Congenital Right to Left Shunts

Healthy Heart
Typically, post-natal systemic and pulmonary circulation run in parallel and maintain separation between low and high oxygen blood. When separation is incomplete, low and high oxygen blood mix, which produces systemic blood with insufficient oxygen concentrations.

Review fetal circulation & adult circulation

Right to Left Shunts

  • Diagnosed prenatally or soon after birth.
  • Are characterized by early cyanosis.
    • So-called “blue babies” have hypoxemia because systemic blood bypasses the lungs.

The Great Vessels

  • The aorta carries blood with a high oxygen concentration to the body tissues.
  • The pulmonary trunk carries blood with a low oxygen concentration to the lungs.
  • Ductus arteriosus
    • Allows blood to flow from pulmonary trunk to aorta, bypassing the lungs.
    • The ligamentum arteriosus is the adult remnant of the ductus arteriosus.

Persistent Truncus Arteriosus

  • Caused by a malformed or absent aortico-pulmonary septum that fails to form separate outflow tracts for systemic and pulmonary circulations.
  • Thus, blood from each ventricle enters the common vessel, largely bypassing the lungs.
  • The defect is attributed to disturbances in secondary heart field or cardiac neural crest formation, and, therefore, is often associated with ventricular septal defect (recall the aorticopulmonary septum contributes to the membranous portion of the interventricular septum.

Transposition of the Great Vessels

  • Occurs when the conotruncal ridges fail to spiral, and the aorticopulmonary septum creates two distinct vessels.
  • The right ventricle continuously pumps low-oxygen blood to the body through the aorta, while the left ventricle continuously pumps high oxygen blood to the lungs through the pulmonary trunk.
  • Thus, blood from the right heart never enters the lungs for re-oxygenation, and blood from the left heart never reaches body tissues.
  • Newborn viability depends on accompanying septal defects and patent ductus arteriosus to provide opportunities for blood mixing.

Tricuspid Atresia

  • The right atrioventricular valve, aka, tricuspid valve, doesn’t properly form. Instead of ensuring unidirectional blood flow from the right atrium to the right ventricle, it blocks flow.
  • Associated characteristics are septal defects and/or patent ductus arteriosus that allow blood mixture, and a hypoplastic (smaller than usual) right ventricle.
  • Corrective surgeries are necessary to establish healthy blood flow.

Tetralogy of Fallot

  • Venous blood from the right heart and mixed blood from the left heart are pumped through the pulmonary trunk and aorta.
    Characterized by 4 defects:
    • Pulmonary stenosis (narrowing of pulmonary trunk)
    • Rightward displaced aorta, with opening over right ventricle (aka, overriding aorta)
    • Ventricular septal defect, specifically, of the membranous portion
    • Right ventricular hypertrophy, which occurs in response to increased work load to pump blood through stenotic pulmonary trunk

Total Anomalous Pulmonary Venous Return

  • Characterized by pulmonary veins that drain into the right atrium via the coronary sinus, superior vena cava, brachiocephalic vein, etc.
    Example:
  • Paired right and left pulmonary veins also draining into the right atrium via the coronary sinus.
    • In this case, viability requires an accompanying shunt that allows oxygenated blood to reach the left side of the heart.

Excision Repair

EXCISION REPAIR MECHANISMS

Mismatch-repair

• Fixes replication errors missed by DNA Pol proofreading(cannot repair damage)

Base excision repair

• Damaged bases removed and replaced
• Deamination & depurination (most common spontaneous damages)
• Alkylation (except for O6-alkyl guanine)
• Oxidation

Nucleotide excision repair

• Damaged nucleotides removed and replaced
• 2 types: global genomic NER (transcriptionally inactive) & transcription-coupled
• Repairs wider variety of lesions that distort DNA helix
• Pyrimidine dimers and other intrastrand adducts
• Bulky group addition (via carcinogen-exposure)

BASE EXCISION REPAIR
• Model: cytosine spontaneously deaminates to uracil

  1. DNA glycosylase excises uracil: cleaves N-glycosidic bond between base & deoxyribose
    • Produces apyrimidinic site (AP site)
  2. AP endonuclease cleaves deoxyribose backbone adjacent to AP site
    • Excises deoxyribose moiety
  3. DNA polymerase and ligase fill/seal gap with cytosine
    • Prokaryotes: DNA Pol. I
    • Eukaryotes: DNA Pol. beta

