Week 5 Random Flashcards
Two types of on/off swithces
Protein kinease / Protein phosphatase (signalling by phosphorylation) = covalent
GTP binding (GEF) / GTP hydrolysis (GAP) (signaling by GTP-binding) = noncovalent
Why molecular swiches are necessary?
Allow integradation of signal at signal processor
Cdk Kinase requirements for activation
Has three conditions in order for Cdk kinease to take signal downstream (2 phsophates and cyclin)
Where can the intracellular signalling complex assemble?
Type and precisioon of response to signalling molecules
Steroid are usually gradual response
Cooperativity might stimulate quicker response (all or nothing like)
Who positive feedback can affect response?
Can accelearate response
How desintization can occur?
Recepotor sequestration
Receptor down-regulation
Receptor inactivation
Inactivation of signalling protein
Productionof inhibitory proteins
How penile erection occur?
Intracellular receptor
Neuron releases ACh -> Activates NO Synthase ->
arginine converted to NO (endothelial) ->
NO diffusion goes to smooth muscle cell activates to gyanylyl cyclase ->
GTP is converted to cGMP and relaxes smooth muscle
Activation of PKG (phosphorylation) to vascular smooth muscle relaxationand blood vessel dilation
PDE inibitors type V
Levitra
Cialis
Viagra
PDE type 5 action
Prevent cGMP conversionto GMP
Some examples of molecules that bind intracellular recepotrs
Cortisol
Estradiol
Testosterone
Thyroxine
Vitamin D3
Retinoic Acid
Machanism of hormone receptor activating transcription
Two types of cellular response to signals
Altered protein function by intracellular singalling pathway (fast sec-min)
Altered gene expression (mins to ours)
Cell membrane receptors
GPCR’s = Ga, Gi, Golf, Gt, Gq
Ras = MAP kinease
Enzyme-linked receptors = PI3 kinease, PLC-g, IP3, Ca2+, SRc, Jak-STATs, NF-kB
(-mab) in the name of the drug
Humainzied monoclone antibody
Drugs inactivating NFkB signalling
Remicaid, Humira, Cimzia, Enbrel, Simponi
Communication in cells is necessary for:
- Regulate development and organization of tissues
- Control their growth and division
- Coordinationof their functionwith each other
Order the signalling pathways from shortest distance to longest
Synaptic
Paracrine
Autocrine
Endocrine
How is the effector different in endocrine vs. synaptic signalling?
In endocrine signalling, the receptor sees mix of signals while in synaptic signalling, the receptor only sees the specific or limited signals.
Why autocrine signalling evolved?
The strength of the signal might be beneficial
Autocrine signalling is important for development and during immune system development
Importance of eicosanoids
Inhibitors of eicosanoids synthesis
Names of enzymes that are involved oxidation
What prostaglanding mediate
Eicosanoids (signaling molecules made by oxidation of 20-carbon fatty acids)
Inhibitors of eicosanoids include cortisone and Nonsteroidal anti-inflammatory drugs (NSAIDs) like ibuprofen
COX1 and COX2 are enzymes incyclooxygenase dependent pathway
Prostaglandin regulates inflamatory response
Problems with COX-2 inibitors?
Multiple sides efects: CELEBREX, and Vioxx (withdrawn)
Endocrine vs. Synaptic signalling affinity / length
Endocrine: Low ligand conc
Synaptic: High lingad conc >10^-4 M; low affinity; quick termination
Prozac
Inhibits seritonin uptake that allows maintenance of seritonin concentration in synapse
Anti-depressant
Selective serotonin reuptake inhibitors (SSRIs)
They act within the brain to increase the amount of serotonin
For anxiety disorders, obsessive-compulsive disorder, and eating disorders.
Effective in treating premature ejaculation in up to 60% of men.
How cells can respond to signals
Survive
Grow + Divide
Differentiate
Die
How same cell can respond to different signals?
