Step 1 First Aid – Biochemistry

Chromatin structure
Negatively charged DNA loops twice around histone octamer (2 each of the positively charged H2A, H2B, H3, and H4) to form nucleosome bead. H1 ties nucleosomes together in a string. (Think of “beads on a string”; H1 is the only histone that is not in the nucleosome core.) In mitosis, DNA condenses to form mitotic chromosomes.

Heterochromatin
Condensed, transcriptionally inactive (“H eteroC hromatin = H ighly C ondensed.”)

Euchromatin
Less condensed, transcriptionally active (Eu = true, “truly transcribed”)

Purines
A, G 2 Rings (“PUR e A s G old = PUR ines”)

Pyrimidines
C, T, U 1 ring (“CUT the PY (pie): PY rimidines”)

Functional groups of the nucleosides
Guanine has a ketone. Thy mine has a methy l. Deamination of cytosine makes uracil.

Base differences btw RNA and DNA
Uracil is found in RNA; Thymine in DNA

Base pair bonds
G-C bond (3 H-bonds) is stronger than A-T bond (2 H-bonds). Incr G-C content —< higher melting temperature.

AA’s necessary for purine synthesis
G lycine A spartate G lutamine

Nucleoside
Base + ribose

Nucleotide
Base + ribose + phosphate; linked by 3′-5′ phosphodiester bond.

Purines are made from…?
IMP precursor (see bottom/right)

Pyrimidines are made from…?
Orotate precursor, with PRPP added later.

Deoxyribonucleotide synthesis
Ribonucleotides are synthesized first and are converted to deoxyribonucleotides by ribonucleotide reductase.

ABX and anti-neoplastic drugs that function by interfering w/ nucleotide synthesis (list)
Hydroxyurea 6-mercaptopurine 5-fluorouracil Methotrexate Trimethoprim

Hydroxyurea
Inhibits ribonucleotide reductase.

6-mercaptopurine (6-MP)
Blocks de novo purine synthesis.

5-Fluorouracil (5-FU)
Inhibits thymidilate synthase (decr dTMP).

Methotrexate
Inhibits dihydrofolate reductase (decr dTMP)

Trimethoprim
Inhibits bacterial dihydrofolate reductase (decr dTMP)

Transition vs. transversion
Transition: Substituting purine for purine or pyrimidine for pyrimidine (“TransI tion = I dentical type”) Transversion: Substituting purine for pyrimidine or vice versa (“TransV ersion = conV ersion btw types”)

Genetic code: unambiguous
Each codon specifies only 1 AA.

Genetic code: degenerate/redundant
< 1 codon may code for the same AA. (Methionine is encoded by 1 codon: AUG)

Genetic code: Commaless, nonoverlapping
Read from a fixed starting point as a continuous sequence of bases. *some viruses are an exception.

Genetic code: universal
Genetic code is conserved throughout evolution. *exceptions include mitochondria, archaebacteria, Mycoplasma , and some yeasts

Silent mutation
Same AA, often base change in 3rd position of codon (tRNA wobble)

Missense mutation
Changed AA (conservative — new AA is similar in chemical structure)

Nonsense mutation
Change resulting in early stop codon (“Stop the nonsense !”)

Frame shift mutation
Change resulting in misreading of all nucleotides downstream, usually resulting in a truncated, nonfunctional protein.

Severity of damage in DNA mutations
Nonsense < missense < silent

Eukaryotic vs. prokaryotic DNA replication.
Eukaryotic DNA replciation is more complex, but uses many analogous enzymes. In both: DNA replication is semiconservative and involves both continuous and discontinuous (Okazaki fragment) synthesis. For eukaryotes, replication begins at a consensus sequence of base pairs.

Origin of replication
Particular sequence in genome where DNA replication begins. May be single (prokaryotes) or multiple (eukaryotes).

Replication fork
Y-shaped region along DNA template where leading and lagging strands are synthesized.

Helicase
Unwinds DNA template at replication fork.

Single-stranded binding protein
Prevents strands from reannealing.

DNA topoisomerases
Create a nick in the helix to relieve supercoils created during replication. *Fluoroquinolones inhibit DNA gyrase (a specific prokaryotic topoisomerase)

Primase
Makes an RNA primer on which DNA polymerase III can initiate replication.

DNA polymerase III
Prokaryotic only. Elongates leading strand by adding deoxynucleotides to the 3′ end. Elongates lagging strand until it reaches primer of preceding fragment. 3′–<5' exonuclease activity "proofreads" each added nucleotide. (5'--<3' synthesis; 3'--<5' proofreading w/ exonuclease)

DNA polymerase I
Prokaryotic only. Degrades RNA primer and fills in the gap w/ DNA. (excises RNA primer w/ 5′–<3' exonuclease)

DNA ligase
Seals.

Single strand nucleotide excision repair
Specific endonucleases release the oligonucleotide-containing damaged bases; DNA polymerase and ligase fill and reseal the gap, respectively. (mutated in xeroderma pigmentosum)

Xeroderma pigmentosum
Mutated single strand nucleotide excision repair gene, which prevents repair of thymidine dimers.; Dry skin w/ melanoma and other cancers (“children of the night”).

Single strand base excision repair
Specific glycosylases recognize and remove damaged bases, AP endonuclease cuts DNA at apyrimidinic site, empyty sugar is removed, and the gap is filled and resealed.

Single strand mismatch repair
Unmethylated, newly synthesized string is recognized, mismatched nucleotides are removed, and the gap is filled and resealed. Mutated in hereditary nonpolyposis colorectal cancer (HNPCC).

Double strand nonhomologous end joining
Brings together 2 ends of DNA fragments. No requirement for homology.

What direction is DNA/RNA made?
They are both synthesized in the 5′–<3' direction. Remember that the 5' of the incoming nucleotide bears the triphosphate (energy source for bond). The 3' hydroxyl of the nascent chain is the target.

What direction is mRNA read?
5′–<3'.

What direction is protein synthesized?
N–

3 Types of mRNA
rRNA is the most abundant mRNA is the longest tRNA is the smalles (“R ampant, M assive, T iny”)

mRNA start codon
AUG (or rarely GUG) (“AUG inAUG urates protein synthesis”) In eukartyotes, codes for methionine, which may be removed before translation is completed. In prokaryotes, codes for formyl-methionine (f-Met).

mRNA stop codons
UGA, UAA, UAG UGA = U G o A way UAA = U A re A way UAG = U A re G one

Functional organization of the gene

Promoter
Site where RNA polymerase and multiple other transcription factors bind to DNA upstream from gene locus (AT-rich upstream sequence w/ TATA and CAAT boxes). Mutation here commonly results in dramatic drop in amount of gene transcribed.

Site where RNA polymerase and multiple other transcription factors bind to DNA upstream from gene locus (AT-rich upstream sequence w/ TATA and CAAT boxes). Mutation here commonly results in dramatic drop in amount of gene transcribed.
Promoter

Enhancer
Stretch of DNA that alters gene expression by binding transcription factors.

Stretch of DNA that alters gene expression by binding transcription factors.
Enhancer

Silencer
Site where negative regulators (repressors) bind.

Site where negative regulators (repressors) bind.
Silencer

Where are enhancers and silencers located?
May be close to, far from, or even within (in an intron) the gene whose expression it regulates.

Eukaryotic RNA polymerases
RNA pol I — makes rRNA RNA pol II — makes mRNA RNA pol III — makes tRNA (I, II, and III are numbered as their products are used in protein synthesis) No proofreading fxns, but can initiate chains. RNA pol II opens DA at promoter site.

Prokaryotic RNA polymerase
One RNA polymerase (a multisubunt complex) makes all of the 3 kinds of RNA.

alpha-amantin
Found in death cap mushrooms. Inhibits RNA pol II.

RNA processing (in eukaryotes)
Occurs in nucleus. After transcription: 1.) Capping on 5′ end (7-methylguanosine) 2.) Polyadenylation on 3′ end (~200 A’s) 3.) Splicing out of introns Only processed RNA is transported out of the nucleus.

hnRNA vs. mRNA
The initial transcript is called heterogeneous nuclear RNA (hnRNA) The capped and tailed transcript is called mRNA.

Polyadenylation signal
AAUAAA

Poly-A polymerase does not require…
…does not require a template.

pre-mRNA splicing (occurs in eukaryotes)
1.) Primary transcript combines w/ snRNPs and other proteins to form spliceosome 2.) Lariat-shaped intermediate is generated 3.) Lariat is released to remove intron precisely and join 2 exons.

Introns vs. exons
Exons contain the actual genetic information coding for protein. Introns are intervening noncoding segments of DNA. (“IN trons stay IN the nucleus, whereas EX ons EX it and are EX pressed”)

Alternative splicing
Different exons are combined to make unique proteins in different tissues (e.g., beta-thalassemia mutations)

tRNA structure
75-90 nucleotides, secondary structure, cloverleaf form, anticodon end is opposite 3′ aminoacyl end. All tRNAs, both eukaryotic and prokaryotic, have CCA at 3′ end along w/ a high percentage of chemically modified bases. The AA is covalently bound to the 3′ end of tRNA.

Charging of tRNA
Aminoacyl-tRNA synthetase (1 per AA, “matchmaker,” uses ATP) scrutinizes AA before and after it binds to tRNA. If incorrect, bond is hydrolyzed. The aa-tRNA bond has energy for formation of peptide bond. A mischarged tRNA reads usual codon but inserts wrong AA.

What is responsible for the accuracy of AA selection?
Aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible for accuracy of AA selection.

Mechanism of tetracyclines
Bind 30S subunit, preventing the attachment of aminoacyl-tRNA.

tRNA wobble
Accurate base pairing is required only in the first 2 nucleotide positions of an mRNA codon, so codons differing in the 3rd “wobble” position may code for the same tRNA/aa (due to degeneracy of genetic code).

Protein synthesis: initiation
Activated by GTP hydrolysis, initiation factors (eIFs) help assemble the 40S ribosomal subunit w/ the initiator tRNA released when the mRNA and the ribosomal subunit assemble w/ the complex. E ukaryotes: 40S + 60S = 80S (E ven) PrO karyotes: 30S + 50S = 70S (O dd)

Protein synthesis: step 1 in elongation
Aminoacyl-tRNA binds to Aa site (except for initiator methionine)

Protein synthesis: step 2 in elongation
Peptidyltransferase catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site.

Protein synthesis: step 3 in elongation
Ribosome advances 3 nucleotides toward the 3′ end of RNA, moving peptidyl RNA to P site (translocation)

mnemonic for the 3 sites in the ribosome
“going APE ” A site = incoming A minoacyl tRNA P site = accommodates growing P eptide E site = holds E mpty tRNA as it E xits

Protein synthesis: termination
Completed protein is released from ribosome thru simple hydrolysis and dissociates.

Aminoglycosides as protein synthesis inhibitors
Inhibit formation of the initiation complex and cause misreading of mRNA.

Chloramphenicol as a protein synthesis inhibitor
Inhibits 50S peptidyltransferase.

Macrolides as protein synthesis inhibitors
Bind 50S, blocking translocation.

Clindamycin as a protein synthesis inhibitor
Binds 50S, blocking translocation.

Energy requirements of translation
tRNA aminoacylation: ATP —< AMP (2 phosphoanhydride bonds) Loading tRNA onto ribosome: GTP --< GDP Translocation: GTP --< GDP Total energy expenditure = 4 high-energy phosphoanhydride bonds

Posttranslational modifications: trimming
Removal of N- or C-terminal propeptides from zymogens to generate mature proteins.

Posttranslational modifications: covalent alterations
Phosphorylation, glycosylation, and hydroxylation.

Posttranslational modifications: Proteasomal degradation
Attachment of ubiquitin to defective proteins to tag them for breakdown.

Enzyme regulation methods
Enzyme concentration alteration (synthesis and/or destruction) Covalent modification (e.g., phosphorylation Proteolytic modification (zymogen) Allosteric regulation (e.g., feedback inhibition) pH Temperature Transcriptional regulation (e.g., steroid hormones)

Cell cycle phases are regulated by what three things?
cyclins, CDKs, and tumor suppressors.

Order (and important lengths) of cell cycle phases
Mitotis (shortest phase): prophase – metaphase – anaphase – telophase. G1 and G0 are of variable duration. G = G ap or G rowth S = S ynthesis

CDKs
Cyclin-dependent kinases; constitutive and inactive.

