Biochemistry:
Symptoms: rickets (kids; bending bones), osteomalacia (adult; soft bones),
Fat Soluble Vitamin: vitamins A,D,E,K; precursor for coenzymes.
o Requires pancreatic enzymes for absorption (ileum): malabsorption syndrome can
cause fat-soluble vitamin deficiency: steatorrhea, cystic fibrosis, sprue
o Stored it fat making toxicity possible (unlike water soluble vitamins)
Vitamin A (Retinol): antioxidant (neutralizes free radical), composes visual pigments
(retinal; B-carotene is converted to cis-retinal which photoisomerizes to trans-retinal
when light is absorbed; cofactor for protein rhodopsin), essential for normal
differentiation of epithelial cells into specialized tissue (pancreatic and mucus-secreting
cells; binds intracellular receptors that regulate transcription at the retinoic acid
response elements), immune system stimulation (vitamin A recommended for treating
measles), retinitis pigmentosa (vitamin A supplementation used as a treatment in
retinitis pigmentosa)
o Source: found in liver, green/yellow veggies, B-carotene (retinal dimer that must
be cleaved and converted to trans-retinol for intestinal absorption), isotretinoin
(form of retinoid acid used to treat acne)
o Deficiency: due to deficient intake, malabsorption, fat-free diet. Symptoms: night
blindness, xerophthalmia, squamous metaplasia of corneal epithelium (spots are
known as Bitot spots), follicular hyperkeratosis (dry skin due to loss of
sebaceous gand), frequent infections
o Excess: due to over supplementation (>15 x RDA), consumption of wild game liver,
isotretinoin treatment. Symptoms: arthralgia (periosteal proliferation), alopecia,
papilledema/seizure (intracranial swelling), skin changes (yellow pigment with
excess B-carotene but sclera remains white so not jaundice), teratogenic (cleft
palate and cardiac abnormalities; pregnancy test must be done prior to
prescribing isotretinoin).
Vitamin D: raises Ca levels (↑: duodenal absorption of Ca and phosphate, reabsorption of
Ca from distal renal tubules, bone resorption via activation of osteoclasts:
bisphosphonates inhibit activation of osteoclasts and ↓ bone resorption e.g., ibandronate,
risedronate, and alendronate). Other functions:
o Bone Remodeling: osteoblasts have vitamin D receptors, binding stimulates
release of alkaline phosphatase (alk-phos), alk-phos dephosphorylates
pyrophosphate which normally inhibits bone mineralization.
o Matures macrophage stem cells into osteoclasts
o Source: pre-formed ingestion in diet (D2: ergocalciferol from plants, used as
supplements; D3: cholecalciferol from fish and milk) formed in sun-exposed skin
(UV catalyzes conversion of 7-dehydrocholesterol → cholecalciferol (D3) in skin
→ 25-hydroxycholecalciferol (25-OH D3) in liver catalyzed by 25-hydroxylase) →
1,25-dihydroxycholecalciferol (1,25-(OH)2 D3) in kidney catalyzed by 1α-
hydroxylase
 Decrease in hepatic function may result in vitamin D deficiency (must
supplement with 25-OH D3 ; occurs in the P450 system.
 1α-hydroxylase upregulated by PTH in response to hypocalcemia
 Decrease in renal function may result in vitamin D deficiency (must supplement
with 1,25-(OH)2 D3 equivalent). Pseudo-vitamin D deficiency rickets:
hereditary deficiency in 1α-hydroxylase.
 25-OH D3 = storage form; 1,25-(OH)2 D3 (calcitriol) = active form
o Deficiency: due to no sun exposure, decrease in hepatic/renal function, fat
malabsorption, induction of P450 which degrades active vitamin D precursors.
hypocalcemic tetany.