Ach signal
1) Skeletal muscle = contraction
2) Heart muscle = relaxation (different receptors)
3) Salivary gland = secretion (same receptor as heart, but different protome)
Extracellular matrix
Components and types
Sugars and Proteins
Interstitial (surrounds in tissues and abundant in connective tissues)
Basement membrane (sheet of fibers that underlie the epithelium)
Functions:
Collagen
Fibronectin
Laminin
Elastin
Glycosaminoglycans
Proteoglycans
Collagen – ropelike fibers that give tissue tensile strength.
Fibronectin and Laminin – glycoproteins that link the extracellular fibers to the cells.
Elastin – thin fibers that give tissue elasticity.
Glycosaminoglycans – extracellular polysaccharides of defined sequence.
Proteoglycans – gel like slimy mucus that hydrates the space between cells.
Collagen Synthesis
Prepocollagen made in ER
(1) Signal sequence is removed -> polyproline helix
(2) Intra chain dilsufide bonds form with a-chains
(3) ER: Various prolines and lysines are hydroxylated by prolyl or lysyl hydroxylases (Vit C)
(4) ER: Collagen is O-glycosylated on hydroxylysines and N-glycosylated on asparagines with galactose or galactosyl-glucose.
(5) ER: The 3 α-chains assemble into a soluble right handed super helix.
(6) Procollagen is secreted
(7) Extracellular proteases cleave the ends generating collagen molecules (tropocollagen).
(8) Fibrils self-assemble into insoluble fiber complexes in a quarter staggered array.
(9) Fibrils are covalently cross-linked
Lysyl oxidase – oxidative deamination of lysine and/or hydroxylysine (Cu2+ dependent ). Followed by an aldol condensation.
More crosslinking = more rigid
Collagen
where is it found?
of types?
examples?
Bones, tendons, and skin
Most common protein in mammals (25-30%)
28 types
examples:
Collagen I, II, and III (90%)
Collagen IV - basal lamina
Non-collagen collagens - C1q, pulmonary surfactant proteins (SPD, SPA)
Pyrimidine synthesis
Source of atoms in pyrimidine synthesis
Name enzymes
ATP + AMP -> 2ADP
ATP + NMP -> ADP + NDP
ATP + NDP -> ADP + NTP
UTP -> CTP
Adenylate kinease
Nucleoside monophosphate kineases
Nucleoside duphosphate kineases
Cytidylate Synthetase
Salvage pathway
Names of enzymes in salvage pathway that form XMP
Defect in one of them
Pyrimidines: orotic acid transferase (existing)
Adenine: adenine phospho ribosyl transferase (A-PRT)
Guanine/Hypoxanthine: G/HX phospho ribosyl transferase (G/HX-PRT)
Lesch-Nyhan syndrome: defect in G/HX-PRT
Source of atoms in purine synthesis
How AMP and GMP are formed from IMP?
Regulation by ATP / GTP levels
How deoxyribonucleotides are synthesized from ribonucleotides?
How the reaction is driven?
By NADH
GMP and AMP syntehsis regulation
General regulation of nucleotide synthesis
Thymidine Synthesis
Cystonie and Uracil degradation
Thymidine degradation
GMP and AMP degradation
Gout
Cause?
Treatments?
The average uric acid concentrations in humans is near the solubility limit. This has a selective advantage because uric acid is a highly effective scavenger of reactive oxygen species. However, if at low pH uric acid crystals form, they can irritate joints and cause gout.
Treatments:
Colchicine: anti-inflammatory
Probenecid: Increases uric acid excretion
Allopurinol: Xanthine oxidase inhibitor
Cobalamine absorption
Reactions that B12 catalyzes
Lack of B12?
B12 (required by methylmalonyl CoA mutase and methionine synthetase)
Megaloblastic anemia. Neurological dysfunction. Deficiency of folate.
Pernicious anemia: autoimmune disease destroys parietal cells.
Treat with B12 supplements or monthly injections.
Folate Importance
B9 vs. B12 deficiency
Folate deficiency
Deficiency? (1) Folate deficiency decreases purine and dTMP synthesis, arresting cell cycle in the S-phase and resulting in megaloblastic anemia. (2) Hyperhomocysteinemia with increased risk for cardiovascular disease. (3) Deficiency in pregnancy can lead to neural tube defects (spina bifida) in baby.