NUCLEOTIDE EXCISION REPAIR
• Model: thymine dimer in E. Coli (prokaryotes)

Global genomic NER: transcriptionally inactive region

• Uvr genes: discovered in E. Coli, mutations conferred extreme UV light sensitivity

  1. UvrA recognizes helical distortion as damage
  2. UvrA recruits uvrB and uvrC

Intertextual variation exists for above step.

  1. Uvr B and C cleave DNA on either side of damage
    • Uvr ABC complex: excinuclease
  2. Helicase unwinds DNA and releases damaged segment
  3. DNA polymerase fills gap and ligase seals it
    • DNA Pol. fills gap in 5′ to 3′ direction
    • Prokaryotes: DNA Pol. I
    • Eukaryotes: DNA Pol. delta and epsilon

Transcription-coupled NER: transcriptionally active region

• Repairs more rapidly than global genomic pathway

  1. NER enzymes recognize RNA polymerase stalled by lesion and displace it
    • E. Coli: NER enzymes are Mfd proteins
    • Humans: RNA polymerase II (prokaryotes only have one RNA Pol.) & CS proteins
  2. Mfd proteins (E. Coli) and CS proteins (humans) recruit other proteins to site
    • E. Coli: Mfd proteins recruit uvr proteins
    • Humans: CS proteins recruit more CS and XP proteins (amongst others)
  3. Recruited proteins cleave and excise damaged oligomer
  4. Identical to Step 5 in global genomic pathway

CLINICAL CORRELATION

Xeroderma pigmentosum (XP)

• Rare genetic defect produces dysfunctional XP proteins
• Patients extremely sensitive to UV light: develop skin cancers in sun-exposed areas

Mismatch Repair

DNA REPLICATION: FACTORS THAT INCREASE FIDELITY
• Increase fidelity to 1 mistake per 10^9 bases added

  1. DNA Pol. proofreading capacity
    • DNA Pol. 3′ to 5′ exonuclease activity: can excise/replace incorrect nucleotides
    • Prokaryotes: DNA Pol. I (replaces RNA primers) & III (daughter strands) are exonucleases
    • Eukaryotes: DNA Pol. delta (lagging strand) & epsilon (leading strand) are exonucleases
  2. Repair mechanisms

Mismatch repair: identifies/fixes replication errors that escape DNA Pol.

• Addresses single-strand breaks in newly replicated DNA
• Cannot repair DNA Damage

MISMATCH REPAIR: E. COLI
• Mismatched nucleotides cannot H-bond –> distorts DNA
• GATC sequence occurs about every thousand nucleotides
• A in GATC is methylated: distinguishes parent strand from daughter strand
• Mut proteins (mismatch repair enzymes in E. Coli)

  1. MutS recognizes mismatched base and initiates repair by binding
  2. MutS forms complex with MutL
  3. MutL binding activates endonuclease MutH
  4. MutH cleaves daughter strand opposite to adenine-methylation (GATC)
  5. Exonuclease and helicase excise portion of daughter strand

Intertextual variation exits regarding above step.

  1. DNA Pol. (III or I) fills gap and ligase seals ends w/ phosphodiester bonds
    • DNA Pol synthesizes gap in 5′ to 3′ direction

MISMATCH REPAIR: HUMANS
• MSH proteins: human homologs for Mut proteins (no MutH homolog)
• Daughter strand specificity poorly understood: not adenine-methylation

Theoretical recognition sites: breaks in lagging strand & lengthening 3′ end of lagging strand.