o Excess: supplementation (>10 x RDA), sarcoidosis (↑ activation of vitamin D by
epithelioid macrophages). Symptoms: polyuria, polydipsia, nocturia,
hypercalcemia/hypercalciuria (high blood Ca concentration promotes
metastatic calcification), loss of appetite, stupor, high levels of vitamin D
promote bone resorption
Vitamin E (a-tocopherol): antioxidant, prevents peroxidation of fatty acids (allows
maintain membrane fluidity), prevents oxidation of LDL, protects against arteriosclerosis
o Deficiency: rare; due to fat malabsorption. Symptoms:↑ fragility of erythrocytes
(hemolytic anemia), muscle weakness, neurodysfunction (poor joint sensation
and ataxia)
o Excess: synergistic ↓ in vitamin K dependent clotting factors with warfarin
(potential hemorrhage in warfarin patients)
Vitamin K: catalyzes γ-carboxylation of glutamic acid residues on blood clotting proteins
(cofactor for γ-glutamyl carboxylase; allows Ca2+ binding site; co-translational
modification in RER; necessary for the synthesis of clotting factors II (prothrombin), VII,
IX, X, and protein C and S)
o Must be activated by epoxide reductase enzymes (inhibited by warfarin, which is a
vitamin K antagonist; an anticoagulant in vivo (not in vitro); 2-3 days required to
achieve anticoagulation thus heparin given for immediate results)
o Source: normal gut flora, greens, breast milk doesn’t contain vitamin K
o Deficiency: due to fat malabsorption, neonates have sterile intestines and are
unable to synthesize vitamin K (aggravated by mothers who took
anticonvulsants during pregnancy; neonates are given vitamin K injection at
birth to prevent hemorrhage), can also occur after prolonged use of broad-
spectrum antibiotics (destruction of normal gut flora) and↓ in hepatic function
can ↓ vitamin K activation. Symptoms: ↑ PT and normal aPTT, but normal
bleeding time (can also see increased aPTT, but as factor VII has the shortest
half-life, PT increases before aPTT), easy bruising and bleeding, hemorrhagic
disease of the newborn
o Excess: rare; symptoms: hemolytic anemia, liver damage
Water Soluble Vitamins: excess is eliminated in urine (except B12 and folate that are
stored in the liver). B-complex deficiencies result in dermatitis, glossitis, diarrhea.
o B1 (thiamine: TPP)
o B2 (riboflavin: FAD and FMN)
o B3 (niacin: NAD+)
o B5 (pantothenic acid: CoA)
o B6 (pyridoxine: PLP)
o B12 (cobalamin)
o C (ascorbic acid)
o Biotin
o Folate
Vitamin B1 (thiamine): a cofactor for several enzymes such as thiamine pyrophosphate
(TPP), pyruvate dehydrogenase (glycolysis), α-ketoglutarate dehydrogenase (TCA cycle),
transketolase (HMP shunt), branched-chain AA dehydrogenase (metabolism of Val, Leu,
Ile)
o Deficiency: due to alcoholism (MC cause in US; EtOH interferes with thiamine
absorption in small intestine), malnutrition (non-enriched rice). Symptoms:
impaired glucose breakdown due to decreased pyruvate dehydrogenase activity
(leads to ATP deplation, highly aerobic tissue are affected first), Wernicke’s
syndrome (ataxia, confusion, nystagmus, ophthalmoplegia), Korsakoff’s
syndrome (confabulation, psychosis, mammillary body hemorrhage), beriberi:
o Dry beriberi: peripheral neuropathy due to
demyelination; symmetrical muscle wasting;
no fluid retention
o Wet beriberi: high-output cardiac failure
(dilated cardiomyopathy), edema
o Diagnosis: by measuring increased transketolase activity after thiamine
administration (thiamine is a cofactor necessary for the function of
transketolase; diagnosis of thiamine deficiency is made by history)
o Treatment: first treat thiamine deficiency, then administer glucose IVF (thiamine
is a cofactor for enzymatic steps in glycolysis, administering glucose before
thiamine could further decrease thiamine levels for enzymes like transketolase
which could exacerbate Wernicke-Korsakoff syndrome)
Vitamin B2 (riboflavin): cofactor (for oxidation and reduction (e.g., FADH2), succinate
dehydrogenase), precursor to FAD and FMN, involved with many dehydrogenase
enzymes
o Deficiency: due to severe malnourishment; symptoms: cheilosis (inflammation
of the lips and scaling and fissures at the corners of the mouth), corneal
vascularization, dry skin, magenta-colored tongue
Vitamin B3 (niacin): constituent of NAD+and NADP+ (used in redox reactions), derived
from tryptophan, involved with many dehydrogenase enzymes, synthesis requires
vitamin B2 and B6
o Deficiency: diets low in tryptophan or niacin (corn staple diets), Hartnup disease
(↓ tryptophan absorption in kidneys and small intestine), malignant carcinoid
syndrome (↑ tryptophan metabolism in production of serotonin), INH therapy (↓
vitamin B6 leading to ↓ niacin synthesis). Symptoms: glossitis, severe deficiency
leads to pellagra (3D’s: diarrhea, dermatitis, dementia)
o Excess: when nicotinic acid given at high doses as hyperlipidemic treatment
(raised HDL). Symptoms: facial flushing (mediated by prostaglandins, treated
with aspirin), intrahepatic cholestasis, hyperglycemia, hyperuricemia
Vitamin B5 (pantothenate): component of coenzyme A required for many enzymatic
processes:
 fatty acid synthase (fatty acid metabolism)
 acyl transferases
 pyruvate dehydrogenase (PDH)
 α-ketoglutarate dehydrogenase (TCA cycle)
o Deficiency: rare; symptoms: dermatitis, enteritis, alopecia, and adrenal
insufficiency
Vitamin B6 (pyridoxine): converted to pyridoxal phosphate, a cofactor used in:
transamination (e.g., ALT and AST in protein catabolism), decarboxylation reactions,
glycogen phosphorylase, cystathionine synthesis, heme synthesis. Required for the
synthesis of niacin from tryptophan.