Cyclins
Regulatory proteins that control cell cycle events; phase specific; activate CDKs

Cyclin-CDK complexes
Must both be activated and inactivated for cell cycle to progress.

Tumor suppressors (and the cell cycle)
Rb and p53 normally inhibit G1-to-S progression; mutations in these genes result in unrestrained cell growth.

Permanent cells
Remain in G0, regenerate from stem cells. (e.g., neurons, skeletal and cardiac muscle, RBCs)

Stable (quiescent) cells
Enter G1 from G0 when stimulated (e.g., Hepatocytes, lymphocytes)

Labile cells
Never go to G0, divide rapidly w/ a short G1 (e.g., Bone marrow, gut epithelium, skin, hair follicles)

Rough Endoplasmic Reticulum (RER)
Site of synthesis of secretory (exported) proteins and of N-linked oligosaccharide addition to many proteins.

Nissl bodies
RER in neurons — synthesize enzymes (e.g., ChAT) and peptide neurotransmitters.

Free ribosomes
unattached to any membrane; site of synthesis of cytosolic and organellar proteins.

2 important examples of cells rich in RER
Mucus-secreting goblet cells of the small intestine, Ab-secreting plasma cells.

Smooth endoplasmic reticulum (SER)
Site of steroid synthesis and detoxification of drugs and poisons.

2 important examples of cells rich in SER
Liver hepatocytes Steroid hormone-producing cells of the adrenal cortex

Golgi apparatus: distribution center of ____ from ___ to ____?
Distribution center of proteins and lipids from ER to the plasma membrane, lysosomes, and secretory vesicles .

Golgi apparatus: modifies N-oligosaccharides on ____?
Asparagine.

Golgi apparatus: adds O-oligosaccharides on ____?
Serine and threonine.

Golgi apparatus: adds mannose-6-phosphate to ____? What does this do?
Specific lysosomal proteins —< targets protein to the lysosome.

Golgi apparatus: assembles ___ from ____?
Assembles proteoglycans from core proteins.

Golgi apparatus: sulfation of ____ and ____?
sulfation of sugars in proteoglycans and selected tyrosine on proteins .

Vesicular trafficking proteins: COPI
Retrograde: Golgi —< ER

Vesicular trafficking proteins: COPII
Anterograde: RER —< cis-Golgi

Vesicular trafficking proteins: Clathrin
trans-Golgi —< lysosomes, plasma membrane --< endosomoes (receptor-mediated endocytosis)

I-cell disease (inclusion cell dz): genetic/molecular basis?
Inherited lysosomal storage d/o; failure of addition of mannose-6-phosphate to lysosome proteins (enzymes are secreted outside the cell instead of being targeted to the lysosome).

I-cell dz (inclusion cell dz): Results?
Coarse facial features, clouded corneas, restricted joint mvmt, and high plasma levels of lysosomal enzymes. Often fatal in childhood.

Microtubules
Cylindrical structure composed of a helical array of polymerized dimers of alpha- and beta-tubulin. Each dimer has 2 GTP bound. Incorporated into flagella, cilia, mitotic spindles. Grows slowly, collapses quickly. Also involved in slow axoplasmic transport in neurons.

Molecular motor
Transport cellular cargo twd opposite ends of MT tracks. Dynein = retrograde to microtubule (+ —< -) Kinesin = anterograde to MT (- ---< +)

Drugs that act on microtubules
1.) Mebendazole/thiabendazole (antihelminthic) 2.) Griseofulvin (antifungal) 3.) Vincristine/vinblastine (anti-cancer) 4.) Paclitaxel (anti-breast cancer) 5.) Colchicine (anti-gout)

Chédiak-Higashi syndrome
Microtubule polymerization defect resulting in decr phagocytosis. Results in recurrent pyogenic infxns, partial albinism, and peripheral neuropathy.

Cilia structure
9 + 2 arrangement of MT’s.

Axonemal dynein
ATPase that links peripheral 9 doublets and causes bending of cilium by differential sliding of doublets.

Kartagener’s syndrome
Immotile cilia due to a dynein arm defect. Results in male and female infertility (sperm immotile), bronchiectasis, and recurrent sinusitis (bacteria and particles not pushed out); associated w/ situs inversus.

Actin and myosin
Microvilli Muscle contraction Cytokinesis Adherens jxn

Microtubules (what structures are they found in?)
Cilia Flagella Mitotic spindle Neurons Centrioles

Intermediate filaments
Vimentin Desmin Cytokeratin Glial fibrillary acid proteins (GFAP) Neurofilaments

Plasma membrane composition
Asymmetric bilayer. Contains XOL (~50%), phospholipid (~50%), sphingolipids, glycolipids, and proteins. High XOL or long saturated FA content —< incr melting temp, decr fluidity.

Vimentin stain
Connective tissue

Desmin stain
muscle

Cytokeratin stain
epithelial cells

GFAP stain
neuroglia

neurofilament stain
neurons.

Polymerase chain reaction (PCR): What is it? What are the steps?
Molecular biology laboratory proccedure used to amplify a desired fragment of DNA. 1.) Denaturation — DNA is denatured by heating to generate 2 separate strands. 2.) Annealing — during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified. 3.) Elongation — heat-stable DNA polymerase replicates the DNA sequence following each primer 3 steps are repeated multiple times for DNA sequence amplification.

Agarose gel electrophoresis
Used for size separation of PCR products (smaller molecules travel further); compared against a DNA ladder

Mnemonic for different blotting procedures
“SN oW DR oP ” S outhern = D NA N orthern = R NA W estern = P rotein

Southern blot
A DNA sample is electrophoresed on a gel and then transferred to a filter. The filter is then soaked in a denaturant and subsequently exposed to a labeled DNA probe that recognizes and anneals to its complementary strand. The resulting ds labeled piece of DNA is visualized when the filter is exposed to film.

Northern blot
Similar technique [to Southern], except that Northern blotting involves radioactive DNA probe binding to sample RNA .

Western blot.
Sample protein is separated via gel electrophoresis and transferred to a filter. Labeled Ab is used to bind to relevant protein .

Microarrays
Thousands of nucleic acid sequences are arranged in grids on glass or silicon. DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amts of complementary binding. Used to profile gene expression levels or to detect single nucleotide polymorphisms (SNPs).

Enzyme-Linked Immunosorbent Assay (ELISA): What is it? What is it used for? How reliable is it?
A rapid immunologic technique for testing Ag-Ab reactivity. Used in many laboratories to determine whether a particular Ab (e.g., anti-HIV) is present in a pt’s blood sample. Both the sensitivity and the specificity of a ELISA approach 100%, but both false (+) and false (-) occur.

Enzyme-Linked Immunosorbent Assay (ELISA): How is it performed?
Pt’s blood sample is probed w/ either: 1.) Test Ag (coupled to color-generating enzyme) — to see if immune system recognizes it OR 2.) Test Ab (coupled to color-generating enzyme) — to see if a certain Ag is present. If the target substance is present in the sample, the test soltn will have an intense color rxtn, indicating a positive test result.

Sodium pump
Na+/K+ ATPase is located in the plasma membrane w/ ATP site on cytoplasmic side. For each ATP consumed, 3 Na+ go out and 2 K+ come in. During cycle, pump is phosphorylated.

Ouabain
Inhibits sodium pump (Na+/K+) by binding the K+ site.

Cardiac glycosides (digoxin and digitoxin)
Directly inhibit Na+/K+ ATPase, which leads to indirect inhibition of Na+/Ca2+ exchange. Incr Ca2+ —< incr cardiac contractility.

Collagen (generally)
Most abundant protein in the human body. Extensively modified. Organizes and strengthens extracellular matrix.

Type I collagen Where is this type of collagen found?
90% Bone, skin, tendon, dentin, fascia, cornea, late wound repair Type I = bONE

Type II collagen Where is this type of collagen found?
Cartilage (including hyaline), vitreous body, nucleous pulposus. Type II = carTWO lage

Type III collagen Where is this type of collagen found?
(Reticulin) Skin, blood vessels, uterus, fetal tissue, granulation tissue

Type IV collagen Where is this type of collagen found?
Basement membrane or basal lamina Type IV = Under the floor (basement membrane)

mnemonic for important tissue types and their respective types of collagen Where is this type of collagen found?
“B e (S o T otally) C ool, R ead B ooks.” I = B one, S kin, T endon II = C artilage III = R eticulin IV = B asement membrane

Step 1: Synthesis (RER) inside fibroblasts
Translation of collagen alpha chains (preprocollagen ) — usually Gly-X-Y polypeptide (X and Y are proline, hydroxyproline, or hydroxylysine)

Step 2: hydroxylation (ER) inside fibroblasts
Hydroxylation of specific proline and lysine residues (requires Vitamin C ) *this step is inhibited in scurvy

Step 3: Glycosylation (ER) inside fibroblasts
Glycosylation of pro-alpha-chain lysine residues and formation of procollagen (triple helix of 3 collagen alpha chains) *this step is inhibited in osteogenesis imperfecta

Step 4: Exocytosis
Exocytosis of procollagen into extracellular space

Step 5: Proteolytic processing outside fibroblast
Cleavage of terminal regions of procollagen transforms it into insoluble tropocollagen

Step 6: cross-linking outside fibroblasts
Reinforcement of many staggered tropocollagen molecules by covalent lysine-hydroxylysine cross-linkage (by lysyl oxidase) to make collagen fibrils *this step is defective in Ehlers-Danlos syndrome

Ehlers-Danlos syndrome: what is it basically? what are the signs/sx?
Faulty collagen synthesis, causing: 1.) Hyperextensible skin 2.) Tendency to bleed (easy bruising) 3.) Hypermobile joints

Ehlers-Danlos syndrome: may be associated with…?
Joint dislocation Berry aneurysms Organ rupture

Types of Ehlers-Danlos
6 types. Inheritance and severity may vary. Can be autosomal dominant or recessive. Type III collagen is most frequently affected.

Osteogenesis imperfecta (generally)
Genetic bone d/o (brittle bone dz) caused by a variety of gene defects. May be confused w/ child abuse. Incidence is 1:10,000

Osteogenesis imperfecta: most common form (autosomal dominant, abnormal type I collagen)
1.) Multiple fractures w/ minimal trauma; may occur during the birthing process. 2.) Blue sclera due to the translucency of the connective tissue over the choroid. 3.) Hearing loss (abnormal middle ear bones) 4.) Dental imperfections due to lack of dentin

Type II osteogenesis imperfecta
Fatal in utero or neonatal period.

Alport’s syndrome: Due to….? Most common form…?
Due to a variety of gene defects resulting in abnormal type IV collagen. (type IV collage is an imp. strxrl component of the basement membrane of the kidney, ears, and eyes) Most common form is X-linked recessive.

Alport’s syndrome: Characterized by…? Associated with…?
Characterized by progressive hereditary nephritis and deafness. May be associated w/ ocular disturbances.

Elastin
Stretchy protein w/in lungs, large arteries, elastic ligaments, vocal cords, ligamenta flava (connect vertebrae —< relaxed and stretched conformations) Rich in proline and glycine, nonglycosylated forms. Tropoelastin w/ fibrillin scaffolding. Broken down by elastase, which is normally inhibited by alpha1-antitrypsin.

Marfan’s syndrome (cause)
Caused by a defect in fibrillin

Emphysema (one cause)
Can be caused by alpha1-antitrypsin deficiency, resulting in excess elastase activity.

Fluoresence in situ Hybridization (FISH)
Fluorescent DNA or RNA probe binds to specific gene site of interest. Used for specific localization of genes and direct visualization of anomalies (e.g., microdeletions) at molecular level (when deletion is too small to be visualized by karyotype). Fluorescence = gene is present; no fluorescence = gene has been deleted.

Cloning methods
Cloning is the production of a recombinant DNA molecule that is self-perpetuating. 1.) DNA fragments are inserted into bacterial plasmids that contain ABX resistance genes. These plasmids can be selected for by using media containing the ABX, and amplified. 2.) Restriction enzymes cleave DNA at 4- to 6-bp palindromic sequences, allowing for insertion of a fragment into the plasmid. 3.) Tissue mRNA is isolated and exposed to reverse transcriptase, forming a cDNA (lacks introns) library.