Why needed? THF; one carbon carrier involved in amino acid metabolism and nucleotide synthesis
Source: Yeast, liver, fruits, green vegetables
ECM functions
Connecting cells together
Guides cell migration (e.g. would healing)
Relay of environmental signals
Collagen I
Charactersitics
Left handed tripple helix of three
Repetitive AA sequence: (Gly-X-Y)n
33% Glycine and about 20.5% Proline/Hydroxyproline at X and Y respectively.
3 α-chains assemble into a right handed super helix.
Mechanism of collagen quaternary structure assembly
Lysyl oxidase – oxidative deamination of lysine and/or hydroxylysine (Cu2+ dependent ).
Followed by an aldol condensation
Diseases associated with collagen mutations
Basal Membrane vs. Basal Lamina
The epithelial ECM the term “basement membrane” is used with light microscopic observation and “basal lamina” is used with electron microscopy.
Basement Membrane = Basal Lamina + Retircular Lamina
Basal lamina characteristics
(about 40–120 nm thick) consists of fine protein filaments embedded in an amorphous matrix.
Membrane proteins of the epithelial cells are anchored in the basal lamina, which is also produced by the epithelial cells.
major component of the basal lamina are two glycoproteins - laminin and (usually type IV) collagen
Reticular lamina charactersitics
consists of reticular fibers embedded in ground substance.
fibers of the reticular lamina connect the basal lamina with the underlying connective tissue.
components of the reticular lamina are synthesized by cells of the connective tissue underlying the epithelium.
Basement membrane compnents
- Collagen IV
- Laminin
- Heparin Sulfate
- Proteoglycans
Laminin (Ln) characteristics
A glycosylated cross-shaped heterotrimer.
A multi-adhesive ECM component enriched at the basal lamina where it binds cells to collagen IV and integrins.
Multiple isoforms.
In general, Ln is associated with cell differentiation.
Laminin vs Lamin A vs Lamina
Laminin 2 – basement membrane protein that links integrin (or dystroglycan) to ECM components.
Lamin A is a nuclear envelope protein that forms filaments.
Lamina is the ECM component of basement membranes seen by electron microscopy (basal lamina).
Elastin + Fibrillin characteristics
Elastic fibers that allow tissue to expand and contract.
Abundant in blood vessels, lung and skin.
Highly cross-linked, insoluble, amorphous structure rich in proline (11%), alanine (22%) and glycine (31%).
Like collagen, lysyl oxidase initiates crosslinking of allysines.
The crosslinks formed are called a desmosine.
Defects in elastin cause Williams-Beuren syndrome and plays a causative role in aortic stenosis.
Elastic Fiber synthesis
Problems with fibrillin?
Marfan Syndrome: defects in fibrillin 1 gene.
Glycosaminoglycans (GAGs) types
Hyaluronan (or hyaluronic acid) at the cell surface
main glycosaminoglycan in connective tissue
high molecular weight (~ MW 1,000,000 )
length of about 2.5 µm hyaluronan
“backbone” for the assembly of other glycosaminoglycans
Hyaluronan is also a major component of the synovial fluid, which fills joint cavities, and the vitreous body of the eye.
Other:
(attach through core and link proteins to hyaluronic acid backbone)
Chondroitin sulphate
Dermatan sulphate
Keratan sulphate
Heparan sulphate (UK sulphate, US sulfate)
What are gags made of?
Unbranched polysaccharides of repeating disaccharide units built from amino sugars and uronic acids.
Formerly: Mucopolysaccharides
How proteoglycans are formed?
GAGs are attached to protein cores.
The proteins can be bound to Hyaluronan
Function of GAGs and PGs
Provide flexible mechanical support to tissues.
Acts as a molecular sieve allowing the diffusion of small molecules but slowing the diffusion of proteins and the movement of cells.
Acts as a lubricant in joints and tissues subject to friction and compression/extension forces.