o Deficiency: due to INH, OCP, goat milk, chronic alcoholism. Symptoms: convulsions,
hyperirritability, peripheral neuropathy, sideroblastic anemias, cheilosis or
stomatitis
Vitamin B12 (cobalamin): cofactor for:
 Homocysteine methyltransferase: transfers CH3 groups as methylcobalamin; cofactor
for homocysteine + N-methyl THF → methionine + THF
 Methylmalonyl-CoA mutase: metabolism of propionate (odd-chain fatty acid
degradation) at the conversion of methylmalonyl CoA → succinyl CoA; folate not
directly involved in this pathway; megaloblastic anemia with an elevated
methylmalonyl CoA indicates B12 deficiency as opposed to folate
 Other: Metabolism of Val, Met, ILe, Thr
 Source: animal products; several years’ reserve stored primarily in liver
 Deficiency: causes pernicious anemia (intrinsic factor required for absorption in the
terminal ileum; B12 not absorbed when intrinsic factor not produced from the
parietal cells of the stomach), gastric bypass surgery (less intrinsic factor produced),
resection of terminal ileum (Crohn’s disease), malabsorption (sprue, enteritis),
bacterial overgrowth of terminal ileum, diphyllobothrium latum (parasite; competes
for B12 absoroption), vegan diet. Symptoms: microcytic megaloblastic anemia (also
found in folate deficiency), hyperhsegmented PMNs, neurologic symptoms due to
abnormal myelin (paresthesia and subacute combined degeneration, dorsal
columns of spinal cord degenerate causing loss of proprioception and vibration
sensation; not found in folate deficiencies; could be reversible with administration
of B12; severe symptoms and longer term B12 deficiency = more residual neurologic
damage and less function regained)
 Schilling test to detect etiology of the deficiency: differential process of radiolabeled
B12
o Oral B12 + IM B12
o B12 + intrinsic factor
o B12 + antibiotics
o B12 + pancreatic enzymes
Folic acid: converted to tetrahydrofolate (THF), a coenzyme for 1-carbon
transfer/methylation reactions; important for the synthesis of nitrogenous bases in DNA
and RNA (thymidylate synthase), recall: uridine + methyl group = thymidine
o Deficiency: MC vitamin deficiency in the US; absorbed in the jejunum via the
action of intestinal conjugase. Caused by dietary insufficiency (elderly, goat
milk), alcoholism/pregnancy (liver store lasts 3 months), phenytoin,
sulfonamides, methotrexate, EtOH
o Findings: macrocytic, megaloblastic anemia, hypersegmented neutrophils,
homocysteinemia (↑ risk of DVT and atherosclerosis), no neurologic symptoms +
normal methylmalonic acid level (as opposed to vitamin B12 deficiency),
deficiency in pregnancy causes fetal neural tube defects
Biotin: cofactor for carboxylation enzymes (adds a 1-carbon group):
 Pyruvate carboxylase: pyruvate (3C) → oxaloacetate (4C); gluconeogenesis
 Acetyl-CoA carboxylase: acetyl-CoA (2C) → malonyl-CoA (4C); fatty acid
synthesis
 Propionyl-CoA carboxylase: propionyl-CoA (3C) → methylmalonyl-CoA (4C);
 odd-carbon fatty acids, Val, Met, Ile, Thr catabolism
o Deficiency: rare; due to antibiotic use, excessive ingestion of raw eggs (contains
avidin which binds biotin). Symptoms: dermatitis, alopecia, enteritis, lactic
acidosis
Vitamin C (ascorbic acid): antioxidant (regenerates vitamin E; ↓ oxidation of LDL), keeps
iron in Fe2+ reduced state (↑ intestinal absorption), collagen synthesis (essential for
hydroxylation of proline and lysine by prolyl and lysyl hydroxylases; addition of
hydroxyl group allows for hydrogen bonding between fibers, without cross-linking triple
helix shape cannot form), synthesis of norepinephrine (necessary for dopamine β-
hydroxylase which converts dopamine to NE), hepatic synthesis of bile acids, keeps THF
in reduced form, protects against nitrosylation of amides (occurs in the stomach with
presence of food preservatives; nitrosamines/amides are carcinogenic)
o Source: fruits and vegetables; british sailors used to carry limes to prevent
scurvy
o Deficiency: diet lacking citrus fruits and greens, infants on formula that is boiled
too long (infantile scurvy; 2-10 months; excessive heat destroys vitamin C),
smoking. Symptoms: scurvy (swollen gums, bruising, perifollicular hemorrhage,
poor wound healing, glossitis,↑ bleeding time, anemia due to combined iron and
folate deficiency.