Sanger DNA sequencing
Dideoxynucleotides halt DNA polymerization at each base, generating sequences of various lengths that encompass the entire original sequence. Terminated fragments are electrophoresed and the original sequence can be deduced.

Transgenic studies in mice involve…
1.) Random insertion of gene into mouse genome (constitutive) 2.) Targeted insertion or deletion of gene thru homologous recombination w/ mouse gene (coditional)

Knock-out vs. Knock-in
Knock-out = removing a gene Knock-in = inserting a gene

Cre-lox system in model systems
A gene can be manipulated at specific developmental points using an inducible Cre-lox system with an ABX-controlled promoter (e.g., to study a gene whose deletion causes an embryonic lethal).

RNAi
dsRNA is synthesized that is complementary to the mRNA sequence of interest. When transfeccted into human cells, dsRNA separates and promotes degradation of target mRNA, knocking down gene expression.

Karyotyping
A process in which metaphase chromosomes are stained, ordered, and numbered according to size, arm-length ratio, and banding pattern. Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue. Used to Dx chromosomal imbalances (e.g., autosomal trisomies, microdeletions, sex chromosome d/o’s).

Genetic terms: Codominance
Neither of 2 alleles is dominant (e.g., blood groups)

Neither of 2 alleles is dominant (e.g., blood groups) What is the genetic term?
Codominance

Genetic terms: Variable expression
Nature and severity of the phenotype varies from 1 individual to another.

Nature and severity of the phenotype varies from 1 individual to another. What is the genetic term?
Variable expression

Genetic terms: Incomplete penetrance
Not all individuals w/ a mutant genotype show the mutant phenotype.

Not all individuals w/ a mutant genotype show the mutant phenotype. What is the genetic term?
Incomplete penetrance

Genetic terms: Pleiotropy
1 gene has < 1 effect on an individual's phenotype.

1 gene has < 1 effect on an individual's phenotype. What is the genetic term?
Pleiotropy

Genetic terms: Imprinting
Differences in phenotype depend on whether the mutation is of maternal or paternal origin (e.g., Prader-Willi syndrome, Angelman’s syndrome)

Differences in phenotype depend on whether the mutation is of maternal or paternal origin (e.g., Prader-Willi syndrome, Angelman’s syndrome) What is the genetic term?
Imprinting

Genetic terms: Anticipation
Severity of dz worsens or age of onset of dz is earlier in succeeding generations (e.g., Huntington’s dz)

Severity of dz worsens or age of onset of dz is earlier in succeeding generations (e.g., Huntington’s dz) What is the genetic term?
Anticipation

Genetic terms: Loss of heterozygosity
If a patient inherits or develops a mutation in a tumor suppressor gene, the complementary allele must be deleted/mutated before cancer develops. This is not true of oncogenes.

If a patient inherits or develops a mutation in a tumor suppressor gene, the complementary allele must be deleted/mutated before cancer develops. This is not true of oncogenes. What is the genetic term?
Loss of heterozygosity

Genetic terms: Dominant negative mutation
Exerts a dominant effect . A heterozygote produces a nonfxnl altered protein that also prevents the normal gene product from functioning.

Exerts a dominant effect . A heterozygote produces a nonfxnl altered protein that also prevents the normal gene product from functioning. What is the genetic term?
Dominant negative mutation

Genetic terms: Linkage disequilibrium
Tendency for certain alleles at 2 linked loci to occur together more often than expected by chance. Measured in a population, not in a family, and often varies in different populations.

Tendency for certain alleles at 2 linked loci to occur together more often than expected by chance. Measured in a population, not in a family, and often varies in different populations. What is the genetic term?
Linkage disequilibrium

Genetic terms: Mosaicism
Occurs when cells in the body have different genetic makeup (e.g., lyonization — random X inactivation in females)

Occurs when cells in the body have different genetic makeup (e.g., lyonization — random X inactivation in females) What is the genetic term?
Mosaicism

Genetic terms: Locus heterogeneity
Mutations at different loci can produce the same phenotype (e.g., albumin)

Mutations at different loci can produce the same phenotype (e.g., albumin) What is the genetic term?
Locus heterogeneity

Genetic terms: Heteroplasmy
Presence of both normal and mutated mtDNA, resulting in variable expression in mitochondrial inherited dz’s.

Presence of both normal and mutated mtDNA, resulting in variable expression in mitochondrial inherited dz’s. What is the genetic term?
Heteroplasmy

Genetic terms: Uniparental disomy
Offspring receives 2 copies of a chromosome from 1 parent and no copies from the other parent.

Offspring receives 2 copies of a chromosome from 1 parent and no copies from the other parent. What is the genetic term?
Uniparental disomy

Hardy-Weinberg equilibrium
If a population is in H-W equilibrium and p and q are separate alleles, then: Dz prevalence: p^2 + 2pq + q^2 = 1 Allele prevalence: p + q = 1 2pq = heterozygote prevalence The prevalence of an X-linked recessive dz in males = q and in females = q^2

Assumptions of Hardy-Weinberg (there are 4)
1.) No mutation occurring at the locus 2.) No selection for any of the genotypes at the locus 3.) Completely random mating 4.) No migration

Imprinting (def.)
At a single locus, only 1 allele is active; the other is inactive (imprinted/inactivated by methylation). Deletion of the active allele —< dz. Most common example: Prader-Willi and Angelman's syndromes

Prader-Willi and Angelman’s syndromes: Location? Mechanism?
Both syndromes due to inactivation or deletion of genes on chromosome 15. Can also occur as a result of uniparental disomy.

P rader-Willi Syndrome
Deletion of normally active P aternal allele. Mental retardation, hyperphagia, obesity,, hypogonadism, hypotonia.

AngelM an’s syndrome
Deletion of normally active M aternal allele. Mental retardation, seizures, ataxia, inappropriate laughter (“happy puppet”).

Autosomal dominant. Often due to defects in structural genes. Many generations, both male and female, affected. Often pleiotropic and, in many cases, present clinically after puberty. Family Hx crucial to Dx.

Autosomal recessive 25% of offspring from 2 carrier parents are affected. Often due to enzyme deficiencies. Usually seen in only 1 generation. Commonly more severe than dominant d/o’s; pts often present in childhood.

X-linked recessive. Sons of heterozygous mothers have a 50% chancce of being affected. No male-to-male transmission. Commonly more severe in males. Heterozygous females may be affected.

X-linked dominant. Transmitted thru both parents. Either male or female offspring of the affected mother may be affected, while all female offspring of the affected father are diseased.

Hypophosphatemic rickets
(Archtypical example of X-linked dominant dz) Formerly known as vitamin D-resistant rickets. Inherited d/o resulting in incr phosphate wasting at proximal tubule. Results in rickets-like presentation.

Mitochondrial inheritance. Transmitted only thru mother. All offspring of affected females may show signs of dz. Variable expression in population due to heteroplasmy.

Mitochondrial myopathies, Leber’s hereditary optic neuropathy
(mitochondrial inheritance dz’s) Degeneration of retinal ganglion cells and axons. Leads to acute loss of central vision.

Autosomal Dominant dz’s: Achondroplasia
Cell-signaling defect of fibroblasts growth factor (FGF) receptor 3. Results in dwarfism; short limbs, but head and trunk are normal size. Associated w/ advanced paternal age.

Autosomal Dominant dz’s: APKD
Formerly known as adult polycystic kidney dz. Always bilateral , massive enlargement of kidneys due to multiple large cysts. Pts p/w flank pain, hemature, HTN, progressive renal failure. 90% cases are due to a mutation in APKD1 (chromosome 16 ; 16 letters in “polycystic kidney”). Associated w/ polycystic liver dz, berry aneurysms, mitral valve prolapse. Infantile form is recessive.

Autosomal Dominant dz’s: Familial adenomatous polyposis
Colon becomes covered w/ adenomatous polyps after puberty. Progresses to colon cancer unless resected. Deletion on chromosome 5 (APC gene ); 5 letters in “polyp”.

Autosomal Dominant dz’s: Familial hypercholesterolemia (hyperlipidemia type IIA)
Elevated LDL due to defective or absent LDL receptor. Heterozygotes (1:500) have cholesterol ~300 mg/dL. Homozygotes (very rare) have cholesterol ~700+ mg/dL, severe athersclerotic dz early in life, and tendon xanthomas (clasically in the Achilles tendon); MI may develop before age 20.

Autosomal Dominant dz’s: Hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu syndrome)
Inherited d/o of blood vessels. Findings: telangiectasia, recurrent epistaxis, skin discolorations, arteriovenous malformations (AVMs).

Autosomal Dominant dz’s: Hereditary spherocytosis
Spheroid erythrocytes due to spectrin or ankyrin defect; hemolytic anemia; Incr MCHC. Splenectomy is curative.

Autosomal Dominant dz’s: Huntington’s dz
Findings: depression, progressive dementia, choreiform mvmts, caudate atrophy, and decr levels of GABA and ACh in the brain. Sx manifest in affected indvls btw the ages of 20-50. Gene located on Chr 4 ; trinucleotide repeat d/o: (CAG) (“Hunting 4 food”)

Autosomal Dominant dz’s: Marfan’s syndrome
Fibrillin gene mutation —< connective tissue d/o affecting skeleton, heart, and eyes. Findings: tall w/ long extremities, pectus excavatum, hyperextensive joints, and long, tapering fingers and toes (arachnodactyly, below); cystic medial necrosis of aorta --< aortic incompetence and dissecting aortic aneurysms; floppy mitral valve. Subluxation of the lenses.

Autosomal Dominant dz’s: Multiple endocrine neoplasias (MEN)
Several distinct syndromes (I, II, III) characterized by familial tumors of endocrine glands, including those of the pancreas, parathyroid, thyroid, and adrenal medula. Men II and III associated w/ ret gene.

Autosomal Dominant dz’s: Neurofibromatosis type 1 (von Recklinghausen’s dz)
Findings: café-au-lait spots, neural tumors, Lisch nodules (pigmented iris hamartomas). Also marked by skeletal d/o’s (e.g., scoliosis), optic pathway gliomas, pheochromocytoma, and incr tumor susceptibility. On long arm of chromosome 17 ; 17 letters in “von Recklinghausen”

Autosomal Dominant dz’s: Neurofibromatosis type 2
Bilateral acoustic neuroma, juvenile cataracts. NF2 gene on chromosome 2 ; (type 2 = 22 )

Autosomal Dominant dz’s: Tuberous sclerosis
Findings: facial lesions (adenoma sebaceum) hypopigmented “ash leaf spots” on skin cortical and retinal hamartomas seizures mental retardation renal cysts and renal angiomyolipomas cardiac rhabdomyomas incr incidence of astrocytomas. Incomplete penetrance, variable presentation.

Autosomal Dominant dz’s: von Hippel-Lindau dz
Findings: Hemangioblastomas of retina/cerebellum/medulla about 1/2 of affected indvls develop multiple bilateral renal cell carcinomas and other tumors. Associated w/ deletion of VHL gene (tumor suppressor) on chromosome 3 (3p). Results in constitutive expression of HIF (transcription factor) and activation of angiogenic growth factors. Von Hippel-Linau = 3 words for chromosome 3.

Autosomal recessive dz’s (list)
Albinism ARPKD (formerly known as infantile polycystic kidney dz) Cystic fibrosis Glycogen storage dz’s Hemochromatosis Mucopolysaccharidoses (except Hunter’s) Phenylketonuria Sickle cell anemias Sphingolipidoses (except Fabry’s) Thalassemias

Genetics of Cystic fibrosis
Autosomal-recessive defect in CFTR gene on chromosome 7, commonly deletion of Phe 508. CFTR channel actively secretes Cl- into lungs and GI tract, and actively reabsorbs Cl- from sweat. *Most common lethal genetic dz of Caucasians

Defective Cl- channel (as in mutCFTR in CF)
Secretion of abnormally thick mucus that plugs lungs, pancreas and liver | Recurrent pulmonary infxns (Pseudomonas species and S. aures), chronic bronchitis, bronchiectasis, pancreatic insufficiency (malabsorption and steatorrhea), meconium ileus in newborns.