Binds and sequesters soluble ECM proteins thereby maintaining high local concentrations at the cell surface.
Mucopolysaccharidoses
Lysosomal storage diseases – cannot degrade GAGs
Fibronectin (Fn)
A glycosylated multi-adhesive protein found in connective tissues and plasma.
Encoded by 1 gene with alternative splicing.
The plasma form is soluble.The ECM form is a fiber of 2 polypeptide chains disulfide linked at the C-terminus.
The RGD sequence binds to both cell surface receptors (integrins) linking cells to ECM components.
Functions of Fibernectin
Development—essential for the migration of cells along fibers.
Wound healing – covalently links to fibrin clots where it attracts fibroblasts and endothelial cells to promote healing.
Cancer – malignant cells lack cell surface Fn and migrate. Their Fn receptors bind to ECM Fn at distant sites facilitating metastasis and cell division.
4.In general, Fn is associated with cell proliferation and migration.
Cell surface receptors that mediate to the ECN and neighbouring cells
Dystroglycan Complex and Integrins
Integrins
Heterodimeric (a-b)trans-membrane glycoprotein receptors.
Modulate ECM deposition.
Modulate gene/protein expression.
Bidirectional signaling
Inside → Out & Outside → In
Integrins have differing affinities for ECM
Activated Integrins functions
Regulate gene expression.
Alter cytoskeletal organization.
Recruit additional integrins to the cell membrane.
Promote cellular growth (hypertrophy).
Influence cell survival. With normal cells Attached = survive Detached = apoptosis.
Promote ECM deposition.
Matrix Metalloproteinases
>20 different MMPs exist in humans
They are zinc containing proteases that degrade all proteins found in the ECM.
They allow cell migration and tissue remodeling during development, in response to injury or as needed.
Degradation of ECM allows the release of growth factors sequestered in the ECM.
Diseases asscoiated with ECM proteins
- *Scurvy** – abnormal collagen-OH (Vitamin C)
- *Osteogenesis Imperfecta** – Collagen
- *Supravalvular aortic stenosis** – Elastin insufficiency
- *Marfan** Syndrome – **Fibrillin **
- *Mucopolysaccharidoses** – **Proteoglycans **
Why anticipation and repeat expansion are non-mendelain inheritance?
Mutations are not stable, they change every generation.
Repeat Expansion and Anticipation examples
**Myotonic Dystrophy **
Decreases RNA stability.
Autosomal Dominant
Huntington Disease
Gain of function mutation.
Fragile X Mental Retardation
Inhibits transcription
Anticipation definition
some dominant disorders manifest at an earlier age of onset and with increasing severity in successive generations
Trinucleotide Repeat Expansion
Triplet repeats in certain regions of the DNA are unstable.
In normal individuals the trinucleotides are repeated a variable
but low number of times (instability is in meiosis)
Each allele varies within the normal range each generation. They are not inherited in a simple Mendelian fashion
If the repeat expands just beyond the normal range, the repeat becomes unstable and expands in subsequent generations (premutations).
Anticipation
Myotonic dystrophy
1/8000 individuals
* * Most common muscular dystrophy in adults** (Skeletal muscle deterioration (starting with face), cardiac and smooth muscle affected as well. ) * * Myotonia** (inability to relax muscles), cataracts, and mild **mental retardation** are also seen.
Disease gets worse in successive generations.
Congenital form seen only in infants of affected mothers.
CTG repeats: Normal range: 5-35 repeats. Premuation 50-100. Premutations: dynamic increases in the number of repeats in succeeding generations. Expansion occurs during gametogenesis in females (only)
Severity of disease is correlated with the number of repeats.
Hunington
Autosomal Dominant
1/20,000
Progressive loss of motor control and psychiatric problems including dementia and affective disorder.
Usually presents between ages 30 and 50. Protracted disease, 15 years from diagnosis to death.
100% Penetrant
Leads to toxic aggregates of huntingtin. Binds to other proteins such as GAPDH and inhibits them. DN
Greater expansion when transmitting parent is male.