o Excess: causes renal calculi made from calcium oxalate (vitamin excreted as
oxalate), diarrhea, nausea, vomiting, excess iron absorption in those predisposed
(hemochromatosis, repeat blood transfusions)
S-Adenosylmethionine (SAM): Not a vitamin but an important cofactor.
o Synthesis: ATP + methionine = SAM; regeneration of methionine (and thus SAM) is
dependent on vitamin B12 and folate
o Function: SAM transfers methyl units (similar to THF); required for the conversion
of NE to epinephrine
Kwashiorkor: due to protein-deficient diet. Presentation: skin lesions, pitting edema (↓
albumin leads to ↓ oncotic pressure in vasculature and loss of fluid into extravascular
space), ascites, liver malfunction (↓ apolipoprotein synthesis; fatty liver),defects in cell-
mediated immunity (↓ in complement protein synthesis), muscle protein relatively
unchanged.
Marasmus: due to protein- and caloric-deficient diet. Presentation: tissue and muscle
wasting (broomstick extremities; breakdown of muscle protein for energy), loss of
subcutaneous fat, variable edema.
Trace metals: micronutrients required in the diet for necessary cellular functions: iron,
copper, zinc, chromium, fluoride, iodide, selenium, etc.
o Function: part of metalloenzyme (enzyme has no activity without the metal. Metal
is fixed, metal:protein is constant. Eg: carbonic anhydrase) and is a part of metal-
containing enzyme (enzyme may have activity without the metal; metal is
reversibly bound, metal:protein ratio is variable. Eg. glycogen phosphorylase
kinase)
o Oxidative stress: organometallic side reactions that damage tissue. Many metals
undergo the Fenton reaction in vivo (oxidation of metal and donation of an
electron to oxygen; most common metals that undergo reaction are Fe2+ and Cu+;
creation of hydroxyl radicals). Heme iron can generate superoxide radicals (O2*)
o Reactions happen frequently, but the body can defend itself with anti-oxidants:
oxidative stress occurs when pro-oxidants > anti-oxidants.
o Examples of oxidative stress damage: stroke, Parkinson’s, Alzheimer’s
Iron: source: diet, recycled erythrocytes. Forms: ferrous iron (Fe2+; dangerous, causes
oxidative stress, found in Hb), ferric iron (Fe3+, less dangerous, methemoglobinemia
(metHb) occurs when Fe3+ is found in hemoglobin).
o Absorption: Fe-containing compounds are solubilized in low stomach pH. Fe3+ is
reduced to Fe2+ (requires vitamin C) in intestine so it can cross gut lumen.
Ferroportin brings Fe3+ into bloodstream from enterocytes, mediates amount of
Fe released into the blood, hepcidin inhibits ferroportin (antibacterial because it
lowers the availability of iron in the plasma).
o Storage: must be immediately used or stored to prevent: bacterial utilization (Fe
required for growth), formation of iron oxides, free radicals (Fe + O2). Site:
hepatocytes (main), enterocytes, macrophages. Stored as ferritin (Fe3+).
Hemosiderin binds excess Fe3+ to prevent from entering the blood.
o Transport: carried as Fe3+ by transferrin in the blood (transferrin chelates the Fe3+
and transports it in the blood to tissues; maintains solubility and keeps
unreactive, transferrin receptors on cells endocytose transferrin:Fe complex,
Fe3+ released into cell triggered by low pH, transferrin returns to cell surface to
be used again), ferroxidase (aka ceruloplasmin) oxidizes Fe2+ to Fe3+ for
transport and storage (ferritin can also oxidize Fe for storage).
o Excretion: no cellular mechanism for iron excretion; lost from blood loss and
removal of skin cells and other epithelial cells
o Toxicity: beyond the sequestration capacity of ferritin; causes oxidative stress
o Disorders in iron handling: hereditary hemochromacytosis
Copper: role: human metabolism. Like other metals, free copper is potentially toxic by
donating electrons (creates hydroxyl radicals and other reactive oxygen species), is a
cofactor for many metalloproteins (eg. lysyl oxidase for collagen synthesis and
tyrosinase for melanin synthesis)
 Transport: albumin and ceruloplasmin carry copper in the blood (similar role to
transferrin in iron transport), metallothionein is a carrier of copper, zinc, and many
other metals (role in preventing oxidative stress in the cell; thiol groups from many
cysteine residues mediate binding)
 Excretion: excess copper removed in the bile unlike iron with no mechanism of
excretion
 Deficiency: causes: excess zinc (metallothionein carries both copper and zinc; copper
is displaced when zinc concentrations rise). Symptoms: a function of what enzymes
require copper (ferroxidase: catalyzes oxidation of iron from Fe2+ to Fe3+,result is
microcytic anemia; lysyl oxidase: crosslinks collagen fibers, result is poor wound
healing, aortic dissection)
 Disorders in copper handling: Wilson’s disease, Menke’s disease: X-linked gene
mutation in ATP7A (ATP-dependent copper efflux protein); aka Ehlers-Danlos
syndrome type IX; inability of enterocytes to release absorbed copper; copper at
toxic levels in small intestine and kidneys; copper in circulation and in brain at low
levels. Symptoms: presents like a copper deficiency: seizures, failure to thrive,
neurodegeneration, steel-colored and brittle hair (due to role of copper in
metalloprotein lysyl oxidase which crosslinks collagen for added strength; at low
serum concentrations of copper, this enzyme cannot function).

