BIOCHEMISTRY Flashcards
DNA exists in the condensed, ________ form in order to fit into the nucleus.
Chromatin
How are nucleosome formed?
Negatively charged DNA loops twice around positively charged histone octamer to form nucleosome “bead.”
What are Histones rich in? and how do they stabilize the chromatin fiber?
Histones are rich in the amino
acids lysine and arginine. H1 binds to the nucleosome and to “linker DNA,” thereby stabilizing the chromatin fiber.
In mitosis, DNA condenses to form _______
Chromosomes
During which phase are DNA and histone synthesized?
In mitosis, DNA condenses to form chromosomes. DNA and histone synthesis occur during S phase.
____is the only histone that is not in the nucleosome core.
H1
Nucleosome core
histones
H2A, H2B, H3, H4
each ×2
Heterocromatin
HeteroChromatin = Highly Condensed.Condensed, transcriptionally inactive, sterically
inaccessible.
Euchromatin
Eu = true, “truly transcribed.”Less condensed, transcriptionally active, sterically accessible.
DNA methylation
CpG Methylation Makes DNA Mute. Template strand cytosine and adenine are methylated in DNA replication, which allows
mismatch repair enzymes to distinguish between old and new strands in prokaryotes. DNA methylation at CpG islands represses transcription.
Histone methylation
Histone Methylation Mostly Makes DNA Mute Usually reversibly represses DNA transcription, but can activate it in some cases.
Histone acetylation
Histone Acetylation makes DNA Active. Relaxes DNA coiling, allowing for transcription
Nucleotides
PURines (A, G)—2 rings.
PYrimidines (C, T, U)—1 ring.
PURe As Gold.
CUT the PY (pie).
Amino acids necessary for purine
synthesis
Glycine
Aspartate
Glutamine
Deamination of cytosine makes
uracil
Difference between nucleotides and nucleosides
NucleoSide = base + (deoxy)ribose (Sugar).
NucleoTides = base + (deoxy)ribose + phosphaTe; linked by 3′-5′ phosphodiester
bond.
Lesch-Nyhan
syndrome
Defective purine salvage due to absent HGPRT(see below),
which converts hypoxanthine to IMP and guanine to GMP. Results in excess uric acid production and de novo purine synthesis. X-linked recessive. Findings: intellectual disability, self-mutilation, aggression, hyperuricemia, gout, dystonia.Treatment: allopurinol or febuxostat (2nd line).
HGPRT: Hyperuricemia Gout Pissed off (aggression, self-mutilation) Retardation (intellectual disability) DysTonia
Adenosine deaminase
deficiency
Excess ATP and dATP imbalances nucleotide pool via feedback inhibition of ribonucleotide
reductase prevents DNA synthesis and thus lymphocyte count.
One of the major causes of autosomal recessive
SCID.
A 6-year-old male child evaluated for treatment of lip lacerations associated with self-mutilating behavior. The parents reported that from infancy the child had been very quite and did not elicit response to many stimuli, though cerebral palsy and mental retardation was not noted. The child had no complain of pain. The patient had chewed most of the fingers leaving a small stump of 1-2 cm. What would you suspect?
LNS- Lesch-Nyhan
syndrome
A 6 years old girl presented with repeated episodes of respiratory infections since the age of 2 years. Frequency of such attacks were variable. However, minimum two such episodes were noticed per month. After observing the patient for a month it was noticed that she was more susceptible infections, particularly those of the skin, respiratory system, and gastrointestinal tract
What would you suspect?
ADA- Adenosine deaminase
deficiency
Genetic code features: Unambiguous
Each codon specifies only 1 amino acid.
Genetic code features: Degenerate/
redundant and exceptions
Most amino acids are coded by multiple codons. Exceptions: methionine and tryptophan encoded by only 1 codon (AUG and UGG, respectively).
Genetic code features: Commaless,
nonoverlapping and exceptions
Read from a fixed starting point as a continuous
sequence of bases. Exceptions: some viruses.
Genetic code features: Commaless,
nonoverlapping and exceptions
Read from a fixed starting point as a continuous
sequence of bases. Exceptions: some viruses.
Genetic code features: Universal and exceptions
Genetic code is conserved throughout evolution. Exception in humans: mitochondria
DNA replication: Origin of replication
Particular consensus sequence of base pairs in genome where DNA replication begins. May be single (prokaryotes) or multiple
(eukaryotes).
DNA replication: Replication fork
Y-shaped region along DNA template where leading and lagging strands are synthesized.
DNA replication: Helicase
Unwinds DNA template at replication fork.
DNA replication: Single-stranded
binding proteins
Prevent strands from reannealing
DNA replication: DNA
topoisomerases
Create a single- or double-stranded break in the
helix to add or remove supercoils.
What inhibits DNA topoisomerase II?
Fluoroquinolones—inhibit DNA gyrase (prokaryotic topoisomerase II).
DNA replication: Primase
Makes an RNA primer on which DNA polymerase III can initiate replication.
DNA 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.
DNA polymerase III: proofreads and synthesis
DNA polymerase III has 5′ 3′ synthesis and proofreads with 3′ 5′ exonuclease.
DNA replication:DNA polymerase I
Prokaryotic only. Degrades RNA primer; replaces it with DNA.Has same functions as DNA polymerase III but also excises RNA primer with 5′ 3′ exonuclease.
DNA replication: DNA ligase
Seals! Catalyzes the formation of a phosphodiester bond within a strand of double-stranded DNA
(i.e., joins Okazaki fragments).
DNA replication: Telomerase
An RNA-dependent DNA polymerase that adds
DNA to 3′ ends of chromosomes to avoid loss of genetic material with every duplication.
Mutations in DNA according to the severity of the damage
silent «_space;missense < nonsense < frameshift
Transition
purine to purine (e.g., A to G) or pyrimidine to pyrimidine (e.g., C to T).
Transversion
purine to pyrimidine (e.g., A to T) or pyrimidine to purine (e.g., C to G).
Mutations in DNA: Silent
Nucleotide substitution but codes for same (synonymous) amino acid; often base change in 3rd position of codon (tRNA wobble).
Missense
Nucleotide substitution resulting in changed amino acid (called conservative if new amino acid is similar in chemical structure).
Nonsense
Nucleotide substitution resulting in early stop codon. (stop the nonsense)
Frameshift
Deletion or insertion of a number of nucleotides not divisible by 3, resulting in misreading of all
nucleotides downstream, usually resulting in a truncated, nonfunctional protein.
12-year-old boy from Guyana who is referred by his family physician for jaundice, normocytic anemia, and recurrent acute bone pains. Blood film revealed numerous sickle cells. Sickle solubility test is positive. What kind of mutation produced this disease?