Normal alleles have 6-30 CAG repeats (6-30 aa poly-glutamine stretch). Each allele varies within this range each generation.
Fragile X Syndrome characteristics
Most common form of inherited mental retardation.
X-linked dominant inheritance; Milder and more variable expression in females than in males.
Overall 80% penetrant in males, 20% penetrant in females. X-inactivation
Name comes from the artifact
NTM: normal transmitting male; no risk of having affected child; expansion occur only in females
CGG 6-55 repeats; Premutation 52-200; Mutation 200+
Expansionoccurs exclusively through mother
100% penetrance in males with full mutation
50% penetrance in heterozygote female with one full mutation
What are the effects of mitochondrial disorder?
The tissues most dependent on oxidative phosphorylation:
heart, skeletal muscles, and CNS.
Mitochondrial disorders often manifest as: myopathies (muscle), neuropathies (neurons), and encephylopathies (brain).
Mitochondrial function are affected by:
Inherited capacity for oxidative phosphorylation-both nuclear and mitochondrial genes.
Tissue specific requirements for oxidative phosphorylation.
Age- capacity for oxidative phosphorylation decreases with age (accumulation of mtDNA mutations).
Accumulation of somatic mtDNA mutations and degree of heteroplasmy.
Mitochondrial disorders are often progressive or do not manifest until adulthood.
Leber’s hereditary optic neuropathy (LHON):
Mitochondrial disorder with rapid loss of central vision due to death of the optic nerve.
Delayed age of onset, 20-30 years old.
95% of cases caused by one of three missense mutations in a mitochondrial protein (mtDNA protein complex I).
MERRF (myoclonic epilepsy with ragged-red fiber)
Four “canonical” features: Myoclonus, generalized epilepsy, ataxia, ragged-red fibers (RRF) in the muscle biopsy
Frequent manifestations: Sensorineural hearing loss, peripheral neuropathy, dementia, short stature, exercise intolerance, optic atrophy
Mutation: single base changes in mitochondrial tRNA molecules that change their codon specificity.
Heteroplasmy
MELAS
mitochondrial encephalomyopathy and stroke-like episodes
Heteroplasmy
mutations in mitochondrial tRNA that change codon specificity
Mitosis requirements
Condensation
Nuclear envelope (phosphorylation of lamins)
ER/Golgi fragmentation
Cells loosens extraceullar adhesions
Cytoskeleton transformed
Cohesins
Condensin
Cohesins cross-link two adjacent sister chromatids, gluing them together.
Centrosome
Microtubule Organizing Center
MTOC
centrosomes at their – ends. + ends grow outwards towards the cell periphery.
Before a cell divides it must duplicate its centrosome to provide one for each daughter cell.
Centriole
Perpendicular cylindrical pairs
Cytoplasmic organelles origin during mitosis
Golgi and ER break up into a set of smaller fragments
ER vesicles seem to associate with microtubules
Organelles like mitochondria cannot assemble spontaneously. They arise from growth and fission of existing organelles.
Microtubulles types
Astral
Kinetochore
Overlap
MAPS
Microtubule associated proteins (allow extension of microtubules)
Catastrophins (Depomylerization)
Separation of the two spindle poles
Kinesin
Overlaping MTs
Anaphase A/B
(A) Shortening of kinetochore microtubules (depolymerization)
(B) Kinesin driven movement over overlap
Checkpoints in cell cycle
Enter mitosis
Exit mitosis
Enter S phase
Two key components of the cell-cycle controlled system
Cyclin-dependent kinease (Cdk)
Cyclin (oscillates)
Regulates of Cdk by ihibitory phosphorylation
Wee1 kinease (mutations cause uncontrolled replications making small cells)
Cdc25 phosphatase
CKI
Regulate Cyclin-cdk complexes
CDK INHIBITOR PROTEINS
p27
Rate limiting step in cyclin destruction
final ubiquitin transfer step by enzymes known as ubiquitin ligases
In G1 and S phase SCF is responsible for the ubiquitylation and destruction of G1/S cyclins and certain CKI proteins that control S phase initiation.