Zinc: hundreds of enzymes require zinc (carbonic anhydrase, ACE (angiotensin I
converting enzyme), RNA and DNA polymerase etc)
 Transport: metallothionein carries zinc (competes with copper)
 Deficiency: due to: poor diet, alcoholism (liver is unable to handle zinc properly).
Symptoms: impaired collagenase (delayed wound healing), impaired zinc finger
transcription factor motifs (hypogonadism; ↓ adult hair: axillary, facial, and pubic), ↓
in senses (dysgeusia: lack of taste and anosmia: lack of smell), diarrhea, hair loss
(alopecia)
Chromium: deficiency: due to total parenteral nutrition (TPN). Symptoms: a function of
what proteins/enzymes require chromium (hypothesized to play a role as part of glucose
tolerance factor): ↓ glucose tolerance
Fluoride: source: fluoridated water; plays a role in thyroid hormone synthesis.
Deficiency: goiter, ↓ thyroid hormone output
Selenium: plays a role in glutathione peroxidase that protects against oxidative stress
thus damage to tissues with high metabolic activity. Deficiency: due to TPN; muscle pain,
cardiomyopathy
Cellular Biochemistry:
RER: composed of a lipid membrane with membrane bound ribosomes (lipid membrane
is continuous with nuclear membrane)
o Function: synthesis of secretory proteins (e.g. peptide hormones) and addition of
N-linked oligosaccharides to peptides
o Found in high concentration in: neurons (termed Nissl bodies; stain basophilic;
synthesize/secrete peptide neurotransmitters in prepackaged vesicles),
pancreatic acinar cells (synthesize/secrete digestive enzymes)
SER: structure: lipid membrane without membrane bound ribosomes.
 Function: steroid synthesis, detox of chemicals (makes compounds water soluble by
two mechanisms hydroxylation via cytochrome P450 hydroxylase complex and
conjugation which involves binding of polar moiety (e.g. glucuronate via glucuronyl
transferase) to toxin), lipid metabolism (release of fatty acids from triglycerides,
assembles lipoproteins for release), carbohydrate metabolism (gluconeogenesis:
allows free glucose to be released into circulation during fasting by removing
phosphate from glucose-6-phosphate mediated by glucose-6-phosphatase; deficient
in von Gierke's disease: glycogen storage disease type I, presents with
accumulation of glycogen in kidney and liver (hepatomegaly) and hypoglycemia).
 Found in high concentration in: hepatocytes, kidney, adrenal cortex, corpus luteum,
muscle (modified SER: sarcoplasmic reticulum which stores and releases Ca to
mediate muscle contraction).
Golgi Apparatus: structure: stacks of cisternae, associated with membrane bound
vesicles, has two faces: cis (receiving face from the ER, input to the Golgi, concave shape)
and trans (exports away from Golgi, location of trans-Golgi network, faces plasma
membrane, convex shape). Function:
o Post-translational peptide modification: (addition of N-oligosaccharides on
asparagine (amino-group), O-oligosaccharides on serine and threonine
(hydroxyl-group), mannose-6-phosphate designates transport to lysosome
(deficient in I-cell disease), sulfation of tyrosines) and cleavage of propeptides
(peptidase cleaves proinsulin to insulin + C-peptide, is deficient
in hyperproinsulinemia: presents similar to NIDDM)
o Packaging/distribution of proteins and lipids received from ER: (adds amino acid
residues that golgi later modifies) via coat protein complex (COP) II: this protein
initiates budding process (RER→ cis-Golgi = anterograde, anterograde
movement mediated by COP II; Golgi → ER = retrograde, retrograde movement
mediated by COP I) leaves Golgi to several destinations: plasma membrane,
lysosome, secretory vesicles, mediated by clathrin
o Proteoglycan assembly (sulfation of sugars).
Lysosome: function: digest endosomal material (via digestive enzymes: glycosylases,
lipases, proteases; all are acid hydrolases which function at low pH); are found in all cells
(higher concentrations in phagocytic cells). Forms: primary lysosomes (newly formed
from the trans-Golgi, waiting to receive endocytosed material) and secondary lysosomes
(aka phagolysosome; formed when primary lysosomes fuse with endocytic vesicles).
