogen Storage Disease Type IV: Andersen’s Disease Enzyme deficiency: glycogen branching enzyme → formation of abnormal glycogen with long and unbranched outer chains and decreased solubility Signs and symptoms • Muscle weakness • Exercise intolerance • Muscle atrophy • Dilated cardiomyopathy • Heart failure • Progressive liver failure • Cirrhosis Dilated cardiomyopathy Commons.wikipedia.org Used with permission • Death by age unless liver transplantation performed • FA 2013: 111.2 • FA 2012: 115.2 • FA 2011: 110.2 • ME 3e: 43 • ME 4e: 43 BC06_7-7 Glycogen Storage Disease Type V: McArdle’s Disease Enzyme deficiency: skeletal muscle glycogen phosphorylase (also known as myophosphorylase) • Affected individuals are unable to break down glycogen to glucose 6-phosphate in muscle Signs and symptoms • Exercise intolerance • Muscle cramping • Myoglobinuria (with strenuous exercise) Characteristic brown discoloration as a result of myoglobinuria Commons.wikipedia.org Used with permission • FA 2013: 111.2 • FA 2012: 115.2 • FA 2011: 110.2 • ME 3e: 43 • ME 4e: 43 BC06_7-8 Glycogen Storage Disease Type VI: Hers Disease Enzyme deficiency: hepatic glycogen phosphorylase Signs and symptoms • Mild hypoglycemia • Hyperlipidemia • Hyperketosis • Hepatomegaly • Growth retardation in early childhood • FA 2013: 111.2 • FA 2012: 115.2 • FA 2011: 110.2 • ME 3e: 43 • ME 4e: 43 BC06_7-9 Gluconeogenesis Insulin ATP Glucagon + PFK-2 + - Fructose 2,6 –bis P - AMP Fructose 2,6-BP Substrates CO2 Malate • Glycerol 3-phosphate (from triacylglycerol in adipose tissue) • Lactate (from anaerobic glycolysis) • Gluconeogenic amino acids (from muscle protein) • FA 2013: 102.2 • FA 2012: 106.2 • FA 2011: 101.1 • ME 3e: 44 • ME 4e: 44 BC06_8-1 Hexose Monophosphate Shunt • Occurs in cytoplasm of all cells • Serves as two major functions • NADPH production • Source of ribose 5-phosphate for nucleotide synthesis • FA 2013: 103.2 • FA 2012: 107.2 • FA 2011: 102.1 • ME 3e: 42 • ME 4e: 42 BC06_9-1 Role of HMP Shunt in Hepatocytes, Phagocytes and RBCs • FA 2013: 103.2 • FA 2012: 107.2 • FA 2011: 102.1 • ME 3e: 42 • ME 4e: 42 BC06_9-2 Glucose 6-Phosphate Dehydrogenase Deficiency • X-linked recessive disease with significant heterogeneity (most common enzyme deficiency) • Heinz bodies due to hemoglobin precipitation in the presence of reactive oxygen species • Episodic hemolysis, characterized by anemia, hemoglobinuria and jaundice • Acute, episodic hemolysis after oxidizing agents: fava beans, sulfonamides, primaquine, anti-TB drugs, dapsone, infections • Malaria resistance (Plasmodium deficient in antioxidant mechanisms) • Increased susceptibility to catalase + infections → tetrazolium test • FA 2013: 104.1 • FA 2012: 108.1 • FA 2011: 102.2 • ME 3e: 42 • ME 4e: 42 negative nitroblue BC06_9-3 Alcohol Metabolism • ↑NADH level favors • Pyruvate → lactate ↓Gluconeogenesis • OAA → malate • DHAP → Glycerol 3-P Lipid accumulation in alcoholic liver disease NAD NADH Lactate Pyruvate Lactate dehydrogenase • FA 2013: 95.3 • FA 2012: 100.1 • FA 2011: 94.3 • ME 3e: 13 • ME 4e: 13 BC06_9-4 Biochemistry Lipid Metabolism Megan Murray M.D., Ph.D Candidate University of Buffalo Fatty Acid Biosynthesis • Fatty acids: long-chain mono-carboxylic acids • Unsaturated FA: contains carbon-carbon double bonds • Saturated FA: no double bonds • Palmitate: only fatty acid humans can synthesize de novo • FA 2013: 113.1 • FA 2012: 117.2 • FA 2011: 112.1 • ME 3e: 50 • ME 4e: 50 BC07_1-2 Lipid Storage • Triglycerides: storage form of fatty acids • Attachment of fatty acids to glycerol • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 51 • ME 4e: 51 BC07_1-3 Glycerophospholipids Functions • Membrane synthesis • Producing a hydrophilic surface layer on lipoproteins • Reservoir of second messengers such as DAG IP3 Arachidonic acid • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: 50 • ME 4e: 50 BC07_1-4 Lipoprotein Metabolism Types of lipoproteins (in order from least dense to most dense) Density • Chylomicrons • VLDL • IDL • LDL • HDL • FA 2013: 116.1 • FA 2012: 121.1 • FA 2011: 115.1 • ME 3e: NA • ME 4e: NA BC07_2-1 Classes of Lipoproteins and Important Apoproteins Lipoprotein Functions Apoproteins Functions Chylomicrons Transport dietary triglyceride and cholesterol from intestine to tissues apo B- 48 apo C- II apo E Secreted by intestine Activates lipoprotein lipase Uptake of remnants by the liver VLDL Transports triglycerides from liver to tissues apo B- 100 apo C- II apo E Secreted by liver Activates lipoprotein lipase Uptake of remnants (IDL) by liver IDL (VLDL remnants) Picks up cholesterol from HDL to become LDL Picked up by liver apo E apo B- 100 Uptake by liver apo B-100 Uptake by liver and other tissues via LDL receptor ( apoB-100 receptor) apoA-1 Activates lecithin cholesterol acetyltransferase (LCAT) to produce cholesterol esters LDL Delivers cholesterol into cells HDL Picks up cholesterol accumulating in blood vessels Delivers cholesterol to liver and steroidogenic tissues via scavenger receptor (SR-B1) Shuttles apoC-II and apoE in blood • FA 2013: 116.1 • FA 2012: 121.1 • FA 2011: 115.1 • ME 3e: 53 • ME 4e: 53 BC07_2-2 Lipoprotein Metabolism: Chylomicrons and VLDL • FA 2013: 115.1 • FA 2012: 120.1 • FA 2011: 114.1 • ME 3e: 53 • ME 4e: 53 BC07_2-3 Lipoprotein Metabolism: LDL and HDL • FA 2013: 115.1 • FA 2012: 120.1 • FA 2011: 114.1 • ME 3e: 53 • ME 4e: 53 BC07_2-4 Regulation of Cholesterol Level in Hepatocytes • FA 2013: 114.2 • FA 2012: 119.2 • FA 2011: 113.1 • ME 3e: 52 • ME 4e: 52 BC07_2-5 LDL, HDL, and Atherogenesis Endothelial damage • Blood flow turbulence • Homocysteinemia • ↑ oxidized LDL particles • HTN • Free radical generation • Diabetes • FA 2013: NA • FA 2012: NA • FA 2011: NA • ME 3e: NA • ME 4e: NA BC07_2-6 Primary Hyperlipidemias Type Deficiency Familial lipoprotein lipase (rare) apo C-II (rare) Autosomal recessive I ↑ lipid in Blood Triglyceride ↑ lipoprotein in Blood Comments Chylomicrons Red-orange eruptive xanthomas Fatty liver Acute pancreatitis Abdominal pain after fatty meal High risk of atherosclerosis and CAD Homozygous condition, usually death