“Other” problems in CF (besides those that result directly from defective Cl- channel)
Infertility in males due to bilateral absence of vas deferens. Fat-soluble vitamin deficiencies (A, D, E, K). Can present as failure to thrive in infancy.

Dx of CF?
Incr concentration of Cl- ions in sweat test.

Tx for CF?
N-acetylcysteine to loosen mucous plugs (cleaves disulfide bonds w/in mucous glycoproteins).

X-linked recessive d/o’s (list)
B ruton’s agammaglobulinemia W iskott-Aldrich syndrome F ragile X G 6PD deficiency O cular albinism L esch-Nyhan syndrome D uchenne’s (and Becker’s) muscular dystrophy H emophilia A and B F abry’s dz H unter’s syndrome “B e W ise, F ool’s GOLD H eeds F alse H ope” Female carriers are rarely affected due to random inactivation of X chromosomes in each cell.

Duchenne’s muscular dystrophy
X-linked frame-shift mutation —< deletion of dystrophin gene --< accelerated muscle breakdown. Weakness begins in pelvic girdle muscles and progresses superiorly. Pseudohypertrophy of calf muscles due to fibrofatty replacement of muscle; cardiac myopathy. Use of Gowers' maneuver, requiring assistance of the upper extremities to stand up, is characteristic. Onset before 5 yrs of age. D uchenne's = D eleted D ystrophin.

Becker’s muscular dystrophy
X-linked mutated dystrophin gene. Less severe than Duchenne’s. Onset in adolescence or early adulthood.

Dystrophin gene (DMD )
(associated w/ Duchenne’s and Becker’s muscular dystrophies) The longest known human gene —< incr rate of spontaneous mutation. Dystrophin helps anchor muscle fibers, primarily in skeletal and cardiac muscle.

Dx of muscular dystrophies (Duchenne’s, Becker’s)
Incr CPK and muscle biopsy.

Fragile X syndrome
X-linked defect affeccting the methylation and expression of the FMR1 gene. Associated w/ chromosomal breakage. The 2nd most common cause of genetic mental retardation (after Down syndrome). Trinucleotide repeat disorder (CGG)

Fragile X syndrome: findings?
Macro-orchidism (enlarged testes), long face w/ a large jaw, large everted ears, autism. “Fragile X = eX tra-large testes, jaw, ears.”

Trinucleotide repeat expansion dz’s (list, specific trinucleotides, shared dz characteristic)
Hunting ton’s dz, my otonnic dystrophy, Fried rich’s ataxia, fragile X syndrome. (“Try [tri nucleotide] hunting for my fried eggs [X ]”) Huntington’s = CAG MyoT onic dystrophy = CT G FraG ile X syndrome = CG G Friedreich’s ataxia = GAA May show genetic anticipation (dz severity incr and age of onset decr in successive generations; germline expansion in females)

D own syndrome (trisomy 21) (D rinking age = 21) Incidence?
1:700 Most common chromosomal d/o and most common cuase of congenital mental retardation.

D own syndrome (trisomy 21) (D rinking age = 21) Findings?
Mental retardation, flat facies , prominent epicanthal folds , simian crease [below], gap btw 1st 2 toes, duodenal atresia, congenital hear dz (most commonly septum primum-type ASD). Assoc w/ incr risk of ALL and Alzheimer’s dz (< 35 yrs of age)

D own syndrome (trisomy 21) (D rinking age = 21) genetic cause in 95% of cases?
Due to meiotic nondisjunction of homogous chromosomes (associated w/ advancced maternal age; from 1:1500 in women >20 to 1:25 in women <45)

Meiotic nondisjunction
Can occur in anaphase I: Or in anaphase II:

D own syndrome (trisomy 21) (D rinking age = 21)
Genetic cause in 4% of cases? Due to robertsonian translocation.

D own syndrome (trisomy 21) Geneti cause in 1% of cases?
Due to Down mosaicism (no maternal association)

Down syndrome: results of pregnancy quad screen? results of ultrasound?
Decr alpha-fetoprotein Incr Beta-hCG Decr estriol Incr Inhibin A Ultrasound shows nuchal translucency.

E dward’s syndrome (trisomy 18 ) (E lection age = 18 ) Incidence?
1:8000 Most common trisomy resulting in live birth after Down syndrome.

E dward’s syndrome (trisomy 18 ) (E lection age = 18 ) Findings?
Severe mental retardation Rocker-bottom feet micrognathia (small jaw) Low-set ears Clenched hands Prominent occiput Congenital heart dz Death usually occurs w/in 1 yr of birth.

P atau’s syndrome (trisomy 13 ) (P uberty ~age 13 ) Incidence?
1:15,000

P atau’s syndrome (trisomy 13 ) (P uberty ~age 13 ) Findings?
Severe mental retardation Rocker-bottom feet Microphthalmia Microcephaly Cleft lip/palate holoProsencephaly Polydactyly Congenital heart dz Death usually occurs w/in 1 yr of birth.

Robertsonian translocation
Nonreciprocal chromosomal transloccation that commonly involves chromosome pairs 13, 14, 15, 21, and 22. One of the most common types of translocation. Occurs when the long arms of two acrocentric chromosomes (chromosomes w/ the centromeres near the ends) fuse at the centromere and the 2 short arms are lost.

Robertsonian translocation: balanced vs. unbalanced translocations?
Balanced translocations normally do not cause any abnormal phenotype. Unbalanced translocations can result in miscarriage, stillbirth, and chromosomal imbalance (e.g., Down syndrome, Patau’s syndrome).

Chromosomal inversions
Chromosome rearrangement in which a segment of a chromosome is reveresed end-to-end. May result in decr fertility.

Chromosomal inversions: Pericentric vs. paracentric?
Pericentric: Involves centromere; proceeds thru meiosis. Paracentric: does not involve centromere; does not proceed thru meiosis.

Cri-du-chat syndrome: genetic basis?
Congenital microdeletion of short arm of Chr 5 (46,XX or XY,5p-)

Cri-du-chat: findings?
Microcephaly Moderate to severe mental retardation High-pitched crying/mewing (Cri-du-chat = “cry of the cat”) Epicanthal folds Cardiac abnormalities

Williams syndrome: genetic basis?
Congenital microdeletion of long arm of Chr 7 (deleted region includes elastin gene).

Williams syndrome: findings?
Distinctive “elfin” facies Mental retardation Well-developed verbal skills Cheerful disposition Extreme friendliness w/ strangers Cardiovascular problems

22q11 deletion syndromes: general characteristics, genetic basis?
Variable presentation, including: C left palate A bnormal facies T hymic aplasia —< T-cell deficiency C ardiac defects H ypocalcemia secondary to parathyroid aplasia Due to microdeletion at chromosome 22 q11. "CATCH-22 "

22q11 deletion syndromes: developmental etiology?
Due to aberrat development of 3rd and 4th branchial pouches

22q11 deletion syndromes: what are they, and what are the main findings?
DiGeorge syndrome: thymic, parathyroid, and cardiac defects. Velocardiofacial syndrome: palate, facial, and cardiac defects.

Fat-soluble vitamins (list + their basic fxns)
Vitamin A – vision Vitamin D – bone calcification, Ca2+ homeostasis Vitamin E – antioxidant Vitamin K – clotting factors

Fat-soluble vitamins: toxicity?
(A, D, E, K) Toxicity more common than for water-soluble vitamins, b/c they accumulate in fat.

Fat-soluble vitamins: absorption?
(A, D, E, K) Absorption dependent on gut (ileum) and pancreas. Malabsorption syndromes (steatorrhea), such as cystic fibrosis and sprue, or mineral oil intake can cause fat-soluble vitamin deficiencies.

Water-soluble vitamins (list and basic fxns)
Vitamin C – antioxidant, collagen synthesis Metabolic: Thiamine (B1) Riboflavin (B2) Niacin (B3) Pantothenic acid (B5) Pyridoxine (B6) Biotin (B7) Folate – blood, neural development Cobalamin (B12) – blood, CNS

Water-soluble vitamins: list with alternative names and related enzymes/cofactors?
B1 (thiamine: TPP) B2 (riboflavin: FAD, FMN) B3 (niacin: NAD+) B5 (pantothenic acid: CoA) B6 (pyridoxine: PLP) B12 (cobalamin) C (ascorbic acid) Also: Biotin, folate

Water soluble vitamin deficiencies
All was out easily from body except B12 and folate (stored in liver). B-complex deficiencies often result in dermatitis, glossitis, and diarrhea.

Vitamin A (retinol): fxn? use? where is it found?
Antioxidant; constituent of visual pigments (retinal). Retinol is vitamin A , so think Retin-A (used topically for wrinkles and acne). Found in liver and leafy vegetables.

Vitamin A (retinol) deficiency?
Night blindness, dry skin.

Vitamin A (retinol) excess?
Arthralgias, fatigue, HAs, skin changes, sore throat, alopecia. Teratogenic (cleft palate, cardiac abnormalities).

Vitamin B1 (thiamine): fxn?
In thiamine pyrophosphate (TPP), a cofactor for several enzymes: 1.) Pyruvate dehydrogenase (glycoslysis) 2.) alpha-ketoglutarate dehydrogenase (TCA cycle) 3.) Transketolase (HMP shunt) 4.) Branched-chain AA dehydrogenase

Vitamin B1 (thiamine) deficiency: causes what? Where do you see this?
Wernicke-Korsakoff syndrome and beriberi (both neurologic and cardiac manifestations). Seen in malnutrition as well as alcoholism (secondary to malnutrition and malabsorption).

Wernicke-Korsakoff syndrome
Due to Vitamin B1 (thiamine) deficiency. confusion, ophthalmoplegia, ataxia + memory loss, confabulation, personality change.

Beri-beri
Due to Vitamin B1 (thiamine) deficiency. (spell it B er1-B er1 ) Dry beriberi – polyneuritis, symmetrical muscle wasting. Wet beriberi – high-output cardiac failure (dilated cardiomyopathy), edema.

Vitamin B2 (riboflavin) fxn?
Cofactor in oxidation and reduction (e.g., FADH2) F AD and F MN are derived from riboF lavin (B2 = 2 ATP)

Vitamin B2 (riboflavin) deficiency?
C heilosis (inflammation of lips, scaling and fissures at corners of mouth), C orneal vascularization. “The 2 C ‘s”

Vitamin B3 (niacin): fxn?
Constituent of NAD+, NADP+ (used in redox rxtns). Derived from tryptophan. Synthesis requires vitamin B6. N AD derived from N iacin (B3 = 3 ATP)

Vitamin B3 (niacin) deficiency?
Glossitis. Severe deficiency leads to pellagra, which can be caused by Hartnup dz (decr tryptophan absorption), malignant carcinoid syndrome (incr tryptophan metabolism), and INH (decr vitamin B6) The 3 D’s of pellagra: D iarrhea, D ermatitis, D ementia.

Vitamin B3 (niacin) excess?
Facial flushing (due to pharmacologic doses for Tx of hyperlipidemia)

Vitamin B4 (pantothenate): fxn?
Essential component of CoA (a cofactor for acyl transfers) and fatty acid synthase. “Pantothen-A is in Co-A “

Vitamin B5 (pantothenate) deficiency?
Dermatitis, enteritis, alopecia, adrenal insufficiency

Vitamin B6 (pyridoxine) fxn?
Converted to pyridoxal phosphate, a cofactor used in transamination (e.g., ALT and AST), decarboxylation rxtns, glycogen phosphorylase, and heme synthesis. Required for the synthesis of niacin from tryptophan.

Vitamin B6 (pyridoxine) deficiency?
Convulsions, hyperirritability, peripheral neuropathy (deficiency inducible by INH and oral contraceptives)

B12 (cobalamin): fxn?
Cofactor for homocysteine methyltransferase (transfers CH3 groups as methylcobalamin) and methylmalonyl-CoA mutase.

B12 (cobalamin): Deficiency?
Macrocytic, megaloblastic anemia; neurologic Sx (paresthesias, subacute combined degeneration) due to abnormal myelin. Prolonged deficiency leads to irreversible nervous system damage.

B12 (cobalamin): what rxtns does it help to proceed?
Homocysteine + N-methyl THF –(B12 + homocysteine methyl transferase)–< Methionine + THF Methylmalonyl-CoA --(B12 + methylmalonyl-CoA mutase)--< Succinyl-CoA

B12 (cobalamin): found where?
Found in animal products. Only synthesized by microorganisms.