• DNA Pol. (delta and epsilon) fill excised gap

CLINICAL CORRELATION

Lynch Syndrome

• Formerly known as hereditary nonpolyposis colorectal cancer (HNPCC)
• Mutation in human Mut homologs: defective mismatch repair pathway
• Increased risk for colorectal and other cancers
• ~ 3 in every 100 cases of colon cancer caused by Lynch syndrome

Mitosis versus Meiosis

KEY DIFFERENCES BETWEEN MITOSIS AND MEIOSIS

  1. Parent cell types
  • Mitosis: diploid somatic cell
  • Meiosis: diploid germ line cell (reproductive cell precursor)
  1. Tetrad formation (Meiosis only)
  • Prophase I
  1. Crossing over (Meiosis only)
  • Chiasmata: site of genetic recombination, occurs in prophase I
  1. Synaptonemal complex (Meiosis only)
  • Zipper-like protein structure that holds homologues together: specific to meiosis I
  1. Kinetochore orientation
  • Mitosis: sister chromatid kinetochores face opposite poles
  • Meiosis I: sister chromatid kinetochores face the same pole
  • Meiosis II: sister chromatid kinetochores face opposite poles
  1. Genetic variability (Meiosis only)
  • Crossing over (genetic recombination)
  • Random fertilization
  • Independent assortment: each tetrad positions itself on the metaphase plate independently of other tetrads
  • Meiosis produces four genetically distinct haploid daughter cells
  • Mitosis produces two genetically identical diploid daughter cells

Meiosis Part II

HUMAN CELLS

  1. Somatic cells: majority of the body’s cells
  • 46 chromosomes – diploid (2n)
  1. Reproductive cells: sperm and egg cells (gametes)
  • 23 chromosomes – haploid (1n)
  • Diploid germ line cells: precursors for reproductive cells; undergo meiosis

FERTILIZATION

  • 1 egg and 1 sperm fuse to form zygote (2n)
  • Followed by repeated cycles of mitosis to produce multicellular organism (2n)

INTERPHASE

  • Parent cell (2n): two sets of 23 chromosomes
  • Homologous chromosomes: contain the same genes in the same order, each from a different parent (contain different alleles)
  • S-phase: each set of 23 chromosomes duplicates (92 chromosomes total), sister chromatids pair at the centromere

PROPHASE I

  • >90% of meiosis
  • Chromosomes condense
  • Tetrad forms via synapsis: each gene aligns with its homologue (4 chromatids)
  • Synaptonemal complex: zipper-like structure holds chromosomes together until crossing over occurs
  • Crossing over: paternal chromosome crosses over to maternal and vice versa
  • Chiasma (site of crossing over) holds tetrad together after synaptonemal complex disassembles

Other features of this phase:

  • Nuclear envelope fragments
  • Nucleolus disperses
  • Centrosomes move to opposite poles
  • Microtubules form spindle & attach kinetochores of homologous chromosomes

METAPHASE I

  • Tetrads align on metaphase plate
  • Sister chromatids face same pole
  • Homologous chromosomes face opposite poles

ANAPHASE I

  • Homologous chromosomes separate

TELOPHASE I AND CYTOKINESIS

  • Two haploid daughter cells: 1 tetrad in each

PROPHASE II

  • Each cell has one duplicated set of 23 chromosomes

METAPHASE II

  • Sister chromatids line up on metaphase plate and face opposite poles

ANAPHASE II

  • Sister chromatids separate

TELOPHASE II

  • Nuclear envelope reforms
  • Nucleolus reappears
  • Mitotic spindles depolymerize
  • Cleavage furrow

CYTOKINESIS

  • 4 haploid daughter cells
  • Daughter cells genetically distinct from each other and parent cells
  • Each develops into reproductive cell (egg or sperm cells)

CLINICAL CORRELATION
Down’s Syndrome (Trisomy 21): aneuploid gametes

  • Nondisjunction: chromosome 21 fails to separate properly during meiosis I
  • 2 daughter cells with extra chromosome 21 copy
  • 2 daughter cells missing chromosome 21
  • Trisomy 21: gamete with extra chromosome fuses with normal gamete during fertilization = zygote with 3 copies of chromosome 21

Meiosis Part I

HUMAN CELLS

  1. Somatic cells: majority of the body’s cells
  • 46 chromosomes – diploid (2n)
  1. Reproductive cells: sperm and egg cells (gametes)
  • 23 chromosomes – haploid (1n)
  • Diploid germ line cells: precursors for reproductive cells; undergo meiosis