Lysosomal storage disease (by deficient enzymes):
 Sphingolipidoses:
o Sphingomyelinase; deficient in Niemann-Pick disease, AR; common in Ashkenazi
Jews; sphingomyelin accumulates (histiocytes look “foamy”). Presentation:
hepatosplenomegaly, anemia, cherry red spot on macula, death <3yrs, FTT,
neurodegeneration.
o α-galactosidase A: deficient in Fabry disease, XR; ceramide trihexose accumulates.
Presentation: peripheral neuropathy (especially in hands and feet),
angiokeratomas (small purple blemishes on skin), cardiovascular disease, renal
disease
o β-galactocerebrosidase: deficient in Krabbe disease, AR;
galactocerebroside accumulates. Presentation: hyperactive reflexes, optic
atrophy, developmental delay
o β-glucocerebrosidase: deficient in Gaucher disease, AR, common in Ashkenazi Jews;
glucocerebroside accumulates in cells of phagocytic cells (histiocytes (dendritic
cells) look like wrinkled tissue paper called Gaucher's cells). Presentation: three
types:
 Type I: MC; hepatosplenomegaly, aseptic necrosis of heads of long bones, mild
anemia, possible to live a normal lifespan
 Type II: "infantile Gaucher”; CNS involved, death <1yr
 Type III: "juvenile Gaucher”; severity < type II
o Hexosaminidase A: deficient in Tay-Sachs disease, AR, common in Ashkenazi Jews;
GM2 ganglioside accumulates, lysosomes with onion skin. Presentation: CNS
degeneration, blindness, cherry red spot on macula, startle reflex, death <4yrs,
similar to Niemann-Pick but without hepatosplenomegaly.
o Arylsulfatase A: deficient in metachromatic leukodystrophy, AR; cerebroside
sulfate accumulates. Presentation: demyelination in CNS and PNS resulting in
ataxia and dementia
 Mucopolysaccharides:
o α-L-iduronidase: deficient in Hurler syndrome, AR; heparan sulfate and dermatan
sulfate accumulates in heart and liver. Presentation: gargoyle-like facies, corneal
clouding, progressive mental retardation
o Iduronate sulfatase: deficient in Hunter syndrome, XR; heparan sulfate and
 dermatan sulfate accumulates. Presentation: severity < than Hurler’s,
aggressive behavior (remember: hunter's are aggressive), corneal clouding
absent
Other lysosomal disorders:
 I (inclusion)-cell disease: cause: proteins marked for localization to lysosomes are
post-translationally modified in the Golgi. Mannose residues are phosphorylated by
N-acetylglucosamine-phosphotransferase enzyme, defect in this enzyme causes I-
cell disease. Without mannose-6-phosphate designation, the enzymes are secreted
instead of being targeted to the lysosome. Cells cannot degrade endocytosed
material and inclusion bodies build up intracellularly.
o Presentation: high plasma levels of lysosomal enzymes, skeletal abnormalities,
restricted joint movement, psychomotor retardation, early death, coarse facial
features. No treatment exists.
 Chédiak–Higashi syndrome: AR; cause: primary lysosomes of leukocytes cannot fuse
with phagosomes; due to inability of microtubles to polymerize; also affects immune
cell chemotaxis. Presentation: ↑ infections (especially S. aureus), partial albinism,
peripheral neuropathy.
Peroxisome: structure: membrane-bound vesicle, contain several important enzymes
(catalase, hydrogen-transferring enzymes etc).
 Deficient peroxisome assembly: infantile refsum disease, AR; brain disorders, skeletal
and craniofacial dysmorphism, liver dysfunction, common presentation to all
peroxisomal disorders
 Function:
 Degradation of very long chain fatty acids (VLCFAs): > 24 carbons; via β-oxidation;
reduces by 14 carbons and then sends to mitochondria
o Deficient in Zellweger syndrome: AR; build-up of VLCFAs in peroxisomes, impaired
myelin synthesis (severe neurological symptoms), hepatomegaly, death < 1 year
(can increase lifespan with diet low in VLCFAs). Other examples of peroxisomal
disorders: neonatal adrenoleukodystrophy (NALD)
 Bile acid synthesis: derived from cholesterol
 Phospholipid modification: alter phosphatidylserine and phosphatidylethanolamine
 Degradation of hydrogen peroxide: via catalase degrades hydrogen peroxide produced
in β-oxidation of fatty acids
Mitochondria: found in high concentrations in metabolically active cells. Structure:
double membrane structure: outer membrane (high permeability due to high
concentration of porins) and inner membrane (impermeable to ions and molecules;
folded into cristae to ↑ surface area; contains enzymes for electron transport chain).
Inner matrix contains circular DNA (mtDNA).