In M Phase, the anaphase–promoting complex (AMP) is responsible for the ubiquitylation of M-cyclins.
Initiation of DNA replication cycle
How is re-replication block insured?
Origin recognitioncomplex recognizes the ORC binding site
Cdc6-MCM forming pre-replicative complex
Cdc degradation
Origin recognitioncomplex becomes phosphorylated
(1) S-Cdk activtity remains high during G2
(2) M-Cdk ensures re-replication by phopshorylating Cdc6 and Mcm
The activation of M-phase
MCdk’s; Cyclin D
Actions of M-Cdk
1) Induce assembly of the mitotic spindle
2) Ensure connection to the spindle
3) Chromosomal condensaion, nuclar envelope ect.
M-cyclin destruction
Destruction of M-cyclin is not required for sister-chromatid separation but is required for the subsequent exit from mitosis.
Mechanisms of sister chromatids separation
Control of G1 progression
G1 is held by active Sic1 and Hct1-APC
Mechanisms of S-phage initiation
DNA damage arrests the cell cycle
Phosphorylation of p53 leading to transcription of CKI p21
Overview of the cell-cycle
What growth factors control?
Example?
Regulation of cell growth or division.
Proliferation of cells.
Survival.
Migration
Physiological function of cells.
e.g. mitogen->RAS->myc
Cell determination
Cells retain a record of signals their ancestors received in early embryonic environment.
Cell determination before differentiation
Two ways of making sister cells different
Asymmetric cell division
Inducitve interactions (also concentration dependent; called morphogens); limited time and space; patterning by sequential induction)
Two ways to create a morphogen gradient
Gradient of inducer source
Gradient of inhibitor source
How embryo can be polar?
Fertilization triggers 2 types of intracellular movements.
Cell cortex rotation through 30 degree relative to the core of the egg in a direction determined by sperm entry.
Active transport of Dishevelled protein, a component of the Wnt signaling pathway.
The resultant Dorsal concentration of Dishevelled protein defines the dorsoventral polarity.
Stem cells
It is not itself terminally differentiated = Self renewal
It can divide without limit
Potency = The capacity to differentiate into any specialized cell.
When it divides, each daughter has a choice: it can either remain a stem cell, or it can embark on a course that commits it to terminal differentiation
Totipotency
Pluripotent
iPS
Multipotent
Totipotency = ALL of the cells in the body
Pluripotent = the potential to divide into any of the three germ layers
iPS = Induced pluripotent stem cells also referred to as iPSCs which are artificially derived from an non-pluripotent cell (somatic cell)
4 cell types in the gut
- Absorptive cells - brush border cells or enterocytes.
- Goblet cells (as in respiratory epithelium) secrete mucus.
- Paneth cells form part of the innate immune defense system.
- Enteroendocrine cells, of more than 15 different subtypes, secrete serotonin and cholecystokinin (CCK).
Wnt/Notch signalling can alter formation of secreting or absorbin cell
deltaG° = RTln(eq)
simply
deltaG° = -1364log(eq) cal/mol
High Energy Compounds values
Esters (amide)
Thiol-Esters (acetyl CoA)
Anhydrides (ATP)
Guanidinium phosphate (CP)
Enoyl Phosphate (PEP)
NADH
FADH2
Esters (amide): -3 kcal/mole
Thiol-Esters (acetyl CoA): -6 to -8 kcal/mole
Anhydrides (ATP): -7 to -10 kcal/mole
Guanidinium phosphate (CP): -10 kcal/mole
Enoyl Phosphate (PEP): -14 kcal/mole
NADH oxidation (to NAD+) -15 kcal/mole
FADH2 oxidation (to FADH+) -15 kcal/mole
Sources / Fates of Pyruvate?
Soruces / Fates of Acteyl CoA?
Co-Factors of Pyruvate Dehydrogenase
When it is inactive?