Missense
James Fenlow, a 19-year old male, is immobile and hospitalized for pneumonia. He has a long history of progressive weakening of his muscles. A Gower’s sign was noted by age four, as was a Trendelenberg gait. James suffers from a condition called Duchenne muscular dystrophy. Which mutation produced his dystrophy?
Frameshift
DNA repair: SS: Nucleotide excision
repair
Specific endonucleases release the oligonucleotide-containing damaged bases; DNA polymerase and ligase fill and reseal the
gap, respectively. Repairs bulky helix-distorting lesions.
DNA repair: SS: Base excision repair
Base-specific glycosylase recognizes altered base and creates AP site (apurinic/apyrimidinic). One or more nucleotides are removed by APendonuclease, which cleaves the 5′ end. Lyase cleaves the 3′ end. DNA polymerase-b fills the gap and DNA ligase seals it.
DNA repair: SS: Mismatch repair
Newly synthesized strand is recognized,
mismatched nucleotides are removed, and the
gap is filled and resealed.
Xeroderma pigmentosum (XP) is a rare disorder, inherited as an autosomal recessive gemodermatosis. It is characterized by photosensitivity, freckly pigmented changes, premature skin ageing, telegiectasis, warty and papillomatous growth and malignant tumor development in later stage. What kind of SS DNA repair is defective?
Nucleotide excision
repair is defective in xeroderma pigmentosum, which
prevents repair of pyrimidine dimers because
of ultraviolet light exposure.
In the human body, deamination takes place primarily in the liver. Deamination is the process by which amino acids are broken down if there is an excess of protein intake. The amino group is removed from the amino acid and converted to ammonia. Ammonia is toxic to the human system. In the case of spontaneous deamination which SS DNA repair is important?
Base excision repair is important in repair of spontaneous/toxic
deamination.
Lynch syndrome (HNPCC or hereditary nonpolyposis colorectal cancer ) is an autosomal dominant genetic condition that has a high risk of colon cancer as well as other cancers including endometrial cancer (second most common), ovary, stomach, small intestine, hepatobiliary tract, upper urinary tract, brain, and skin. The increased risk for these cancers is due to inherited mutations that impair _________.
DNA Mismatch repair is defective in hereditary nonpolyposis colorectal
cancer (HNPCC).
DNA repair :DS: Nonhomologous end
joining
Brings together 2 ends of DNA fragments to repair double-stranded breaks. No requirement
for homology.
Ataxia-telangiectasia is a rare inherited disorder that affects the nervous system, immune system, and other body systems. This disorder is characterized by progressive difficulty with coordinating movements (ataxia) beginning in early childhood, usually before age 5. Which DNA repair method is affected.
Mutated in ataxia telangiectasia
DNA/RNA/protein
synthesis direction
DNA and RNA are both synthesized 5′ 3′.
The 5′ end of the incoming nucleotide bears
the triphosphate (energy source for bond).
Protein synthesis is N-terminus to C-terminus
mRNA synthesis direction
mRNA is read 5′ to 3′.
The triphosphate bond is the target of the
_____ attack.
The triphosphate bond is the target of the
3′ hydroxyl attack. Drugs blocking DNA
replication often have modified 3′ OH,
preventing addition of the next nucleotide
(“chain termination”).
mRNA start codons
AUG (or rarely GUG). AUG inAUGurates protein synthesis
mRNA stop codons
UGA, UAA, UAG. UGA = U Go Away.
UAA = U Are Away.
UAG = U Are Gone.
Regulation of gene expression:Promoter
Site where RNA polymerase and multiple other
transcription factors bind to DNA upstream
from gene locus (AT-rich upstream sequence
with TATA and CAAT boxes).Promoter mutation commonly results in dramatic lower in level of gene transcription.
Regulation of gene expression: Enhancer
Stretch of DNA that alters gene expression by
binding transcription factors.Enhancers and silencers may be located close to, far from, or even within (in an intron) the gene whose expression it regulates
Regulation of gene expression: Silencer
Site where negative regulators (repressors) bind.
RNA polymerases:Eukaryotes
RNA polymerase I makes rRNA (most
numerous RNA, rampant).
RNA polymerase II makes mRNA (largest RNA,
massive).
RNA polymerase III makes tRNA (smallest
RNA, tiny).
No proofreading function, but can initiate
chains. RNA polymerase II opens DNA at promotor site.
RNA polymerases: Prokaryotes
1 RNA polymerase (multisubunit complex)
makes all 3 kinds of RNA.
RNA processing (eukaryotes)
Initial transcript is called heterogeneous nuclear
RNA (hnRNA). hnRNA is then modified and
becomes mRNA.The following processes occur in the nucleus following transcription:
Capping of 5′ end (addition of 7-methylguanosine cap)
Polyadenylation of 3′ end (≈ 200 A’s)
Splicing out of introns Capped, tailed, and spliced transcript is called mRNA.
Splicing of pre-mRNA
1)Primary transcript combines with small
nuclear ribonucleoproteins (snRNPs) and
other proteins to form spliceosome.
2)Lariat-shaped (looped) intermediate is
generated.
3)Lariat is released to precisely remove intron
and join 2 exons.
Antibodies to spliceosomal snRNPs (anti-
Smith antibodies) are highly specific for SLE.
Anti-U1 RNP antibodies are highly associated
with mixed connective tissue disease.
Introns vs. exons
Exons contain the actual genetic information
coding for protein.
Introns are intervening noncoding segments of
DNA.
Different exons are frequently combined by
alternative splicing to produce a larger number
of unique proteins.
tRNA: Structure
75–90 nucleotides, 2º structure, cloverleaf form, anticodon end is opposite 3′ aminoacyl end. All tRNAs, both eukaryotic and prokaryotic, have CCA at 3′ end along with a high percentage of chemically modified bases. The amino acid is covalently bound to the 3′ end of the tRNA. CCA
Can Carry Amino acids.
T-arm: contains the TΨC (thymine, pseudouridine, cytosine) sequence necessary for tRNAribosome
binding.
D-arm: contains dihydrouracil residues necessary for tRNA recognition by the correct aminoacyltRNA
synthetase.
Acceptor stem: the 3′ CCA is the amino acid acceptor site.
tRNA: Charging
Aminoacyl-tRNA synthetase (1 per amino acid; “matchmaker”; uses ATP) scrutinizes amino acid
before and after it binds to tRNA. If incorrect, bond is hydrolyzed. The amino acid-tRNA bond
has energy for formation of peptide bond. A mischarged tRNA reads usual codon but inserts
wrong amino acid.
Aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible for
accuracy of amino acid selection.