 Maternal inheritance as mitochondria in the zygote come from the egg.
 Non-Mendelian inheritance examples of mtDNA disorders: Leber’s optic neuropathy
(degeneration of retinal ganglion cells, progressive loss of central vision, eventual
blindness), Pearson marrow-pancreas syndrome (some protein synthesis occurs
here; most proteins are synthesized in cytoplasm and translocated to mitochondria)
 Function: oxidizes substrates (NADH/FADH2 → NAH+/FAD), generates ATP (converts
NADH/FADH2 into ATP via ETC), stores Mg2+ and Ca2+
 Mitochondrial inheritance pedigree: Affected males (transmit to none of their
children), affected females (transmit to all of their children)
Plasma membrane: structure: bilayer of phospholipids, asymmetrical in respect to
intracellular and extracellular faces, ”fluid mosaic”. Composition:
 Cholesterol: 50%; adds thermal stability to membrane (↑ melting temperature), ↓
membrane flexibility; amount in plasma membrane tightly regulated
 Phospholipids: 50%; sphingolipids: fatty acid chain attached to a sphingosine.
Disorders of sphingolipid metabolism: Fabry’s and Gaucher’s disease.
 Glycolipids
 Proteins:
 Pumps: move substances against their concentration gradient, requires energy. E.g.
Na+-K+ ATPase:
o Transmembrane pump, ATP binding-site accessible from cytoplasm, process: 3 Na+
bind pump intracellularly, ATP binds and phosphorylates the pump, pump
changes conformation which releases 3 Na+ extracellularly, 2 K+ bind pump
extracellularly, phosphate ion removed. Pump changes conformation which
releases 2 K+ intracellularly, 3 Na+ bind pump intracellularly (repeat). Net: each
ATP results in 3 Na+ out and 2 K+ in.
o Pharmacological importance: cardiac glycosides (digoxin and digitoxin; mechanism
of action: inhibits pump, depolarization of cell membrane, ↑ intracellular [Na+], ↓
Na+ gradient required for Na+/Ca2+ exchange, ↑ [Ca2+] intracellularly, ↑ cardiac
contractility) and ouabain (inhibits by binding to K+ site, similar response to
cardiac glycosides)
 Channels: move substances down their concentration gradient. 3 types:
 Ungated: always open (eg. K channel)
 Voltage gated: open in response to changes in membrane voltage (found in excitable
tissue)
 Ligand-gated: open in response to a ligand (e.g. post-synaptic membrane receptors)
 Function: selective permeability (controls an intracellular environment distinct from
extracellular environment), signalling, localization of enzymes to promote or inhibit
interaction
Membrane physiology:
 Electrochemical potential determined by: conductance (G; ability of ions to
move across a membrane; controlled by opening/closing channels, ↑ channels =
↑ conductance) and net force (combination of concentration force:
concentration difference of a substance across a membrane and electrostatic
force: attraction of unlike charges/repulsion of like charges)
 Equilibrium potential: defined as the electrical potential across a membrane
that would prevent the diffusion of a substance via its concentration force for a
given concentration difference across a membrane
o Measured in millivolts (mV)
o For a single substance, calculated by: Nernst equation: Ex+ = 60/Z log(
[X+]extracell / [X+]intracell ); Z = absolute value of ionic charge; K+, Cl-, Na+ =
1, Ca2+ = 2. Answers the question: what is the voltage that exists across a
membrane when a certain ion is at its equilibrium. Another way of
thinking of this: what is the voltage required so that there will be no
net flow of a certain ion? For example: -80 mV is the Nernst potential
for potassium. This means that if the inside of the cell was -80 mV, K+
would not leave or enter the cell. The -80 mV of the cell pulling K+ in is
equal to the concentration gradient that wants to pull K+ out (remember
that K+ is low outside the cell and wants to travel down its ion gradient).