B12 (cobalamin): etiology of deficiency?
Very large reserve pool (several yrs) stored primarily in liver. Deficiency is usually caused by malabsorption (sprue, enteritis, Diphyllobothrium latum ), lack of intrinsic factor (pernicious anemia, gastric bypass surgery), or absence of terminal ileum (Crohn’s dz).

Schilling test
Used to detect the etiology of B12 (cobalamin) deficiency.

Folic acid: fxn?
Converted to tetrahydrofolate (THF), a coenzyme for 1-carbon transfer/methylation rxtns. Important for the synthesis of nitrogenous bases in DNA and RNA.

Folic acid: deficiency?
Macrocytic, megaloblastic anemia; no neurologic Sx (as opposed to vitamin B12 deficiency). Most common vitamin deficiency in the USA. Seen in alcoholism and pregnancy.

Folic acid: where is it found?
FOL ate is from FOL iage (green leaves)

Etiology of folic acid deficiency?
Small reserve pool stored primarily in liver (eat green leaves!) Deficiency can be caused by several drugs (e.g., phenytoin, sulfonamides, MTX).

Folic acid and pregnancy
Supplemental folic acid in early pregnancy reduces neural tube defects.

S-adenosyl-methionine (SAM): formation?
ATP + methionine —< SAM

S-adenosyl-methionine (SAM): fxn?
SAM transfers methyl units. Regeneration of methionine (and thus SAM) is dependent on vitamin B12 and folate. (“SAM the methyl donor man”)

Biotin: fxn?
Cofactor for carboxylation enzymes: 1.) Pyruvate carboxylase : Pyruvate —< oxaloacetate 2.) Acetyl-CoA carboxylase : Acetyl-CoA --< malonyl-CoA 3.) Propionyl-CoA carboxylase : Propionyl-CoA --< methylmalonyl-CoA

Biotin: deficiency?
Relatively rare. Dermatitis, alopecia, enteritis. Caused by ABX use or excessive ingestion of raw eggs. “AVID in in egg whites AVID ly binds biotin.”

Vitamin C (ascorbic acid): fxn?
Antioxidant. Also: 1.) Facilitates iron absorption by keeping iron in Fe2+ state (more absorbable) 2.) Necessary for hydroxylation of proline and lysine in collagen synthesis. 3.) Necessary for dopamine Beta-hydroxylase, which converts dopamine to NE

Vitamin C (ascorbic acid): deficiency?
Scurvy: swollen gums, bruising, anemia, poor wound healing.

Vitamin C (ascorbic acid): where is it found?
Found in fruits and vegetables. British sailors carried limes to prevent scurvy (thus the origin of the word “limey”)

Vitamin D: forms?
D2 = ergocalciferol — ingested from plants, used as pharmacologic agent. D3 = cholecalciferol — consumed in milk, formed in sun-exposed skin. 25-OH D3 = storage form. 1,25-(OH)2-D3 (calcitriol) = active form

Vitamin D: fxn?
Incr intestinal absorption of calcium and phosphate, incr bone resorption

Vitamin D: deficiency?
Rickts in children (bending bones), osteomalacia in adults (soft bones), hypocalcemic tetany. Drinking milk (fortified w/ vitamin D) is good for bones.

Vitamin D: excess?
Hypercalcemia, hypercalciuria, loss of appetite, stupor. Seen in sarcoidosis (incr activation of vitamin D by epithelioid macrophages)

Vitamin E: fxn?
Antioxidant (protects erythrocytes and membranes from free-radical damage). “E is for E rythrocytes”

Vitamin E: deficiency?
Incr fragility of erythrocytes (hemolytic anemia), muscle weakness, neurodysfxn.

Vitamin K: fxn?
Catalyzes gamma-carboxylation of glutamic acid residues on various proteins concerned w/ blood clotting. “K for K oagulation.” Necessary for synthesis of factors II, VII, IX, X, and protein C and S.

Vitamin K: synthesis?
Synthesized by intestinal flora.

Vitamin K antagonist?
Warfarin.

Vitamin K: deficiency?
Neonatal hemorrhage w/ incr PT and incr aPTT, but normal bleeding time (neonates have sterile intestines and are unable to synthesize vitamin K). Neonates are give vitamin K injection at birth to prevent hemorrhage. Can also occur after prolonged use of broad-spectrum ABX.

Zinc: fxn?
Essential for the activity of 100+ enzymes. Important in the formation of Zinc fingers (a transcription motif)

Zinc: deficiency?
Delayed wound healing, hypogonadism, decr adult hair (axillary, facial, pubic). May predispose to alcoholic cirrhosis.

Ethanol metabolism: chemical rxtns?

Ethanol metabolism: kinetics?
NAD+ is the limiting reagent. Alcohol dehydrogenase operates via zero-order kinetics.

Fomepizole
Inhibits alcohol dehydrogenase

Disulfiram (antabuse)
Inhibits acetaldehyde dehydrogenase (acetaldehyde accumulates, contributing to Sx of hangover)

Ethanol hypoglycemia
Ethanol metabolism increases NADH/NAD+ ratio in liver, causing a diversion of pyruvate to lactate and OAA to malate, thereby inhibiting gluconeogenesis and stimulating FA synthesis. Leads to hypoglycemia and hepatic fatty change (hepatocellular steatosis) seen in chronic alcoholics.

Kwashiorkor
Protein malnutrition resulting in skin lesions, edema, liver malfxn (fatty change). Clinical picture is small child w/ swollen belly. “Kwashiorkor results from a protein-deficient MEAL : M alnutrition, E dema, A nemia, L iver (fatty)”

Marasmus
Energy malnutrition resulting in tissue and muscle wasting, loss of subcutaneous fat, and variable edema. “M arasmus results in M uscle wasting”

Metabolism sites: Mitochondria
Fatty acid oxidation (beta-oxidation) Acetyl-CoA production TCA cycle Oxidative phosphorylation

Metabolism sites: cytoplasm
Glycolysis Fatty acid synthesis HMP shunt Protein synthesis (RER) Steroid synthesis (SER)

Metabolism sites: both mitochondria and cytoplasm
H eme synthesis U rea cycle G luconeogenesis “HUG s take two “

Process: Glycolysis What is the rate-limiting enzyme?
Phosphofructokinase-1 (PFK-1)

Rate-limiting enzyme: Phosphofructokinase-1 (PFK-1) What is the process?
Glycolysis

Process: Gluconeogenesis What is the rate-limiting enzyme?
Fructose bisphosphatase-2

Rate-limiting enzyme: Fructose bisphosphatase-2 What is the process?
Gluconeogenesis

Process: TCA cycle What is the rate-limiting enzyme?
Isocitrate dehydrogenase

Rate-limiting enzyme: Isocitrate dehydrogenase What is the process?
TCA cycle

Process: Glycogen synthesis What is the rate-limiting enzyme?
Glycogen synthase

Rate-limiting enzyme: Glycogen synthase What is the process?
Glycogen synthesis

Process: Glycogenolysis What is the rate-limiting enzyme?
Glycogen phosphorylase

Rate-limiting enzyme: Glycogen phosphorylase What is the process?
Glycogenolysis

Process: HMP shunt What is the rate-limiting enzyme?
Glucose-6-phosphate dehydrogenase (G6PD)

Rate-limiting enzyme: Glucose-6-phosphate dehydrogenase (G6PD) What is the process?
HMP shunt

Process: De novo pyrimidine synthesis What is the rate-limiting enzyme?
Aspartate transcarbamoylase (ATCase)

Rate-limiting enzyme: Aspartate transcarbamoylase (ATCase) What is the process?
De novo pyrimidine synthesis

Process: De novo purine synthesis What is the rate-limiting enzyme?
Glutamine-PRPP amidotransferase

Rate-limiting enzyme: Glutamine-PRPP amidotransferase What is the process?
De novo purine synthesis

Process: Urea cycle What is the rate-limiting enzyme?
Carbamoyl phosphate synthetase

Rate-limiting enzyme: Carbamoyl phosphate synthetase What is the process?
Urea cycle

Process: Fatty acid synthesis What is the rate-limiting enzyme?
Acetyl-CoA carboxylase (ACC)

Rate-limiting enzyme: Acetyl-CoA carboxylase (ACC) What is the process?
Fatty acid synthesis

Process: Fatty acid oxidation What is the rate-limiting enzyme?
Carnitine acyltransferase I

Rate-limiting enzyme: Carnitine acyltransferase I What is the process?
Fatty acid oxidation

Process: Ketogenesis What is the rate-limiting enzyme?
HMG-CoA synthase

Rate-limiting enzyme: HMG-CoA synthase What is the process?
Ketogenesis

Process: Cholesterol synthesis What is the rate-limiting enzyme?
HMG-CoA reductase

Rate-limiting enzyme: HMG-CoA reductase What is the process?
Cholesterol synthesis

Summary of biochemical pathways

Glycolysis/ATP production
Aerobic metabolism of glucose produces 32 ATP via malate-aspartate shuttle (heart and liver), 30 ATP via glycerol-3 phosphate shuttle (muscle) Anaerobic glycolysis produces only 2 net ATP per glucose molecule. ATP hydrolysis can be coupled to energetically favorable rxtns.

Structure of ATP (what are the 3 important moieties?)

Subtance: Phosphoryl What is the activated carrier for this substance?
ATP

Activated carriers: ATP What does substance does this carry?
Phosphoryl

Subtance: Electrons What is the activated carrier for this substance?
NADH, NADPH, FADH2

Activated carriers: NADH, NADPH, FADH2 What does substance does this carry?
Electrons

Subtance: Acyl What is the activated carrier for this substance?
Coenzyme A, lipoamide

Activated carriers: Coenzyme A, lipoamide What does substance does this carry?
Acyl

Subtance: CO2 What is the activated carrier for this substance?
Biotin

Activated carriers: Biotin What does substance does this carry?
CO2

Subtance: 1-carbon units What is the activated carrier for this substance?
Tetrahydrofolate

Activated carriers: Tetrahydrofolate What does substance does this carry?
1-carbon units

Subtance: CH3 groups What is the activated carrier for this substance?
SAM

Activated carriers: SAM What does substance does this carry?
CH3 groups

Subtance: Aldehydes What is the activated carrier for this substance?
TPP

Activated carriers: TPP What does substance does this carry?
Aldehydes

Universal electron acceptors (list)
Nicotinamides (NAD+, NADP+) and flavin nucleotides (FAD+)

NAD+ vs. NADP+
NAD+ is generally used in catabolic processes to carry reducing equivalents away as NADH. NADPH is used in anabolic processes (steroid and FA synthesis) as a supply of reducing equivalents.

NADPH: Product of…? Used in… (3 things)?
Product of the HMP shunt. Used in: 1.) Anabolic processes 2.) Respiratory burst 3.) P-450

Hexokinase vs. glucokinase: why are the 2 enzymes similar?
Phosphorylation of glucose to yield glucose-6-phosphate serves as the 1st step of glycolysis (also serves as the first step of glycogen synethsis in the liver). Rxtn is catalyzed by either hexokinase or glucokinase, depending on location.

Hexokinase vs. glucokinase: Location?
Hexokinase: ubiquitous. Glucokinase: Liver and Beta-cells of pancreas only.

Hexokinase vs. glucokinase: Affinity / Capacity?
Hexokinase: high affinity (low Km), low capacity (low Vmax) Glucokinase: Low affinity (high Km), high capacity (high Vmax)

Hexokinase vs. glucokinase: response to insulin?
Hexokinase: uninduced by insulin Glucokinase: induced by insulin

Hexokinase vs. glucokinase: Feedback?
Hexokinase: Feedback inhibited by glucose-6-phosphate. Glucokinase: No direct feedback inhibition.

Hexokinase vs. glucokinase: Role in blood sugar hemostasis?
Glucokinase phosphorylates excess glucose (e.g., after a meal) to sequester it in the liver. Allows liver to serve as a blood glucose “buffer”.

Net glycolysis rxtn (cytoplasm)
Glucose + 2 Pi + 2 ADP + 2 NAD+ | [yields] | 2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2H2O

Steps in glycolysis that require ATP: What are the substrates/products/enzymes?