Thiamine PyroPhosphate
Lipoic Acid
FAD
* inactive when phosporylated
contains kinease and phosphatase
Pyruvate Dehydrogenase Deficiency
symptoms?
diet?
treatment?
elevated serum levels of lactate, pyruvate, and alanine.
acidotic
low in carbohydrates
dichloroacetate (phosphatase)
Order of substrates and enzymes in Krebs cycle
At which step is GTP / FADH2 / NADH produced?
Which steps are irreversible?
Which is the rate limiting step?
Which moleucules of Krebs cycles are exported out of the mitochondria and used as a regulatory signals?
Where and what signals control Krebs cycle?
Electron flow
Reduction potential, Eo
deltaG° = -nFdeltaE°
Flavoproteins
examples?
of electrons that they can accept?
Ubiquinone
nickname?
of electrons it can accept?
location?
Coenzyme Q or just Q
1-2 electrons
Freely diffusible within inner membrane billayer
Heme in cytochromes
oxidation?
Iron Sulfur Centers :)
Fe2+ Fe3+
Respirasome
Recent studies support the idea that complex I, III, and IV are associated
Complex 1,2,3,4 composition
Disease associated with heme b (complex 2)
(1) six iron-sulfur centers and FMN-containing flavoprotein
(2) heme b (binding site for Q) - prevent e- leak, iron-sulfur centers
(3) bc1 ‘ ubiquinone:cytochrome c oxidoreductase
(4) Cu ions, heme groups
paraganglioma (tumor in head and neck)
Which part is F1 and which F0?
Which one is rotating and which one synthesizes ATP?
The top is F1, and the bottom F0
F0 rotating
F1 ATP synthesis
How NADH is transported to mitochondria from cystol? (in liver, kidney, and heart)
Malate-Aspartate shuttle
How NADH is transported to mitochondria from cystol? (in muscle and brain)
glycerol 3 phosphate shuttle
Molecule that is found in brown adipose tissue
Thermogenin
Uncouples reacitons
Drugs that interfere with oxidative phosphorylation?
Cyanide, carbon monoxide – cytochrome oxidase inhibition
Rotenone, amytal – prevent e- transfer from Fe-S to Q
Oligomycin – inhibitor of ATP synthase
Loss of a copies in thalassemia a 2, 3, 4?
2 = a thalassemia trait: asymptomatic, but can detect biochemically, reduced RBC size.
3 = Hemoglobin H disease: moderate to marked anemia.
a0 thalassemia (Hb Barts, HYDROPS FETALIS)- lethal.
Loss of alleles in thalassemia b?
b-thalassemia minor 1 mutant allele
b-thalassemia trait intermediate expression
b-thalassemia major 2 mutant allele, 0 or little expression
Direct testing vs. linkage analysis vs. biochemical testing vs. cytogenetic testing.
Direct testing - analyzing DNA base by base
Linkage analysis - looking at the marker and linkage association
Biochemical tests are screen for enzymes proteins (karyotyping, FISH, CHIP, CFFDNA)
Which types of tests are used for prenatal and neonatal screening?
Biochemical and Cyotgenetic testing
Define sequence analysis
When it is used?
Difficulties? (BRAC1)
Sequencing
Selected regions possibly causing problems are selected.
Difficulties (BRAC1) can identify unknown benign polymorphism or mutation that increases risk. Likely to reveal numerous variants. Ambigiuity.
Sequence analysis vs. Mutation analysis
Sequencing a segment of DNA identifies most variations from the wild-type.
In contrast, mutation analysis identifies only specific targeted mutations within a given segment of DNA.)
Reasons why sequence alteration might not be detected
Not covered by lab test
Mutation that cannot be detected (large deletion)
Mutation causing disease might be in another gene (locus heterogenity)
Define mutation scanning (exon scanning)
When use it?
Examples?
Exons (coding regions within a gene) are subjected to physical tests to confirm the presence of a mutation before sequencing is used to delineate the exact mutation
When use it: If a gene has many possible mutations; reduces amount of DNA to be sequenced
Methods used include: conformation sensitive gel electrophoresis (CSGE), single-stranded conformational-polymorphism (SSCP), and denaturing gradient gel electrophoresis (DGGE).