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/amino acid (as a result
of degeneracy of genetic code).
Protein synthesis:Initiation
Initiated by GTP hydrolysis; initiation factors
(eukaryotic IFs) help assemble the 40S
ribosomal subunit with the initiator tRNA
and are released when the mRNA and the
ribosomal 60S subunit assemble with the
complex.
Protein synthesis:Elongation
- Aminoacyl-tRNA binds to A site (except for
initiator methionine) - rRNA (“ribozyme”) catalyzes peptide bond
formation, transfers growing polypeptide to
amino acid in A site - Ribosome advances 3 nucleotides toward 3′
end of mRNA, moving peptidyl tRNA to P
site (translocation)
Protein synthesis:Termination
Stop codon is recognized by release factor,
and completed polypeptide is released from
ribosome.
Posttranslational modifications:Trimming
Removal of N- or C-terminal propeptides from zymogen to generate mature protein (e.g., trypsinogen to trypsin).
Posttranslational modifications:Covalent alterations
Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and ubiquitination.
Chaperone protein
Intracellular protein involved in facilitating and/or maintaining protein folding.
In yeast, some are heat shock proteins (e.g., Hsp60) that are expressed at high temperatures to
prevent protein denaturing/misfolding.
Cell cycle phases
Checkpoints control transitions between phases of cell cycle. This process is regulated by cyclins, cyclin-dependent kinases (CDKs), and tumor suppressors. Mitosis (shortest phase of cell cycle) includes prophase, metaphase, anaphase, and telophase. G1 and G0 are of variable duration
REGULATION OF CELL CYCLE: CDKs
Constitutive and inactive.
REGULA TION OF CELL CYCLE: Cyclins
Regulatory proteins that control cell cycle events; phase specific; activate CDKs
REGULA TION OF CELL CYCLE:Cyclin-CDK complexes
Must be both activated and inactivated for cell
cycle to progress.
REGULA TION OF CELL CYCLE:Tumor suppressors
p53 and hypophosphorylated Rb normally
inhibit G1-to-S progression; mutations in these
genes result in unrestrained cell division (e.g.,
Li-Fraumeni syndrome).
CELL TYPES: Permanent
Remain in G0, regenerate from stem cells. Neurons, skeletal and cardiac muscle, RBCs
CELL TYPES: Stable (quiescent)
Enter G1 from G0 when stimulated. Hepatocytes, lymphocytes.
CELL TYPES: Labile
Never go to G0, divide rapidly with a short G1.
Most affected by chemotherapy.
Bone marrow, gut epithelium, skin, hair follicles,
germ cells.
Rough endoplasmic reticulum
Site of synthesis of secretory (exported) proteins
and of N-linked oligosaccharide addition to
many proteins. Nissl bodies (RER in neurons)—synthesize
peptide neurotransmitters for secretion.
Free ribosomes—unattached to any membrane;
site of synthesis of cytosolic and organellar
proteins.
Smooth endoplasmic reticulum
Site of steroid synthesis and detoxification of
drugs and poisons. Lacks surface ribosomes
Cell trafficking
Golgi is the distribution center for proteins and lipids from the ER to the vesicles and plasma membrane. Modifies N-oligosaccharides on asparagine. Adds O-oligosaccharides on serine and threonine. Adds mannose-6-phosphate to proteins for trafficking to lysosomes. Endosomes are sorting centers for material from outside the cell or from the Golgi, sending it to lysosomes for destruction or back to the membrane/Golgi for further use.
I-cell disease
(inclusion cell disease)—inherited lysosomal storage disorder; defect in phosphotransferase failure of the Golgi to phosphorylate mannose residues (i.e., mannose-
6-phosphate) on glycoproteins proteins are secreted extracellularly rather than delivered to lysosomes. Results in coarse facial features, clouded corneas, restricted joint movement, and high plasma levels of lysosomal enzymes. Often fatal in childhood.
Signal recognition particle (SRP)
Abundant, cytosolic ribonucleoprotein that traffics proteins from the ribosome to the RER. Absent or dysfunctional SRP proteins accumulate in the cytosol
Vesicular trafficking proteins
COPI: Golgi Golgi (retrograde); Golgi ER. COPII: Golgi Golgi (anterograde); ER Golgi. Clathrin: trans-Golgi lysosomes; plasma membrane endosomes (receptormediated endocytosis [e.g., LDL receptor activity]).
Peroxisome
Membrane-enclosed organelle involved in catabolism of very-long-chain fatty acids, branched-chain
fatty acids, and amino acids.
Proteasome
Barrel-shaped protein complex that degrades damaged or ubiquitin-tagged proteins. Defects in the
ubiquitin-proteasome system have been implicated in some cases of Parkinson disease.
Microtubule
Cylindrical structure composed of a helical
array of polymerized heterodimers of α- and
β-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.
Drugs that act on microtubules
(Microtubules Get Constructed Very Poorly): Mebendazole (anti-helminthic) Griseofulvin (anti-fungal) Colchicine (anti-gout) Vincristine/Vinblastine (anti-cancer) Paclitaxel (anti-cancer)
Microtubules: Molecular motor proteins
Molecular motor proteins—transport cellular
cargo toward opposite ends of microtubule
tracks.
Dynein = retrograde to microtubule (+ -).
Kinesin = anterograde to microtubule
(- +).
Cilia structure
9 + 2 arrangement of microtubules A.
Axonemal dynein—ATPase that links peripheral
9 doublets and causes bending of cilium by
differential sliding of doublets
Kartagener syndrome (1° ciliary dyskinesia)—
immotile cilia due to a dynein arm defect.
Results in male and female infertility due to
immotile sperm and dysfunctional fallopian
tube cilia, respectively; ↑ risk of ectopic
pregnancy. Can cause bronchiectasis, recurrent
sinusitis, and situs inversus (e.g., dextrocardia
on CXR).
Cytoskeletal elements:Actin and myosin
Muscle contraction, microvilli, cytokinesis, adherens junctions. Actins are long, structural
polymers. Myosins are dimeric, ATP-driven motor proteins that move along actins.
Cytoskeletal elements:Microtubule
Movement. Cilia, flagella, mitotic spindle, axonal trafficking, centrioles
Cytoskeletal elements:Intermediate
filaments
Structure. Vimentin, desmin, cytokeratin, lamins, glial fibrillary acid proteins (GFAP),
neurofilaments.
Plasma membrane
composition
Asymmetric lipid bilayer.
Contains cholesterol, phospholipids, sphingolipids, glycolipids, and proteins. Fungal membranes
contain ergosterol.