Steps in glycolysis that require ATP: Regulation?
Hexokinase (ubiquitous)/glucokinase (liver) — (-) feedback from Glucose-6-Phosphate Phosphofructokinase 1 (rate limiting step): (-) feedback from ATP , citrate (+) feedback from AMP, fructose-2,6-bisphosphate

Steps in glycolysis that produce ATP: What are the substrates/products/enzymes?

Steps in glycolysis that produce ATP: Regulation?
Pyruvate kinase: (-) feedback from ATP, alanine (+) feedback from fructose-1,6-bisphosphate

Pyruvate dehydrogenase
Takes pyruvate (the product of glycolysis) and produces Acetyl-CoA, which can enter TCA/Krebs cycle. (-) feedback from: ATP, NADH, Acetyl-CoA

Regulation by fructose-2,6-bisphosphate
F2,6BP is the most potent activator of PFK-1 (overrides inhibition by ATP and citrate) and is a potent regulator of glycolysis and gluconeogenesis

Glycolytic enzyme deficiency: Associated with…? Why? Due to deficiencies in…?
Associated w/ hemolytic anemia . Inability to maintain activity of Na+/K+ ATPase leads to RBC swelling and lysis. (RBCs metabolize glucse anaerobically – no mitochondria – and thus depend solely on glycolysis. Due to deficiencies in pyruvate kinase (95%), phosphoglucose isomerase (4%), and other glycolytic enzymes.

Pyruvate dehydrogenase: net rxtn?
Pyruvate + NAD+ + CoA | [yields] | acetyl-CoA + CO2 + NADH

Pyruvate dehydrogenase complex: 3 enzymes that require what 5 cofactors?
1.) Pyrophosphate (B1, thiamine; TPP) 2.) FAD (B2, riboflavin) 3.) NAD (B3, niacin) 4.) CoA (B5, pantothenate) 5.) Lipoic acid

How does exercise active the pyruvate dehydrogenase complex?
Incr NAD+/NADH ratio Incr ADP Incr Ca2+

Pyruvate dehydrogenase complex vs. alpha-ketoglutarate complex
PD complex is similar to the a-KG complex (same cofactors, similar substrate and action), which converts alpha-ketoglutarate —< succinyl-CoA (TCA cycle)

Arsenic: inhibits…? Findings w/ poisoning?
Inhibits lipoic acid (cofactor for pyruvate dehydrogenase complex and alpha-KG complex) Findings: vomiting, rice water stools, garlic breath.

Pyruvate dehydrogenase deficiency: Causes…? Etiology?
Causes backup of substrate (pyruvate and alanine), resulting in lactic acidosis. Can be congenital or acquired (as in alcoholics due to B1 deficiency).

Pyruvate dehydrogenase deficiency: findings?
Neurologic defects

Pyruvate dehydrogenase deficiency: Tx?
Incr intake of ketogenic nutrients (e.g., high fat content or incr lysine and leucine) *L ysine and L eucine are the only purely ketogenic amino acids.

Pyruvate metabolism: Alanine?
Alanine (made via ALT): carries amino groups to the liver from muscle [#1 above]

Pyruvate metabolism: oxaloacetate?
OAA (formed via PC) can replenish TCA cycle or be used in gluconeogenesis [#2 above]

Pyruvate metabolism: Acetyl-CoA
#3: transition from glycolysis to the TCA cycle

Pyruvate metabolism: Lactate?
#4: End of anaerobic glycolysis (major pathway in RBCs, leukocytes, kidney medulla, lens, testes, and cornea)

Cori cycle
Allows lactate generated during anaerobic metabolism to undergo hepatic gluconeogenesis and become a source of glucose for muscle/RBCs. This comes at a cost of a net loss of 4 ATP/cycle. Shifts metabolic burden to the liver.

Pyruvate —< acetyl-CoA produces what?
1 NADH + 1 CO2

The TCA cycle (Krebs) produces what?
3 NADH, 1 FADH2, 2 CO2, 1 GTP per acetyl-CoA = 12 ATP/acetyl-CoA (2x everything per glucose).

Where does the TCA cycle rxtn take place?
In the mitochondria.

alpha-ketoglutarate dehydrogenase complex
Part of TCA cycle. Requires the same cofactors as the pyruvate dehydrogenase complex (B1, B2, B3, B5, lipoic acid)

Enzymes of TCA (Krebs cycle) + schematic
“C itrate I s K rebs’ S tarting S ubstrate F or M aking O xaloacetate.” *note the irreversible enzymes above in bold

What does the ETC do, and how does it relate to oxidative phosphorylation?
NADH electrongs from glycolysis and the TCA cycle enter mitochondria via the malate-aspartate or glycerol-3-phosphate shuttle. FADH2 electrons are transferred to complex II (at a lower energy level than NADH). The passage of electrons results in the formation of a proton gradient that, coupled to oxidative phosphorylation, drives the production of ATP.

ATP produced via ATP synthase
1 NADH —< 3 ATP 1 FADH2 --< 2 ATP

Oxidative phosphorylation poisons: ETC inhibitors?
Directly inhibit electron transport, causing a lower proton gradient and block of ATP synthesis. E.g., Retenone, CN-, antimycin A, CO

Oxidative phosphorylation poisons: ATPase inhibitors
Directly inhibit mitochondrial ATPase, causing an incr proton gradient. No ATP is produced b/c electron transport stops. E.g., Oligomycin

Oxidative phosphorylation proteins: uncoupling agents
Incr permeability of membrane, causing a decr proton gradient and incr O2 consumption. ATP synthesis drops, but electron transport continues. E.g., 2,4-DNP, aspirin, thermogenin in brown fat.

Gluconeogenesis, irreversible enzymes: Pyruvate carboxylase Location? Rxtn? Requires…?
In mitochondria. Pyruvate —< Oxaloacetate. Requires biotin, ATP. Activated by acetyl-CoA.

Gluconeogenesis, irreversible enzymes: PEP carboxykinase Location? Rxtn? Requires…?
In cytosol. Oxaloacetate —< phosphoenolpyruvate. Requires GTP.

Gluconeogenesis, irreversible enzymes: Fructose-1,6-bisphosphatase Location? Rxtn? Requires…?
In cytosol. Fructose-1,6-bisphosphate —< fructose-6-phosphate

Gluconeogenesis, irreversible enzymes: Glucose-6-phosphatase Location? Rxtn? Requires…?
In ER. Glucose-6-P —< glucose.

Mnemonic for the irreversible enzymes of gluconeogenesis?
“P athway P roduces F resh G lucose” P yruvate carboxylase P EP carboxykinase F ructose-1,6-bisphosphatase G lucose-6-phosphatase

Where does gluconeogenesis occur (anatomically)?
Occurs primarily in liver. Enzymes also found in kidney, intestinal epithelium.

Deficiency of gluconeogenesis enzymes causes…?
Causes hypoglycemia. (Muscle cannot participate in gluconeogenesis.)

Fatty acids and gluconeogenesis
Odd-chain FA’s yield 1 propionyl-CoA during metabolism, which can enter the TCA cycle, undergo gluconeogenesis, and serve as a glucose source. Even-chain FA’s cannot produce new glucose, sine they yield only acetyl-CoA equivalents.

HMP shunt (pentose phosphate pathway): What does it do? Where does it occur (in the cell)? What is the net ATP requirement or yield?
Produces NADPH, which is req’d for FA and steroid biosynthesis and for glutathione reduction inside RBCs. 2 distinct phases (oxidative and non-oxidative), both of which occur in the cytoplasm. No ATP is used or produced.

HMP shunt (pentose phosphate pathway): Where does it occur (anatomically)?
Lactating mamary glands, liver, adrenal cortex (sites of FA or steroid synthesis), RBCs

HMP shunt (pentose phosphate pathway): Oxidative rxtns (irreversible) — key enzymes? products?
Key enzymes: Glucose-6-phosphate dehydrogenase (rate-limiting step) Products: NADPH (for FA and steroid synthesis, glutathione reduction, and cytochrome P-450)

HMP shunt (pentose phosphate pathway): Nonoxidative (reversible) — key enzymes? products?
Key enzymes: Transketolases (require thiamine). Products: Ribose-5-phosphate (for nucleotide synthesis); G3P, F6P (glycolytic intermediates)

Respiratory burst (oxidative burst)
Involves activation of membrane-bound NADPH oxidase (e.g., in neutrophils, macrophages). Plays an important role in the immune response —< results in the rapid release of reactive oxygen species. [note enzymes/rxtns in schematic below]

Glucose-6-Phosphate dehydrogenase deficiency: molecular explanation?
NADPH is necessary to keep glutathione reducced, which in turn detoxifies free radicals and peroxides. Decr NADPH in RBCs leads to hemolytic anemia due to poor RBC defense against oxidizing agents (e.g., fava beans, sulfonamides, primaquine, antituberculosis drugs).

Glucose-6-phosphate dehydrogenase deficiency: Genetics? Findings in blood?
X-linked recessive d/o; most common human enzyme deficiency; more prevalent among blacks. Incr malarial resistance. H einz bodies — altered H emoglobin precipitated w/in RBCs Bite cells — result from the phagocytic removal of Heinz bodies by macrophages.

Fructose intolerance: Deficiency? Genetics? Biochem/molecular problem?
Hereditary deficiency in aldolase B . Autosomal recessive. Fructose-1-phosphate accumulates, causing a decr in available phosphate, which results in inhibition of glycogenolysis and gluconeogenesis. [fructose metabolism in liver — above]

Fructose intolerance: Sx?
Hypoglycemia, jaundice, cirrhosis, vomiting.

Fructose intolerance: Tx?
Decr intake of both fructose and sucrose (glucose + fructose)

Essential fructosuria: Defect? Genetics? Biochem/molecular problem?
Involves a defect in fructokinase . Autosomal recessive. A benign, asymptomatic condition, since fructose doesn’t enter cells. [above: fructose metabolism in liver]

Essential fructosuria: Sx?
Fructose appears in blood and urine (benign)

D/o of fructose metabolism vs. galactose metabolism?
D/o’s of fructose metabolism cause milder Sx than analogous d/o’s of galactose metabolism.

Classic galactosemia: Deficiency? Genetics? Biochem/molec problem?
Absence of galactose-1-phosphate uridyltransferase . Autosomal recessive. Damage is caused by accumulation of toxic substances (including galactitol, which accumulates in the lens of the eye).

Classic galactosemia: Sx?
Failure to thrive, jaundice, hepatomegaly, infantile cataracts, mental retardation.

Classic galactosemia: Tx?
exclude galactose and lactose (galactose + glucose) from diet.

Galactokinase deficiency: Deficiency? Genetics? Biochem/molec problem?
Hereditary deficiency of galactokinase . Autosomal recessive. Galactitol accumulates if galactose is present in diet. Relatively mild condition.

Galactokinase deficiency: Sx?
Galactose appears in blood and urine, infantile cataracts. May initially present as failure to track objects or to develop a social smile.

Lactase deficiency: what is it?
Age-dependent and/or hereditary lactose intolerance (blacks, Asians) due to loss of brush-border enzyme.

Lactase deficiency: Sx? Tx?
Bloating, cramps, osmotic diarrhea. Tx by avoiding dairy products or adding lactase pills to diet.

Amino acids: which form is found in proteins?
Only L-form AA’s are found in proteins.

Essential Glucogenic amino acids
Met, Val, Arg, His Glucogenic AA’s can be converted into glucose via gluconeogenesis.

Essential AA’s
Met, Val, Arg, His Ile, Phe, Thr, Trp Leu, Lys All essential AA’s need to be supplied in diet. (classified as glucogenic and/or ketogenic)

Glucogenic/ketogenic AA’s
Ile, Phe, Thr, Trp Glucogenic AA’s can be converted into glucose via gluconeogenesis. Ketogenic AA’s form ketone bodies.

Ketogenic AA’s
Leu, Lys Ketogenic AA’s form ketone bodies.

Acidic AA’s
Asp and Glu (negatively charged at body pH)

Basic AA’s
Arg, Lys, and His Arg is the most basic. His has no charge at body pH. Arg and His are req’d during periods of growth. Arg and His are elevated in histones, which bind (-) charged DNA.