FERTILIZATION

  • 1 egg and 1 sperm fuse to form zygote (2n)
  • Followed by repeated cycles of mitosis to produce multicellular organism (2n)

INTERPHASE

  • Parent cell (2n): two sets of 23 chromosomes
  • Homologous chromosomes: contain the same genes in the same order, each from a different parent (contain different alleles)
  • S-phase: each set of 23 chromosomes duplicates (92 chromosomes total), sister chromatids pair at the centromere

PROPHASE I

  • >90% of meiosis
  • Chromosomes condense
  • Tetrad forms via synapsis: each gene aligns with its homologue (4 chromatids)
  • Synaptonemal complex: zipper-like structure holds chromosomes together until crossing over occurs
  • Crossing over: paternal chromosome crosses over to maternal and vice versa
  • Chiasma (site of crossing over) holds tetrad together after synaptonemal complex disassembles

Other features of this phase:

  • Nuclear envelope fragments
  • Nucleolus disperses
  • Centrosomes move to opposite poles
  • Microtubules form spindle & attach kinetochores of homologous chromosomes

METAPHASE I

  • Tetrads align on metaphase plate
  • Sister chromatids face same pole
  • Homologous chromosomes face opposite poles

ANAPHASE I

  • Homologous chromosomes separate

TELOPHASE I AND CYTOKINESIS

  • Two haploid daughter cells: 1 tetrad in each

PROPHASE II

  • Each cell has one duplicated set of 23 chromosomes

METAPHASE II

  • Sister chromatids line up on metaphase plate and face opposite poles

ANAPHASE II

  • Sister chromatids separate

TELOPHASE II

  • Nuclear envelope reforms
  • Nucleolus reappears
  • Mitotic spindles depolymerize
  • Cleavage furrow

CYTOKINESIS

  • 4 haploid daughter cells
  • Daughter cells genetically distinct from each other and parent cells
  • Each develops into reproductive cell (egg or sperm cells)

CLINICAL CORRELATION

Down’s Syndrome (Trisomy 21): aneuploid gametes

  • Nondisjunction: chromosome 21 fails to separate properly during meiosis I
  • 2 daughter cells with extra chromosome 21 copy
  • 2 daughter cells missing chromosome 21
  • Trisomy 21: gamete with extra chromosome fuses with normal gamete during fertilization = zygote with 3 copies of chromosome 21

Membrane Asymmetry

MEMBRANE COMPONENTS

  • Phospholipids: synthesized on the cytosolic face of the ER
  • Glycolipids
  • Membrane proteins

MEMBRANE SYNTHESIS

  1. Phospholipids are synthesized on the cytosolic face of the ER and glycolipids on the lumenal face of the Golgi apparatus
  2. Vesicles bud from organelles and transport them to cell membrane
  3. Vesicles fuse with cell membrane the lipids they transport retain same orientation unequal distribution of molecules generates curvature
  4. Flippases flip some phospholipids to the extracellular face of bilayer
  5. Glycolipids remain on extracellular face (no flippase action)

TOPOLOGICALLY EQUIVALENT SPACES
Endoplasmic reticulum
Golgi Apparatus
Vesicles
Extracellular space

MEMBRANE LIPIDS DISTRIBUTION

Extracellular layer

  • Phosphatidylcholine: most common, structural
  • Sphingomyelin: less abundant, variable head groups
  • Glycolipids: carbohydrate attached to membrane lipid

Cytosolic layer

  • Phosphatidylethanolamine: small head group that generates curvature
  • Phosphatidylinositol: minor lipid, binds proteins (signal transduction)
  • Phosphatidylserine: binds proteins to membrane
    Variable head groups & fatty acid tail length/saturation

CLINICAL CORRELATION

Apoptosis (programmed cell death)

  • Phosphatidylserine in extracellular leaflet of bilayer is signal for phagocytosis

Membrane Proteins Overview

FREEZE-FRACTURE METHOD

  • Freeze cell and fracture it along cell membrane’s hydrophobic interior
  • Proteins associate with either layer after fracturing
  • More proteins associate with cytosolic layer