Immunohistochemical stains for intermediate filaments
STAIN CELL TYPE Vimentin Connective tissue DesMin Muscle Cytokeratin Epithelial cells GFAP NeuroGlia Neurofilaments Neurons
Sodium-potassium pump
Na+-K+ ATPase is located in the plasma
membrane with ATP site on cytosolic side.
For each ATP consumed, 3 Na+ go out
and 2 K+ come in.
___________ inhibits by binding to K+ site.
___________ directly inhibit the Na+-K+ ATPase,
which leads to indirect inhibition of Na+/ Ca2+ exchange [Ca2+] cardiac contractility.
Ouabain inhibits by binding to K+ site.
Cardiac glycosides (digoxin and digitoxin) directly inhibit the Na+-K+ ATPase, which leads to indirect inhibition of Na+/ Ca2+ exchange [Ca2+]i cardiac
contractility.
Collagen
Most abundant protein in the human body.
Extensively modified by posttranslational
modification.
Organizes and strengthens extracellular matrix.
Collagen: Type I
Most common (90%)—Bone (made by osteoblasts), Skin, Tendon, dentin, fascia, cornea, late wound repair.
Collagen: Type II
Cartilage (including hyaline), vitreous body,
nucleus pulposus.
Collagen: Type III
Reticulin—skin, blood vessels, uterus, fetal
tissue, granulation tissue
Collagen:Type IV
Basement membrane, basal lamina, lens.
Collagen synthesis and structure: Inside fibroblasts: Synthesis (RER)
Translation of collagen α chains
(preprocollagen)—usually Gly-X-Y (X and Y are proline or lysine). Glycine content best reflects collagen synthesis (collagen is 1⁄3 glycine).
Collagen synthesis and structure: Inside fibroblasts:Hydroxylation (RER)
Hydroxylation of specific proline and lysine
residues (requires vitamin C; deficiency
scurvy).
Collagen synthesis and structure: Inside fibroblasts: Glycosylation (RER)
Glycosylation of pro-α-chain hydroxylysine residues and formation of procollagen via
hydrogen and disulfide bonds (triple helix of 3 collagen α chains). Problems forming triple
helix osteogenesis imperfecta.
Collagen synthesis and structure: Inside fibroblasts: Exocytosis
Exocytosis of procollagen into extracellular
space.
Collagen synthesis and structure: Proteolytic
processing
Cleavage of disulfide-rich terminal regions of
procollagen, transforming it into insoluble tropocollagen.
Collagen synthesis and structure:Cross-linking
Reinforcement of many staggered tropocollagen
molecules by covalent lysine-hydroxylysine cross-linkage (by Cu2+-containing lysyl
oxidase) to make collagen fibrils. Problems with cross-linking Ehlers-Danlos.
Osteogenesis
imperfecta
Genetic bone disorder (brittle bone disease A) caused by a variety of gene defects. Most common form is autosomal dominant with production of otherwise normal type I
collagen. OI manifestations can include: Multiple fractures with minimal trauma; may occur during the birth process,
Blue sclerae B due to the translucency of the connective tissue over the choroidal
veins, Hearing loss (abnormal ossicles), Dental imperfections due to lack of dentin
Ehlers-Danlos
syndrome
Faulty collagen synthesis causing hyperextensible skin, tendency to bleed (easy
bruising), and hypermobile joints. 6+ types. Inheritance and severity vary. Can be autosomal dominant or recessive. May be
associated with joint dislocation, berry and aortic aneurysms, organ rupture.
Menkes disease
Connective tissue disease caused by impaired copper absorption and transport. Leads to activity of lysyl oxidase (copper is a necessary cofactor). Results in brittle, “kinky” hair, growth retardation
and hypotonia
Elastin
Stretchy protein within skin, lungs, large arteries, elastic ligaments, vocal cords, ligamenta flava (connect vertebrae relaxed and stretched conformations). Rich in proline and glycine, nonhydroxylated
forms. Tropoelastin with fibrillin scaffolding. Cross-linking takes place extracellularly and
gives elastin its elastic properties.
Broken down by ______, which is normally inhibited by __________
elastase; α1-antitrypsin.
What causes marfan syndrome?
caused by a defect in
fibrillin, a glycoprotein that forms a sheath around elastin.
Emphysema can be caused by?
can be caused by α1-antitrypsin
deficiency, resulting in excess elastase activity.
Wrinkles of aging are due to____ collagen and
elastin production.
Low
Polymerase chain
reaction
Molecular biology laboratory procedure used to amplify a desired fragment of DNA. Useful as a diagnostic tool (e.g., neonatal HIV, herpes encephalitis).
Steps of PCR
Steps:
- Denaturation—DNA is denatured by heating to generate 2 separate strands
- Annealing—during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified.
- Elongation—heat-stable DNA polymerase replicates the DNA sequence following each
primer. These steps are repeated multiple times for DNA sequence amplification.
_______—used for size separation of PCR products (smaller molecules travel
further); compared against DNA ladder
Agarose gel electrophoresis
Southern blot
A DNA sample is enzymatically cleaved into smaller pieces, electrophoresed on a gel, and
then transferred to a filter. The filter is then soaked in a denaturant and subsequently
exposed to a radiolabeled DNA probe that recognizes and anneals to its complementary
strand. The resulting double-stranded, labeled piece of DNA is visualized when the filter is
exposed to film.
Northern blot
Similar to Southern blot, except that an RNA sample is electrophoresed. Useful for studying mRNA levels, which are reflective of gene
expression.
Western blot
Sample protein is separated via gel electrophoresis and transferred to a filter. Labeled antibody is used to bind to relevant protein. Confirmatory test for HIV after positive ELISA.
Southwestern blot
Identifies DNA-binding proteins (e.g., transcription factors) using labeled oligonucleotide probes.
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 amounts of complementary binding.
What are microarrays used for
Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments. Able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) for a variety of applications including genotyping, clinical genetic testing, forensic analysis, cancer mutations, and genetic linkage analysis
Enzyme-linked
immunosorbent assay
Used to detect the presence of either a specific
antigen (direct) or a specific antibody (indirect) in a patient’s blood sample.
During an enzyme-linked
immunosorbent assay a patient’s blood sample is probed with either:
Patient’s blood sample is probed with either:
Indirect ELISA: uses a test antigen to see if a specific antibody is present in the patient’s blood; a secondary antibody coupled to a color-generating enzyme is added to detect the first antibody.
Direct ELISA: uses a test antibody to see if a specific antigen is present in the patient’s blood; a secondary antibody coupled to a
color-generating enzyme is added to detect the antigen.
What indicates an enzyme-linked immunosorbent assay test is positive?
If the target substance is present in the sample,
the test solution will have an intense color
reaction, indicating a positive test result.
Fluorescence in situ
hybridization
Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes.