Urea cycle: fxn/purpose?
Amino acid catabolism results in the formation of common metabolites (e.g., pyruvate, acetyl-CoA), which serve as metabolic fuels. Excess nitrogen (NH4+) generated by this process is converted to urea and excreted by the kidneys.

Mnemonic to remeber the important molecules in the urea cycle
“O rdinarily, C areless C rappers A re A lso F rivolous A bout U rination” O rnithine C arbamoyl phosphate C itrulline A spartate A rginosuccinate F umarate A rginate U rea

What do the atoms of urea come from?

Transport of ammonium by alanine and glutamine

Hyperammonemia: etiology?
Can be acquired (e.g., liver dz) or hereditary (e.g., urea cycle enzyme deficiencies)

Hyperammonemia: Result?
Results in exccess NH4+, which depletes alpha-ketoglutarate, leading to inhibition of the TCA cycle. Ammonia intoxication : tremor, slurring speech, somnolence, vomiting, cerebral edema, blurring of vision.

Tx for hyperammonemia
Benzoate or phenylbutyrate to lower serum ammonia levels.

Phenylalanine derivatives
1.) Phenylalanine —< Tyrosine --< Thyroxine + Dopa 2.) Dopa --< Melanin + Dopamine 3.) Dopamine --< NE --< Epi

Tryptophan derivatives
Tryptophan —< Niacin + Serotonin Niacin --< NAD+/NADP+ Serotonin --< Melatonin

Histadine derivatives
Histadine —< Histamine

Glycine derivatives
Glycine —< Porphyrin --< heme

Arginine derivatives
Arginine —< Creatinine, urea, NO

Glutamate derivatives
Glutamate —< GABA (glutamate decarboxylase -- requires B6) Glutamate --< Glutathione

Phenylketonuria: due to…? What does this mean molecularly?
Due to decr phenylalanine hydroxylase or decr tetrahydrobiopterin cofactor. Tyrosine becomes essential. Incr phenylalanine leads to excess phenylketones in urine.

Findings w/ PKU
Mental retardation, growth retardation, seizures, fair skin, eczema, musty body odor (d/o of aromatic AA metabolism —< musty body odor ).

Tx for PKU
Decr phenylalanine (contained in aspartame, e.g., NutraSweet) and incr tyrosine in diet.

Maternal PKU
Lack of proper dietary therapy during pregnancy. Findings in infant: microcephaly, mental retardation, growth retardation, congenital heart defects.

PKU: genetics? incidence? screening?
Autosomal recessive. Incidence ~ 1:10,000 Screened for 2-3d after birth.

Phenylketones
Phenylacetate, phenyllactate, and phenylpyruvate. Present in excess in urine w/ PKU.

Alkaptonuria (ochronosis): What is it? Genetics? Prognosis?
Congenital deficiency of homogentisic acid oxidase in the degradative pathway of tyrosine. Autosomal recessive. Benign dz.

Alkaptonuria (ochronosis): findings?
Dark connective tissue, pigmented sclera, urine turns black on standing. May have debilitating arthralgias.

Albinism: etiologies (+ genetics, when applicable)?
Congenital deficiency of either of the following: 1.) Tyrosinase (inability to synthesize melanin from tyrosine) — Autosomal recessive 2.) Defective tyrosine transporters (decr amounts of tyrosine, and thus melanin) Can result from lack of migration of neural crest cells. Variable inheritance due to locus heterogeneity (vs. ocular albinism — X-linked recessive)

Risk of what dz w/ albinism?
Lack of melanin results in an incr risk of skin cancer.

3 Forms of homocystinuria
1.) Cystathionine synthase deficiency (Tx: decr Met and Incr Cys, and incr B12 and folate in diet) 2.) Decr affinity of cystathionine synthase for pyridoxal phosphate (Tx: incr (++) vitamin B6 in diet) 3.) Homocysteine methyltransferase deficiency

Commonalities for all 3 forms of homocystinuria
All are autosomal recessive. All forms result in excess homocysteine. Cystine becomes essential.

Findings w/ homocystinuria
(++) homocysteine in urine, mental retardation, osteoporosis, tall stature, kyphosis, lens subluxation (downward and inward), and atherosclerosis (stroke and MI)

Cystinuria: Etiology? Genetics/incidence?
Hereditary defect of renal tubular AA transporter for cysteine, ornithine, lysine, and arginine in the PCT of the kidneys. Autosomal recessive and common (1:7,000)

Cystinuria: findings? Tx?
Excess cystine in urine can lead to precipitation of cystine kidney stones (cystine staghorn calculi). Tx: acetazolamide to alkalinize urine *Cystine is make of 2 cysteines connected by a disulfide bond.

Maple syrup urine dz: etiology?
Blocked degradation of branched amino acids (I le, L eu, V aline) due to decr alpha-ketoglutarate dehydrogenase. (“I L ove V ermont maple syrup from maple trees with branches .”)

Maple syrup urine dz: findings?
Causes severe CNS defects, mental retardation, and death. Urine smells like maple syrup.

Purine salvage deficiencies: Adenosine deaminase deficiency
Excess ATP and dATP imbalances nucleotide pool via feedback inhibition of ribonucleotide reductase. | Prevents DNA synthesis and thus decr lymphocyte count. One of the major causes of SCID. [ADA is #3 below]

SCID (sever combined immunodeficiency dz)
SCID happens to kids (e.g., “bubble boy”). 1st dz to be Tx w/ experimental human gene therapy. One of the major causes of SCID: ADA deficiency [defect in purine salvage — #3, below]

Purine salvage deficiencies: Lesch-Nyhan syndrome
Defective purine salvage owing to absence of HGPRT [#1, below], which converts hypoxanthine to IMP and guanine to GMP. Results in excess uric acid production.

Lesch-Nyhan syndrome: findings?
Retardation, self-mutilation, aggression, hyperuricemia, gout, choreoathetosis.

Lesch-Nyhan syndrome: genetics?
X-linked recessive. HGPRT gene: “H e’s G ot P urine R ecovery T rouble”

Orotic aciduria: etiology? genetics?
Inability to convert orotic acid UMP (de novo pyrimidine synthesis pathway) due to defect in either orotic acid phosphoribosyltransferase or orotidine 5′-phosphate decarboxylase. Autosomal recessive.

Orotic aciduria: findings?
Incr orotic acid in urine, megaloblastic anemia (does not improve w/ administration of vitamin B12 or folic acid), failure to thrive. No hyperammonemia (vs. OTC deficiency — incr orotic acid w/ hyperammonemia).

Orotic aciduria: Tx?
Oral uridine administration.

Insulin: when/where is it made? What are its basic effects?
Made in Beta-cells of panccreas in response to ATP from glucose metabolism acting on K+ channels and depolarizing cells. Required for adipose and skeletal muscle uptake of glucose. Inhibits glucagon release by alpha-cells of pancreas.

C-peptide
Serum C-peptide is not present w/ exogenous insulin intake (proinsulin —< insulin + C-peptide)

Anabolic effects of inuslin (list of 6)
1.) Incr glucose transport (“In sulin moves glucose In to cells.” 2.) Incr glycogen synthesis and storage 3.) Incr TG synthesis and storage 4.) Incr Na+ retention (kidneys) 5.) Incr protein synthesis (muscles) 6.) Incr cellular uptake of K+

Tissues that don’t need insulin for glucose uptake
“BRICK L ” B rain R BCs I ntestine C ornea K idney L iver

GLUT1 transporter
RBCs, brain

GLUT2 transporter
(bidirectional) Beta-islet cells, liver, kidney

GLUT4 transporter
(insulin responsive) Adipose tissue, skeletal muscle

Glycogen synthase: metabolism and activity?
Fed state: *GS* (active) vs. Fasting state: GS-P (phosphorylated, inactive)

Glycogen synthase: regulation in liver and muscle?
Regulation in liver: (+): Insulin, Glucose (-): Glucagon, epinephrine Regulation in muscle: (+): Insulin (-): Epinephrine

Glycogen phosphorylase (in muscle, V): metabolism and activity?
Is phosphorylated/dephosphorylated similarly to glycogen synthase, with the opposite resulting activity: GP (inactive) in fed state vs. *GP-P* (active, phosphorylated) in fasting state

Glycogen phosphorylase (in muscle, V): regulation in liver and muscle?
Regulation in liver: (+): Epinephrine, Glucagon (-): Insulin Regulation in muscle: (+): AMP, Epinephrine (-): ATP, Insulin

Phosphorylation and Insulin vs. Glucagon
Insulin de phosphorylates (decr cAMP —< decr PKA) Glucagon phosphorylates (incr cAMP --< incr PKA)

Glycogen structure
Branches have alpha(1,6) bonds; Linkages have alpha(1,4) bonds.

Glycogen in skeletal muscle
Glycogen undergoes glycogenolysis to form glucose, which is rapidly metabolized during exercise.

Glycogen in hepatocytes
Glycogen is stored and undergoes glycogenolysis to maintain blood sugar at appropriate levels.

Glycogen synthesis

Glycogenolysis/glycogen synthesis cycle

Glycogen synthesis dz’s: generallly How many types? Molec path? List?
12 types, all resulting in abnormal glycogen metabolism and an accumulation of glycogen w/in cells. V ery P oor C arbohydrate M etabolism: V on Gierke’s dz (type 1) P ompe’s dz (type 2) C ori’s dz (type 3) M cArdle’s dz (type 5)

Glycogen storage dz’s: Von Gierke’s dz (type I) Findings? Deficient enzyme? Comments?
Findings: severe fasting hypoglycemia, (++) glycogen in liver, incr blood lactate, hepatomegaly. Deficient enzyme: Glucose-6-phosphatase [see below]

Glycogen storage dz’s: Pompe’s dz (type II) Findings? Deficient enzyme? Comments?
Findings: cardiomegaly and systemic findings leading to early death. Deficient enzyme: Lysosomal alpha-1,4 glucosidase (acid maltase) [see below] “P ompe’s trashes the P ump (heart, liver, and muscle).”

Glycogen storage dz’s: Cori’s dz (type III) Findings? Deficient enzyme? Comments?
Findings: milder form of type I w/ normal blood lactate levels. Deficient enzyme: Debranching enzyme (alpha-1,6-glucosidase) [see below] Gluconeogenesis is intact.

Glycogen storage dz’s: McArdle’s dz (type V) Findings? Deficient enzyme? Comments?
Findings: incr glycogen in muscle, but cannot break it down, leading to painful muscle cramps, myoglobinuria w/ strenuous exercise. Deficient enzyme: Skeletal muscle glycogen phosphorylase. [see below] M cArdle’s = M uscle

Lysosomal storage dz’s (generally)
Each is caused by a deficiency in one of the many lysosomal enzymes. Results in an accumulation of abnormal metabolic products. Include sphingolipidoses and mucopolysaccharidoses.