Define Targeted Mutation Analysis
When use it?
Difficulties? (CF)
Testing for a specific muation (Glu6Val for sickle cell anemia), OR specific type of mutation (trinucleotide repeat expansion in HD or MD, deletions in DMD), OR set of mutations (CF e.g. microarray)
When use it: for diseases with common disease causing allele
Difficulties: The mutational analysis may not identify uncommon alleles (2%)
Define deletion/duplication analysis: (copy number analysis)
Examples of methods?
Clinical mportance?
A process to detect deletions/duplications of an entire exon, multiple exons, or the whole gene that typically are not identifiable by sequence analysis of genomic DNA.
Examples: Methods include: quantitative PCR, real-time PCR, multiplex ligation dependent probe amplification (MLPA; multiplex quantitative PCR- up to 40 sequences), and array CGH (comparative genomic hybridization; gene CHIPs).
Clinical importance: testing heterozygotes ex. Williams, spinal muscular atrophy, X-linked disorder
Clinical uses of genetic testing
Diagnostic testing- confirm/rule out a genetic disorder in symptomatic individual.
Predictive testing- offered to asymptomatic individuals with a family history of genetic disorder (presymptomatic vs. predispositional).
Carrier testing- performed to identify individuals who carry a mutation for an autosomal or x-linked recessive disorder.
Prenatal testing- performed during a pregnancy to assess the health status of a fetus.
Preimplantation testing- performed on early embryos resulting from in vitro fertilization.
Newborn screening- to help identify individuals with genetic diseases to start treatment as soon as possible
Austism
Heritable causes?
Chromosomal: prader-willi/angelman, down
Single gene: Fragile-X, Rett syndrome, Tuberous sclerosis, Sotos
Mitochondrial:
Techniques that can be used to detect disease causing genetic mutations
DNA testing- detection of variation at the DNA level. sequencing, Southern blotting, PCR
RNA detection- changes in transcription of specific genes (promotor mutations). Northern blotting, RT-PCR
Protein electrophoresis- hemoglobinapathies; changes in protein structure (charge or size).
Protein detection- antibodies used to detect changes in protein abundance. ELISA, western blotting, immuno-histochemistry
Biochemical assays- inborn errors of metabolism (neonatal screening). Measure analytes, enzyme assays
Cytogenetics- changes in chromosomes (# or structure). Spectral karyotyping, FISH, G-band karyotyping
What can be used to detect promoter mutations?
Protein assays and RNA assay
Use of Northern
RNA size and abundance
Use of Southern
Genomic DNA followed by restirction digest
Southern/Northern vs. PCR
S/N: require specific probe; 1-20kb; only large differences
PCR: small amount of DNA; no probe;
Duchene Muscular Dystrophy
Causing mutation?
Issues with detection?
How is the disease analyzed DNA/protein level?
Heterozygote issues?
Only 1/10 is disease causing; large gene to sequence
Multiplex PCR of 9 exons; Immunofluoresence
Cannot detect heterozygote just by normal PCR
Hemoglobinopathies
qualitative (SCA) and quantitative disorders of hemoglobin (all forms abab).
Thalassemias
Globin chain imbalances
Two ways that sickle cells can be detected
Protein electrophoresis
Allelic specific nucleotides
Hemoglobin lepore:
Testing?
It is a type of β thalassemia allele
Unequal crossing over between d and b genes generates hybrid db and bd genes.
Hb lepore (db) is functionally active, but expressed at low levels due to fetal promotor
1) Targeted mutation analysis (population appropriate),
2) Mutation scanning or sequence analysis.
Repeat Expansion and Anticipation
diseases and their cause
Myotonic Dystrophy - after stop codon 3’UTR decreases mRNA stability
Hunington - expansion in coding region
Fragile X - before 5’ UTR -> inhibits transcription due to hypermethylation
How is Myotonic dystrophy assayed?
Southern bloot
How Huntington disease is assayed
Fragile X synrome
Fragile X = Most common form of inherited mental retardation.
Down syndrome is the most common genetic cause of mental retardation.