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
Cloning methods steps
Steps:
- Isolate eukaryotic mRNA (post-RNA processing steps) of interest.
- Expose mRNA to reverse transcriptase to produce cDNA (lacks introns).
- Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes.
- Transform recombinant plasmid into bacteria.
- Surviving bacteria on antibiotic medium produce cDNA.
Gene expression
modifications
Transgenic strategies in mice involve:
Random insertion of gene into mouse genome
Targeted insertion or deletion of gene through homologous recombination with mouse gene
Cre-lox system
Can inducibly manipulate genes at specific developmental points (e.g., to study a gene whose deletion causes embryonic death).
RNA interference (RNAi)
dsRNA is synthesized that is complementary to the mRNA sequence of interest. When
transfected 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 morphology, size, arm-length ratio, and banding pattern.
Karyotyping: can be performed on? and diagnoses what?
Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental tissue. Used to diagnose chromosomal imbalances (e.g.,
autosomal trisomies, sex chromosome disorders).
Codominance
Both alleles contribute to the phenotype of the
heterozygote.
Variable expressivity
Phenotype varies among individuals with same
genotype.
Incomplete
penetrance
Not all individuals with a mutant genotype show the mutant phenotype.
Pleiotropy
One gene contributes to multiple phenotypic
effects.
Anticipation
Increased severity or earlier onset of disease in
succeeding generations.
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.
Dominant negative
mutation
Exerts a dominant effect. A heterozygote produces a nonfunctional altered protein that also prevents the normal gene product from functioning
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.
Mosaicism
Presence of genetically distinct cell lines in the same individual. Arises from mitotic errors
after fertilization.
Somatic mosaicism
mutation propagates
through multiple tissues or organs
Gonadal mosaicism
mutation only in egg or
sperm cells.
McCune-Albright syndrome
is lethal if the
mutation is somatic, but survivable if mosaic
Locus heterogeneity
Mutations at different loci can produce a similar
phenotype.
Allelic heterogeneity
Different mutations in the same locus produce the same phenotype.
Heteroplasmy
Presence of both normal and mutated mtDNA, resulting in variable expression in
mitochondrial inherited disease.
Uniparental disomy
Offspring receives 2 copies of a chromosome from 1 parent and no copies from the other
parent.
Heterodisomy (heterozygous) indicates
a ______
meiosis I error.
Isodisomy (homozygous)
indicates a_______.
meiosis II error or postzygotic
chromosomal duplication of one of a pair of chromosomes and loss of the other of the
original pair.
Hardy-Weinberg
population genetics
If a population is in Hardy-Weinberg equilibrium and if p and q are the frequencies
of separate alleles, then: p2 + 2pq + q2 = 1 and
p + q = 1, which implies that:
p2 = frequency of homozygosity for allele p
q2 = frequency of homozygosity for allele q
2pq = frequency of heterozygosity (carrier
frequency, if an autosomal recessive disease).
The frequency of an X-linked recessive disease
in males = q and in females = q2.
Hardy-Weinberg law assumptions include:
No mutation occurring at the locus
Natural selection is not occurring
Completely random mating
No net migration
Imprinting
At some loci, only one allele is active; the other is inactive (imprinted/inactivated by
methylation). With one allele inactivated, deletion of the active allele disease.
Prader-Willi syndrome
Maternal imprinting: gene from mom is normally silent and Paternal gene is deleted/
mutated. Results in hyperphagia, obesity, intellectual disability, hypogonadism, and hypotonia.
AngelMan syndrome
Paternal imprinting: gene from dad is normally silent and Maternal gene is deleted/mutated. Results in inappropriate laughter (“happy
puppet”), seizures, ataxia, and severe intellectual disability.
Modes of inheritance: Autosomal dominant
Often due to defects in structural genes. Many
generations, both male and female, affected
Modes of inheritance:Autosomal recessive
25% of offspring from 2 carrier parents are
affected. Often due to enzyme deficiencies.
Usually seen in only 1 generation.
Modes of inheritance: X-linked recessive
Sons of heterozygous mothers have a 50%
chance of being affected. No male-to-male
transmission.
Modes of inheritance: X-linked dominant
Transmitted through both parents. Mothers
transmit to 50% of daughters and sons; fathers
transmit to all daughters but no sons.
Modes of inheritance: Mitochondrial
inheritance
Transmitted only through the mother. All offspring of affected females may show signs of
disease.
Hypophosphatemic rickets
formerly known as
vitamin D–resistant rickets. Inherited disorder
resulting in phosphate wasting at proximal
tubule. Results in rickets-like presentation (x-Linked dominant)
Mitochondrial myopathies
—rare disorders;
often present with myopathy, lactic acidosis and CNS disease. 2° to failure in oxidative phosphorylation. Muscle biopsy often shows
“ragged red fibers.” (mitochondrial inheritance)
Autosomal dominant diseases: Autosomal dominant
polycystic kidney
disease (ADPKD)
Formerly known as adult polycystic kidney disease. Always bilateral, massive enlargement of kidneys due to multiple large cysts. 85% of cases are due to mutation in PKD1 (chromosome 16; 16 letters in “polycystic kidney”); remainder due to mutation in PKD2 (chromosome 4).
Autosomal dominant diseases: Familial adenomatous
polyposis
Colon becomes covered with adenomatous polyps after puberty. Progresses to colon cancer unless colon is resected. Mutations on chromosome 5 (APC gene); 5 letters in “polyp.”
Autosomal dominant diseases:Familial
hypercholesterolemia
Elevated LDL due to defective or absent LDL receptor. Leads to severe atherosclerotic disease early in life, and tendon xanthomas (classically in the Achilles tendon).
Autosomal dominant diseases: Hereditary hemorrhagic
telangiectasia
Inherited disorder of blood vessels. Findings: telangiectasia, recurrent epistaxis, skin discolorations, arteriovenous malformations (AVMs), GI bleeding, hematuria. Also known as Osler-Weber-Rendu syndrome.
Autosomal dominant diseases: Hereditary
spherocytosis
Spheroid erythrocytes due to spectrin or ankyrin defect; hemolytic anemia; MCHC. Treatment: splenectomy.
Autosomal dominant diseases: Huntington disease
Findings: depression, progressive dementia, choreiform movements, caudate atrophy, and levels
of GABA and ACh in the brain. Gene on chromosome 4; trinucleotide repeat disorder: (CAG)n. repeats age of onset. “Hunting 4 food.”
Autosomal dominant diseases: Marfan syndrome
Fibrillin-1 gene mutation connective tissue disorder affecting skeleton, heart, and eyes. Findings: tall with long extremities, pectus excavatum, hypermobile joints, and long, tapering fingers and toes (arachnodactyly); cystic medial necrosis of aorta aortic incompetence and dissecting aortic aneurysms; floppy mitral valve. Subluxation of lenses, typically upward and temporally.