Lysosomal storage dz’s, sphingolipidoses: Fabry’s dz Findings? Deficient Enzyme? Accumulated substrate? Inheritance?
Findings: peripheral neuropathy of hands/feet, angiokeratomas, CV/renal dz. Def. enzyme: alpha-galactosidease A Accum substrate: Ceramide trihexose Inheritance: XR

What sphingolipidosis (lysosomal storage dz) does this describe? [don’t look at the picture if you want to guess] Findings: peripheral neuropathy of hands/feet, angiokeratomas, CV/renal dz. Def. enzyme: alpha-galactosidease A Accum substrate: Ceramide trihexose Inheritance: XR
Fabry’s dz

Lysosomal storage dz’s, sphingolipidoses: Gaucher’s dz (most common) Findings? Deficient Enzyme? Accumulated substrate? Inheritance?
Findings: hepatosplenomegaly, aseptic necrosis of femur, bone crises, Gaucher’s cells (macrophages that look like crumpled tissue paper) Def enzyme: Beta-glucocerebrosidase Accum substrate: glucocerebroside Inheritance: AR

What sphingolipidosis (lysosomal storage dz) does this describe? [don’t look at the picture if you want to guess] Findings: hepatosplenomegaly, aseptic necrosis of femur, bone crises, Gaucher’s cells (macrophages that look like crumpled tissue paper) Def enzyme: Beta-glucocerebrosidase Accum substrate: glucocerebroside Inheritance: AR
Gaucher’s dz (most common)

Lysosomal storage dz’s, sphingolipidoses: Niemann-Pick dz Findings? Deficient Enzyme? Accumulated substrate? Inheritance?
Findings: progressive neurodegeneration, hepatosplenomegaly, cherry-red spot on macula, lysosomes w/ onion skin. Def. enzyme: sphingomyelinase Accum substrate: sphingomyelin Inheritance: AR

What sphingolipidosis (lysosomal storage dz) does this describe? [don’t look at the picture if you want to guess] Findings: progressive neurodegeneration, hepatosplenomegaly, cherry-red spot on macula, lysosomes w/ onion skin. Def. enzyme: sphingomyelinase Accum substrate: sphingomyelin Inheritance: AR
Niemann-Pick dz

Lysosomal storage dz’s, sphingolipidoses: Tay-Sachs dz Findings? Deficient Enzyme? Accumulated substrate? Inheritance?
Findings: progressive neurodegeneration, developmental delay, optic atrophy, globoid cells. Def. enzyme: hexosaminidase A Accum substrate: GM2 ganglioside. Inheritance: AR

What sphingolipidosis (lysosomal storage dz) does this describe? [don’t look at the picture if you want to guess] Findings: progressive neurodegeneration, developmental delay, optic atrophy, globoid cells. Def. enzyme: hexosaminidase A Accum substrate: GM2 ganglioside. Inheritance: AR
Tay-Sachs dz

Lysosomal storage dz’s, sphingolipidoses: Krabbe’s dz Findings? Deficient Enzyme? Accumulated substrate? Inheritance?
Findings: Peripheral neuropathy, developmental delay, optic atrophy, globoid cells Def. enzyme: Galactocerebrosidase Accum substrate: Galactocerebroside Inheritance: AR

What sphingolipidosis (lysosomal storage dz) does this describe? [don’t look at the picture if you want to guess] Findings: Peripheral neuropathy, developmental delay, optic atrophy, globoid cells Def. enzyme: Galactocerebrosidase Accum substrate: Galactocerebroside Inheritance: AR
Krabbe’s dz

Lysosomal storage dz’s, sphingolipidoses: Metachromatic leukodystrophy Findings? Deficient Enzyme? Accumulated substrate? Inheritance?
Findings: central and peripheral demyelination w/ ataxia, dementia. Def. enzyme: Arylsulfatase A Accum substrate: Cerebroside sulfate Inheritance: AR

What sphingolipidosis (lysosomal storage dz) does this describe? [don’t look at the picture if you want to guess] Findings: central and peripheral demyelination w/ ataxia, dementia. Def. enzyme: Arylsulfatase A Accum substrate: Cerebroside sulfate Inheritance: AR
Metachromatic leukodystrophy

Lysosomal storage dz’s, mucopolysaccharidoses: Hurler’s syndrome Findings? Deficient Enzyme? Accumulated substrate? Inheritance?
Findings: developmental delay, gargoylism, airway obstruction, corneal clouding, hepatosplenomegaly. Def. enzyme: alpha-L-iduronidase Accum substrate: Heparan sulfate, dermatan sulfate Inheritance: AR

What mucopolysaccharidosis (lysosomal storage dz) does this describe? [don’t look at the picture if you want to guess] Findings: developmental delay, gargoylism, airway obstruction, corneal clouding, hepatosplenomegaly. Def. enzyme: alpha-L-iduronidase Accum substrate: Heparan sulfate, dermatan sulfate Inheritance: AR
Hurler’s syndrome

Lysosomal storage dz’s, mucopolysaccharidoses: Hunter’s syndrome Findings? Deficient Enzyme? Accumulated substrate? Inheritance?
Findings: Mild Hurler’s + aggressive behavior, no corneal clouding. Def. enzyme: Iduronate sulfatase Accum substrate: Heparan sulfate, dermatan sulfate Inheritance: XR

What mucopolysaccharidosis (lysosomal storage dz) does this describe? [don’t look at the picture if you want to guess] Findings: Mild Hurler’s + aggressive behavior, no corneal clouding. Def. enzyme: Iduronate sulfatase Accum substrate: Heparan sulfate, dermatan sulfate Inheritance: XR
Hunter’s syndrome

Mnemonic for Niemann-Pick dz?
No man picks (Niemann-Pick ) his nose with his sphinger (sphingo myelinase).

Mnemonic for Tay-Sachs?
Tay-SaX lacks heX osaminidase

Mnemonic for Hunter’s syndrome?
Hunters see cclearly (no corneal clouding) and aim for the X (X -linked recessive).

Lysosomal storage dz’s: population at risk?
Incr incidence of Tay-Sachs, Niemann-Pick, and some forms of Gaucher’s dz in Ashkenazi Jews.

Fatty acid metabolism: synthesis
“SY trate = SY nthesis”

Fatty acid metabolism: degradation
“CAR nitine = CAR nage of FA’s”

Carnitine deficiency
Inability to utilize LCFAs and toxic accumulation

Acyl-CoA dehydrogenase deficiency
Incr dicarboxylic acids, decr glucose and ketones

Where does FA degradation take place?
Occurs where its products will be consumed: in the mitochondrion.

Ketone bodies: in liver?
In the liver, FAs and AAs are metabolized to acetoacetate and Beta-hydroxybutyrate (to be used in muscle and brain)

Ketone bodies: in prolonged starvation or diabetic ketoacidosis? … in alcoholism? Why are these two cases related?
In prolonged starvation or diabetic ketoacidosis, oxaloacetate is depleted for gluconeogenesis. In alcoholism, excess NADH shunts oxaloacetate to malate. Both processes stall the TCA cycle, which shunts glucose and FFA twd the production of ketone bodies.

Ketone bodies: made from…? How are they metabolized in brain? How are they excreted?
Made from HMG-CoA. Metabolized in brain to 2 molecules of acetyl-CoA. Excreted in urine.

Ketone bodies: findings w/ elevated levels?
Breath smells like acetone (fruity odor). Urine test for ketones does not detect beta-hydroxybutyrate (favored by high redox state).

Metabolic fuel use: 1g protein = ? 1g fat = ?
1g protein = 4 kcal 1g fat = 9 kcal

Metabolic fuel use: in exercise (generally)
As distances increase, ATP is obtained from additional sources.

Metabolic fuel use: in exercise — 100-meter sprint (seconds)
Stored ATP, creatine phosphate, anaerobic glycolysis

Metabolic fuel use: in exercise — 1000-meter run (minutes)
Stored ATP, creatine phosphate, anaerobic glycolysis (>— as used in sprint) + Oxidative phosphorylation

Metabolic fuel use: in exercise — marathon (hours)
Glycogen and FFA oxidation; glucose conserved for final sprinting.

Metabolic fuel use: Fasting and starvation (generally)
Priorities are to supply sufficient glucose to brain and RBCs and to preserve protein.

Metabolic fuel use: fasting and starvation — days 1-3
Blood glucose is maintained by: 1.) Hepatic glycogenolysis and glucose release 2.) Adipose release of FFA 3.) Muscle and liver shifting fuel use from glucose to FFA 4.) Hepatic gluconeogenesis from peripheral tissue lactate and alanine, and from adipose tissue glycerol and propionyl-CoA from odd-chain FFA metabolism (the only TG components that can contribute to gluconeogenesis)

Metabolic fuel use: fasting and starvation — after day 3
Muscle protein loss is maintained by hepatic formation of ketone bodies, supplying the brain and heart.

Metabolic fuel use: fasting and starvation — after several weeks
Ketone bodies become main source of energy for brain, so less muscle protein is degraded than during days 1-3. Survival time is determined by amount of fat stores. After this is depleted, vital protein degradation accelerates, leading to organ failure and death.

Cholesterol synthesis
Rate-limiting step is catalyzed by HMG-CoA reductase* , which converts HMG-CoA —< mevalonate. 2/3 of plasma XOL is esterified by lecithin-cholesterol acyltransferase (LCAT). *Statins (e.g., lovastatin) inhibit HMG-CoA reductase

Essential fatty acids
Linoleic and linolenic acids. Arichidonic acid, if linoleic acid is absent. Eicosanoids are dependent on essential FA’s.

Lipid transport overall flow-chart/schematic

Lipid transport enzymes: Pancreatic lipase
Degradation of dietary TG in small intestine [not labeled in image below]

Lipid transport enzymes: Lipoprotein lipase (LPL)
Degradation of TG circulating in chylomicrons and VLDLs [active at several points in the schematic below]

Lipid transport enzymes: Hepatic TG lipase (HL)
Degradation of TG remaining in IDL [labeled at two points in the schematic below]

Lipid transport enzymes: Hormone-sensitive lipase
Degradation of TG stored in adipocytes [not labeled in schematic below]

Lipid transport enzymes: Lecithin-cholesterol acyltransferase (LCAT)
Catalyzes esterification of XOL

Lipid transport enzymes: Cholesterol ester transfer protein (CETP)
Mediates transfer of XOL-esters to other lipoprotein particles.

Major apolipoproteins: A-I
A -I A ctivates LCAT

Major apolipoproteins: B-100
B -100 B inds to LDL recceptor, mediates VLDL secretion.

Major apolipoproteins: C-II
C -II is a C ofactor for lipoprotein lipase.

Major apolipoproteins: B-48
Mediates chylomicron secretion.

Major apolipoproteins: E
E mediates E xtra (remnant) uptake

Lipoprotein fxns (generally)
Lipoproteins are composed of varying proportions of XOL, TGs, and phospholipids. LDL and HDL carry most XOL.

Mnemonic for LDL vs. HDL
LDL transports XOL from liver to tissues: “L DL is L ousy” HDL transports XOL from periphery to liver: “H DL is H ealthy”

Type of lipoprotein: Chylomicron Function and route? Apolipoproteins?
Deligers dietary TGs to peripheral tissue. Delivers XOL to liver in the form of chylomicron remnants, which are mostly depleted of their triaclyglycerols. Secreted by intestinal epithelial cells. Apolipoproteins: B-48, A-IV, C-II, and E

Type of lipoprotein: VLDL Function and route? Apolipoproteins?
Delivers hepatic TGs to peripheral tissue. Secreted by liver. Apo’s: B-100, C-II, and E

Type of lipoprotein: LDL Function and route? Apolipoproteins?
Delivers hepatic XOL to peripheral tissues. Formed by lipoprotein lipase modification of VLDL in the peripheral tissue. Taken up by target cells via receptor-mediated endocytosis. Apo’s: B-100

Type of lipoprotein: HDL Function and route? Apolipoproteins?
Mediates reverse XOL transport from periphery to liver. Acts as a repository for apoC and apoE (which are needed for chylomicron and VLDL metabolism). Secreted from both liver and intestine.

Familial dyslipidemia: Type I – hyperchylomicronemia What’s increased? Elevated blood levels of…? Pathophys?
Increased: chylomicrons Elevated blood levels: TG, XOL Pathophys: lipoprotein lipase deficiency or altered apoplipoprotein C-II

Increased: chylomicrons Elevated blood levels: TG, XOL Pathophys: lipoprotein lipase deficiency or altered apoplipoprotein C-II What familial dyslipidemia does this describe?
Type I – hyperchylomicronemia

Familial dyslipidemia: Type IIa – familial hypercholesterolemia What’s increased? Elevated blood levels of…? Pathophys?
Increased: LDL Elevated blood levels: XOL Pathophys: Autosomal dominant; absent or decr LDL receptors.

Increased: LDL Elevated blood levels: XOL Pathophys: Autosomal dominant; absent or decr LDL receptors. What familial dyslipidemia does this describe?
Type IIa – familial hypercholesterolemia

Familial dyslipidemia: Type IV – hypertriglyceridemia What’s increased? Elevated blood levels of…? Pathophys?
Increased: VLDL Elevated blood levels: TG Pathophys: hepatic overproduction of VLDL

Increased: VLDL Elevated blood levels: TG Pathophys: hepatic overproduction of VLDL What familial dyslipidemia does this describe?
Type IV – hypertriglyceridemia

Abetalipoproteinemia: what is it? Genetics? Sx? Findings?
Hereditary inability to synthesize lipoproteins due to deficiencies in apoB-100 and apoB-48. Autosomal recessive. Sx appear in the first few months of life. Findings: failure to thrive, steatorrhea, acanthocytosis, ataxia, night blindness.

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