INTEGRAL PROTEINS

  • Embedded in the bilayer

Transmembrane proteins: amphipathic, pass through both membrane layers

  • Single pass or multi-pass
  • Alpha helices: hydrophobic side chains
  • Beta barrel: multi stranded beta sheet (i.e. porin proteins)

Monolayer associated

  • Alpha helix
  • Lipid-linked

PERIPHERAL PROTEINS

  • Do not extend into the bilayer
  • Protein-attached: non-covalently bound to transmembrane protein
  • Oligosaccharide-attached: bound to carbohydrate head group of glycolipid

Glycocalyx

  • Oligosaccharide side chains and glycolipids form carbohydrate coat on external surface of cell

MEMBRANE PROTEIN FLUIDITY

  1. Fuse mouse and human cells with surface marker proteins
  2. Marker proteins mix on hybrid cell surface
  • Conclusion: membrane proteins are fluid

MEMBRANE PROTEIN FUNCTIONS

  • Transport ions, nutrients and other substances across membrane
  • Anchor cells to each other, to extracellular matrix or basement membrane
  • Transduce external signals to inside of cell
  • Mediate cell-cell recognition of glycoproteins on adjacent cell surfaces
  • Enzymatically catalyze metabolic pathways

Membrane Structure Overview

PLASMA MEMBRANE

  • Phospholipid bilayer: bilayer that comprises mostly phospholipids
  • Fluid mosaic: mosaic of proteins embedded within a fluid phospholipid bilayer
  • Selectively permeable: some substances move through passively, others use proteins for transport

MEMBRANE COMPONENTS

  • Phospholipids
  • Proteins
  • Cholesterol
  • Carbohydrates

PHOSPHOLIPIDS

  • Amphipathic: hydrophilic head and hydrophobic fatty acid tails
  • Form liposomes in aqueous environment
  • Weak hydrophobic interactions = membrane fluidity
  • Saturated phospholipids: maximize hydrogens in fatty acid tails, no kinks
  • Unsaturated phospholipids: double bond produces kink, increases fluidity

CHOLESTEROL

  • Temperature buffer
  • Moderate temperature: decreases fluidity, lessens lateral movement
  • Low temperature: increases fluidity, prevents solidification

PROTEINS

  • Includes transmembrane proteins that span the bilayer (other types exist)
  • Proteins provide about half the mass of the membrane

CARBOHYDRATES

  • Glycoproteins: branched carbohydrates covalently bound to proteins
  • Glycolipids: carbohydrates covalently bound to lipids (extracellular only)

CLINICAL CORRELATION:

Blood types

  • Carbohydrates on surface of red blood cells must be compatible between donor & recipient in blood transfusion

FUNCTIONS OF THE CELL MEMBRANE

  • Cell communication
  • Import and export of molecules
  • Cell growth
  • Cell motility

Eukaryotes have internal membranes within the cell, prokaryotes do not.

Fluorescence Microscopy

FLUORESCENCE MICROSCOPY APPLICATIONS

Fluorescence recovery after photobleaching (FRAP)

  • Used to study membrane fluidity

Fluorescence resonance energy transfer (FRET)

  • Used to study protein-protein interactions

METHOD

  1. Light passes through excitation filter
  2. Excitation filter filters out undesired wavelengths of light
  3. Mirror deflects light downward toward sample
  4. Light passes through the objective lens and onto specimen of interest
  5. Molecules in sample absorb light & emit light with longer wavelength (fluoresce)
  6. Fluorescent light travels back upward and passes through mirror w/o deflecting
  7. Barrier filter above the mirror lets fluorescent light through
  8. Fluorescence observed

FRAP

  1. Tag membrane proteins with fluorophore (i.e. GFP)
  2. Irreversibly bleach portion of membrane with laser (photobleaching)
  3. Measure rate at which membrane recovers fluorescence (proportional to rate at which tagged molecules diffuse back into bleached area)

FRET

  1. Tag one protein with blue GFP and another with green GFP
  2. Shine violet light on sample

If the proteins interact (i.e they come in close proximity):

  • Blue light from blue GFP excites green GFP
  • Green light observed

If the proteins do not interact:

  • Blue light observed (not absorbed and reemitted by green GFP)