Autosomal dominant diseases: Multiple endocrine
neoplasias (MEN)
Several distinct syndromes (1, 2A, 2B) characterized by familial tumors of endocrine glands, including those of the pancreas, parathyroid, pituitary, thyroid, and adrenal medulla. MEN 2A and 2B are associated with ret gene.
Autosomal dominant diseases: Neurofibromatosis type 1 (von Recklinghausen disease)
Neurocutaneous disorder characterized by café-au-lait spots and cutaneous neurofibromas. Autosomal dominant, 100% penetrance, variable expression. Caused by mutations in the NF1 gene
on chromosome 17; 17 letters in “von Recklinghausen.”
Autosomal dominant diseases:Neurofibromatosis
type 2
Findings: bilateral acoustic schwannomas, juvenile cataracts, meningiomas, and ependymomas.
NF2 gene on chromosome 22; type 2 = 22.
Autosomal dominant diseases: Tuberous sclerosis
Neurocutaneous disorder with multi-organ system involvement, characterized by numerous benign hamartomas. Incomplete penetrance, variable expression.
Autosomal dominant diseases: von Hippel-Lindau disease
Disorder characterized by development of numerous tumors, both benign and malignant. Associated with deletion of VHL gene (tumor suppressor) on chromosome 3 (3p). Von Hippel-Lindau = 3 words for chromosome 3.
Mention some autosomal recessive diseases
Albinism, ARPKD (formerly known as infantile polycystic kidney disease), cystic fibrosis, glycogen
storage diseases, hemochromatosis, Kartagener syndrome, mucopolysaccharidoses (except
Hunter syndrome), phenylketonuria, sickle cell anemia, sphingolipidoses (except Fabry disease), thalassemias, Wilson disease.
Cystic fibrosis : Genetics
Autosomal recessive; defect in CFTR gene on chromosome 7; commonly a deletion of Phe508. Most common lethal genetic disease in Caucasian population.
Cystic fibrosis: Pathophysiology
CFTR encodes an ATP-gated Cl- channel that secretes Cl- in lungs and GI tract, and reabsorbs Cl- in sweat glands. Mutations –> misfolded protein –> protein retained in RER and not transported to cell membrane, causing lower Cl- (and H2O) secretion; increase intracellular Cl- results
in compensatory increase Na+ reabsorption via epithelial Na+ channels –> H2O reabsorption –>
abnormally thick mucus secreted into lungs and GI tract. increase Na+ reabsorption also causes more
negative transepithelial potential difference.
Cystic fibrosis: diagnosis
increase Cl- concentration (>60 mEq/L) in sweat is diagnostic. Can present with contraction alkalosis
and hypokalemia (ECF effects analogous to a patient taking a loop diuretic) because of ECF
H2O/Na+ losses and concomitant renal K+/H+ wasting.
Cystic fibrosis: complications
Recurrent pulmonary infections (e.g., Pseudomonas), chronic bronchitis and bronchiectasis–>
reticulonodular pattern on CXR, pancreatic insufficiency, malabsorption and steatorrhea, nasal polyps, and meconium ileus in newborns. Infertility in males (absence of vas deferens, absent sperm). Fat-soluble vitamin deficiencies (A, D, E, K).
Cystic fibrosis: treatment
N-acetylcysteine to loosen mucus plugs (cleaves disulfide bonds within mucus glycoproteins), dornase alfa (DNAse) to clear leukocytic debris.
Mention some X-linked recessive disorders
Bruton agammaglobulinemia, Wiskott-Aldrich syndrome, Fabry disease, G6PD deficiency, Ocular albinism, Lesch-Nyhan syndrome, Duchenne (and Becker) muscular dystrophy, Hunter Syndrome, Hemophilia A and B, Ornithine transcarbamylase deficiency.
Muscular dystrophies: Duchenne
X-linked frameshift mutation–> truncated dystrophin protein–> accelerated muscle breakdown. Weakness begins in pelvic
girdle muscles and progresses superiorly. Pseudohypertrophy of calf muscles due to fibrofatty replacement of muscle A. Gower
maneuver—patients use upper extremity to help them stand up. Onset before 5 years of
age. Dilated cardiomyopathy is common cause of death
Muscular dystrophies: Becker
Usually, X-linked point mutation in dystrophin gene (no frameshift). Less severe than
Duchenne. Onset in adolescence or early adulthood.
Muscular dystrophies: Myotonic type 1
CTG trinucleotide repeat expansion in the
DMPK gene –> abnormal expression of
myotonin protein kinase–> myotonia, muscle
wasting, frontal balding, cataracts, testicular
atrophy, and arrhythmia.
Fragile X syndrome
X-linked defect affecting the methylation and expression of the FMR1 gene. The 2nd most
common cause of genetic intellectual disability (after Down syndrome). Findings: postpubertal macroorchidism (enlarged testes),long face with a large jaw, large everted ears,
autism, mitral valve prolapse
Trinucleotide repeat
expansion diseases
Huntington disease, myotonic dystrophy, Friedreich ataxia, fragile X syndrome. Fragile X syndrome = (CGG)n. Friedreich ataxia = (GAA)n. Huntington disease = (CAG)n. Myotonic dystrophy = (CTG)n.
Autosomal trisomies: Down syndrome
(trisomy 21), 1:700. Findings: intellectual disability, flat facies,
prominent epicanthal folds, single palmar crease, gap between 1st 2 toes, duodenal
atresia, Hirschsprung disease, congenital heart disease (most commonly ostium primum-type
atrial septal defect [ASD]), Brushfield spots.
Autosomal trisomies: Edwards syndrome
(trisomy 18), 1:8000. Findings: severe intellectual disability, rockerbottom feet, micrognathia (small jaw), low-set Ears, clenched hands, prominent occiput, congenital heart disease. Death usually occurs within 1 year of birth
Autosomal trisomies: Patau syndrome
(trisomy 13),
1:15,000. Findings: severe intellectual disability, rockerbottom feet, microphthalmia, microcephaly,
cleft liP/Palate, holoProsencephaly,
Polydactyly, congenital heart disease. Death usually occurs within 1 year of birth.
Robertsonian
translocation: balanced and imbalanced
Nonreciprocal chromosomal translocation that commonly involves chromosome pairs 13, 14, 15, 21, and 22.Balanced translocations normally do not cause any abnormal
phenotype. Unbalanced translocations can result in miscarriage, stillbirth, and chromosomal imbalance (e.g., Down syndrome, Patau syndrome).
Cri-du-chat syndrome
Congenital microdeletion of short arm of chromosome 5 (46,XX or XY,5p-).
Findings: microcephaly, moderate to severe intellectual disability, high-pitched
crying/mewing, epicanthal folds, cardiac abnormalities (VSD).
Williams syndrome
Congenital microdeletion of long arm of chromosome 7 (deleted region includes elastin gene).Findings: distinctive “elfin” facies, intellectual disability, hypercalcemia ( high sensitivity to vitamin D), well-developed verbal skills, extreme friendliness with strangers, cardiovascular problems
22q11 deletion
syndromes
Variable presentation, including Cleft palate, Abnormal facies, Thymic aplasia –> T-cell deficiency, Cardiac defects, Hypocalcemia 2° to parathyroid aplasia, due to microdeletion at
chromosome 22q11.
22q11 deletion
syndromes: DiGeorge syndrome—organs affected
thymic, parathyroid, and
cardiac defects.
22q11 deletion
syndromes:Velocardiofacial syndrome—organs affected
palate, facial, and
cardiac defects.
Vitamins: fat soluble
A, D, E, K. Absorption dependent on gut and
pancreas. Toxicity more common than for
water-soluble vitamins because fat-soluble vitamins accumulate in fat
Vitamins: water soluble
B1 (thiamine: TPP) B2 (riboflavin: FAD, FMN) B3 (niacin: NAD+) B5 (pantothenic acid: CoA) B6 (pyridoxine: PLP) B7 (biotin) B9 (folate) B12 (cobalamin) C (ascorbic acid)
Vitamin A (retinol): Funcion
Antioxidant; constituent of visual pigments (retinal); essential for normal differentiation of epithelial cells into specialized tissue (pancreatic cells, mucus-secreting cells); prevents squamous metaplasia
Vit A: Uses
Used to treat
measles and AML, subtype M3.
Vit A: Deficiency
Night blindness (nyctalopia); dry, scaly skin (xerosis cutis); alopecia; corneal degeneration (keratomalacia); immune suppression
Vit A: Excess
Arthralgias, skin changes (e.g., scaliness), alopecia, cerebral edema, pseudotumor cerebri, osteoporosis, hepatic abnormalities.Teratogenic (cleft palate, cardiac abnormalities), so a negative pregnancy test and
reliable contraception are needed before isotretinoin is prescribed for severe acne.
Vitamin B1 (thiamine): funcion
In thiamine pyrophosphate (TPP), a cofactor for
several dehydrogenase enzyme reactions:
-Pyruvate dehydrogenase (links glycolysis to TCA cycle)
- α-ketoglutaratedehydrogenase (TCA cycle)
- Transketolase (HMP shunt)
- Branched-chain ketoacid dehydrogenase
Vitamin B1: deficiency
Impaired glucose breakdown ATP depletion worsened by glucose infusion; highly aerobic
tissues (e.g., brain, heart) are affected first. Wernicke-Korsakoff syndrome and beriberi. Seen in malnutrition and alcoholism (2° to malnutrition and malabsorption). Diagnosis made by in RBC transketolase activityfollowing vitamin B1 administration
Wernicke-Korsakoff syndrome—
confusion, ophthalmoplegia, ataxia (classic triad) + confabulation, personality change, memory loss (permanent). Damage to medial dorsal nucleus of thalamus, mammillary bodies.
Dry beriberi—
polyneuritis, symmetrical muscle wasting
Wet beriberi—
high-output cardiac failure
(dilated cardiomyopathy), edema.
Function of Vit B2
Component of flavins FAD and FMN, used as
cofactors in redox reactions, e.g., the succinate
dehydrogenase reaction in the TCA cycle.
Deficiency of Vit B2
Cheilosis (inflammation of lips, scaling and
fissures at the corners of the mouth), Corneal
vascularization.
Function of Vitamin B3 (niacin)
Constituent of NAD+, NADP+ (used in redox
reactions). Derived from tryptophan. Synthesis
requires vitamins B2 and B6. Used to treat
dyslipidemia; lowers levels of VLDL and raises
levels of HDL.
Deficiency of Vit B3
Glossitis. Severe deficiency leads to pellagra,
which can be caused by Hartnup disease
( low tryptophan absorption), malignant carcinoid
syndrome ( high tryptophan metabolism),
and isoniazid ( low vitamin B6). Symptoms
of pellagra: Diarrhea, Dementia (also
hallucinations), Dermatitis (e.g., Casal
necklace or hyperpigmentation of sun-exposed
limbs).
Excess of Vit B3
Facial flushing (induced by prostaglandin, not histamine), hyperglycemia, hyperuricemia
Vitamin B5 (pantothenate): Function
Essential component of coenzyme A (CoA,
a cofactor for acyl transfers) and fatty acid
synthase.
Vit B5 deficiency
Dermatitis, enteritis, alopecia, adrenal
insufficiency.
Vit B6 function
Converted to pyridoxal phosphate, a cofactor used in transamination (e.g., ALT and AST),
decarboxylation reactions, glycogen phosphorylase. Synthesis of cystathionine, heme, niacin,
histamine, and neurotransmitters including serotonin, epinephrine, norepinephrine, dopamine,
and GABA.
Vit B6 deficiency
Convulsions, hyperirritability, peripheral neuropathy (deficiency inducible by isoniazid and oral
contraceptives), sideroblastic anemias due to impaired hemoglobin synthesis and iron excess.
Function Vit B7
Cofactor for carboxylation enzymes (which add
a 1-carbon group):
Pyruvate carboxylase: pyruvate (3C)
oxaloacetate (4C)
Acetyl-CoA carboxylase: acetyl-CoA (2C)
malonyl-CoA (3C)
Propionyl-CoA carboxylase: propionyl-CoA
(3C) methylmalonyl-CoA (4C)
Deficiency Vit B7
Relatively rare. Dermatitis, alopecia, enteritis.
Caused by antibiotic use or excessive ingestion
of raw egg whites.
Vitamin B9 (folic acid): Function
Converted to tetrahydrofolate (THF), a
coenzyme for 1-carbon transfer/methylation
reactions.
Important for the synthesis of nitrogenous bases
in DNA and RNA.
Vit B9: deficiency
Macrocytic, megaloblastic anemia;
hypersegmented polymorphonuclear cells
(PMNs); glossitis; no neurologic symptoms
(as opposed to vitamin B12 deficiency). Labs:
homocysteine, normal methylmalonic
acid. Most common vitamin deficiency in
the United States. Seen in alcoholism and
pregnancy.
Vitamin B12 (cobalamin): function
Cofactor for homocysteine methyltransferase
(transfers CH3 groups as methylcobalamin)
and methylmalonyl-CoA mutase
Vit B12: deficiency
Macrocytic, megaloblastic anemia; hypersegmented PMNs; paresthesias, and subacute combined degeneration
(degeneration of dorsal columns, lateral corticospinal tracts, and spinocerebellar tracts) due to abnormal myelin. Associated with high serum homocysteine and methylmalonic acid levels. Prolonged deficiency irreversible nerve damage.
Vit C Function
Antioxidant. Also facilitates iron absorption by reducing it to
Fe2+ state. Necessary for hydroxylation of proline and
lysine in collagen synthesis. Necessary for dopamine β-hydroxylase, which converts dopamine to NE.
Vit C Deficiency
Scurvy—swollen gums, bruising, hemarthrosis,
anemia, poor wound healing, perifollicular
and subperiosteal hemorrhages, “corkscrew”
hair.
Weakened immune response.
Vit C Excess
Nausea, vomiting, diarrhea, fatigue, calcium
oxalate nephrolithiasis. Can risk of iron toxicity in predisposed individuals (e.g., those with transfusions, hereditary hemochromatosis).
Vit D: types
D2 = ergocalciferol—ingested from plants.
D3 = cholecalciferol—consumed in milk,
formed in sun-exposed skin (stratum basale).
25-OH D3 = storage form.
1,25-(OH)2 D3 (calcitriol) = active form
Vit D: function
increase intestinal absorption of calcium and
phosphate, increase bone mineralization.
Vit D: deficiency
Rickets in children (bone pain and deformity), osteomalacia in adults (bone pain and muscle weakness), hypocalcemic tetany. Breastfed infants should receive oral vitamin D. Deficiency is exacerbated by low sun exposure, pigmented skin, prematurity.
Vit D: excess
Hypercalcemia, hypercalciuria, loss of appetite, stupor. Seen in sarcoidosis ( activation of vitamin D by epithelioid macrophages).
Vitamin E (tocopherol/tocotrienol): Function
Antioxidant (protects erythrocytes and
membranes from free radical damage).
Vitamin E (tocopherol/tocotrienol): deficiency
Hemolytic anemia, acanthocytosis,
muscle weakness, posterior column and
spinocerebellar tract demyelination.
Vitamin K: function
Cofactor for the γ-carboxylation of glutamic
acid residues on various proteins required for
blood clotting. Synthesized by intestinal flora.
Vitamin K: deficiency
Neonatal hemorrhage with increase PT and Increase aPTT
but normal bleeding time (neonates have
sterile intestines and are unable to synthesize
vitamin K). Can also occur after prolonged
use of broad-spectrum antibiotics
Zinc: function
Essential for the activity of 100+ enzymes. Important in the formation of zinc fingers (transcription
factor motif).
Zinc: deficiency
Delayed wound healing, hypogonadism, decrease adult hair (axillary, facial, pubic), dysgeusia, anosmia, acrodermatitis enteropathica. May predispose to alcoholic cirrhosis.
Malnutrition: Kwashiorkor
Protein malnutrition resulting in skin lesions, edema, liver malfunction (fatty change due to low apolipoprotein synthesis). Clinical picture is small child with swollen belly
Malnutrition: Marasmus
Total calorie malnutrition resulting in tissue and
muscle wasting, loss of subcutaneous fat, and
variable edema.
Metabolism sites:Mitochondria
Fatty acid oxidation (β-oxidation), acetyl- CoA production,TCA cycle, oxidative phosphorylation.
Metabolism site:Cytoplasm
Glycolysis, fatty acid synthesis, HMP shunt,
protein synthesis (RER), steroid synthesis
(SER), cholesterol synthesis.
Metabolism site: Both (mitochondria and cytoplasm)
Heme synthesis, Urea cycle, Gluconeogenesis.
Kinase
Uses ATP to add high-energy phosphate group onto substrate (e.g., phosphofructokinase).
Phosphorylase
Adds inorganic phosphate onto substrate without using ATP (e.g., glycogen phosphorylase).
Phosphatase
Removes phosphate group from substrate (e.g., fructose-1,6-bisphosphatase).
Dehydrogenase
Catalyzes oxidation-reduction reactions (e.g., pyruvate dehydrogenase).
Hydroxylase
Adds hydroxyl group (-OH) onto substrate (e.g., tyrosine hydroxylase).
Carboxylase
Transfers CO2 groups with the help of biotin (e.g., pyruvate carboxylase).
Mutase
Relocates a functional group within a molecule (e.g., vitamin B12–dependent methylmalonyl-CoA
mutase).
Rate-determining enzymes of metabolic processes
Read Pag 99 and 100
ATP production
Aerobic metabolism of glucose produces 32 net
ATP via malate-aspartate shuttle (heart and
liver), 30 net ATP via glycerol-3-phosphate
shuttle (muscle).
Anaerobic glycolysis produces only 2 net ATP
per glucose molecule.
ATP hydrolysis can be coupled to energetically
unfavorable reactions
Universal electron
acceptors
Nicotinamides (NAD+ from vitamin B3,
NADP+) and flavin nucleotides (FAD+ from
vitamin B2).
NAD+ is generally used in catabolic processes
to carry reducing equivalents away as NADH.
NADPH is used in anabolic processes (steroid
and fatty acid synthesis) as a supply of reducing
equivalents.
Hexokinase vs.
glucokinase
Phosphorylation of glucose to yield glucose-6-P serves as the 1st step of glycolysis (also serves as the 1st step of glycogen synthesis in the liver). Reaction is catalyzed by either hexokinase or glucokinase, depending on the tissue. At low glucose concentrations, hexokinase sequesters
glucose in the tissue. At high glucose concentrations, excess glucose is stored in the liver.
Pyruvate
dehydrogenase
complex
Mitochondrial enzyme complex linking glycolysis and TCA cycle. Differentially regulated in fed/fasting states (active in fed state). Reaction: pyruvate + NAD+ + CoA acetyl- CoA + CO2 + NADH. The complex contains 3 enzymes that require 5 cofactors: 1. Pyrophosphate (B1, thiamine; TPP) 2. FAD (B2, riboflavin) 3. NAD (B3, niacin) 4. CoA (B5, pantothenate) 5. Lipoic acid Activated by exercise, which increase: NAD+/NADH ratio ADP Ca2+
What does the Pyruvate dehydrogenase complex deficiency cause? Findings? Treatment?
Causes a buildup of pyruvate that gets shunted
to lactate (via LDH) and alanine (via ALT).
Neurologic defects, lactic acidosis, increase serum
alanine starting in infancy.
increase intake of ketogenic nutrients (e.g., high fat
content or increase lysine and leucine).