 If the voltage became -81 mV then K+ would want to travel into the cell as to dynein arm defect. Presentation: male and female infertility: sperm
this negative charge would overpower the drive for K+ to travel down its are immotile (no functional flagellar tail), fallopian tubes cannot
concentration gradient out of the cell. If the voltage was -79 mV then K+ sweep egg and sperm towards each other; bronchiectasis, recurrent
would leave the cell as the concentration gradient pulling K+ out is sinusitis (mucus with bacteria and particles cannot be removed;
greater than the negative voltage attracting/holding K+ in associated with situs inversus)
o Does NOT determine rate of ionic diffusion → only whether diffusion is • Mitotic spindles
favorable • Molecular motor proteins, mediates intracellular transport. 2 types: kinesin (cell
 Resting membrane potential (Emem): equilibrium potential of most cells = -90 center → periphery; anterograde to microtubule e.g. transports
mV; calculated by: sum of individual membrane potentials for neurotransmitter vesicles down axon towards synapse) and dynein (periphery
all permeable ions proportional to their conductances, for ions X+, Y+, and Z: → cell center; retrograde to microtubule e.g. lipid transport from synapse back
Emem = Gx(Ex+) + Gy(Ey+) + Gz(Ez-). Note: the closer the resting membrane to Golgi apparatus)
potential is to the equillibrium potential of an individual ion, the greater the • Pharmacologic importance:
membrane conductance is for that ion. When Emem = Ex+ , there is no net • Mebendazole: antihelminthic; mechanism of action: ↓ microtubule synthesis in worms
movement of ions and net force = 0. Example: • Griseofulvin: antifungal; moa: deposits in new keratin and disrupts microtubule
 Emem = -77 mV polymerization; uses: active against dermatophytes only
 EK+ = -95 mV • Vincristine/vinblastine: anti-cancer; moa: ↓ microtubule polymerization, inhibits
mitosis
Type Filament name Locations Stain • Paclitaxel (taxol): anti-breast cancer; moa: ↑ stability of microtubule and does not
I-II Keratin Epithelial cells Cytokeratin allow disassembly; inhibits mitosis
III Desmin Muscle (smooth, cardiac, and skeletal) Desmin • Colchicine: anti-gout; moa: binds free tubulin, ↓ microtubule polymerization, inhibits
Peripherin Peripheral nerve axons - leukocyte/granulocyte migration
Vimentin Fibroblasts and endothelium Vimentin
Glial fibrillary acidic Astrocytes and Schawnn cells GFAP Intermediate filaments:
protein (GFAP) • Structure: stable polymers; unlike microtubules and actin → does not undergo
IV Neurofilaments Neurons Neurofilament dynamic treadmilling; must be degraded via the ubiquitin pathway (sends to
(NF) proteasomes). Intermediate in size between microfilaments (7 nm) and
V Lamins Nuclear envelope of all cells - microtubules (25 nm)
 Is diffusion of K+ across this membrane favorable? Yes, given open • Function: give strength to the cytoskeleton: 5 types:
channels (G) to K+ it will diffuse until Emem = -95 mV  Clinical importance: alcoholic liver disease: ubiquitinated cytokeratin filaments
o Inside cells (as compared to extracellular environment) accumulate in hepatocytes (eosinophilic inclusions called Mallory bodies) and
 ↑ K+: EK+ = -95 mV; G is high for K+→ changes in [K+]extracellular will have parkinson's disease: alpha-synuclein and ubiquitinated neurofilaments accumulate
a large impact on Emem. ↑ G will hyperpolarize cell (efflux from cell) in neurons of substantia nigra eosinophilic inclusions called Lewy bodies
 ↓ Na+: ENa+ = +45 mV; G is low for Na+ → changes in [Na+]extracellular will
NOT have a large impact on Emem. ↑ G will depolarize cell (influx into Microfilament: examples: actin:
cell)  Structure: helical polymers of G-actin; has polarity (+ end is site of formation, - end
 ↓ Cl-: ECl- = -90 mV; since in most cells Emem = -90 mV → Cl- is is site of degradation); "treadmilling": formation requires ATP; dynamic structure
at equilibrium and will not diffuse that is constantly forming and breaking down , can sample environment, G-actin has
intrinsic ATPase capabilities.
Microtubule: cellular structural protein with a hollow tubular structure  Function: can interact with myosin to mediate: contractile force, motile force,
• Structure: composed of polymerized dimers of α- and β-tubulin, each dimer has 2 GTP cytokinesis and can interact with cell membrane to: form core of microvilli (function
molecules bound; constant assembly (slow) and disassembly (fast) to increase absorptive surface area), anchor tight junction and zonula adherens
• Clinical importance: Chédiak–Higashi syndrome (CHS): AR; disease caused by a
microtubule polymerization defect resulting in decreased chemotaxis, Cell surface protein: purpose: allow cells to attach to: each other (via different types of
degranulation, phagocytosis. Presentation: recurrent pyogenic infections cell-cell junctions), basement membrane (complex interaction between cells and
(particularly S. aureus), partial albinism, peripheral neuropathy supporting matrix, two divisions: basal lamina (provides attachment for most types of
• Function: component of many important cellular structures: epithelium; composition: type IV collagen, laminin, heparan sulfate) and reticular lamina
• Cilia: 9+2 arrangement of microtubules; axonemal dynein: ATPase that attaches the (supports lymphoid and adipose tissues; composition: type III collagen [reticular
peripheral 9 doublets causes bending of cilium by binding differentially to fibers]). Types of molecules:
doublets; also forms the core of flagella
◦ Clinical importance: Kartagener syndrome: immotile cilia disease due
  Cadherin: structure: calcium dependent, binds cadherin dimer on another cell
extracellularly and actin intracellularly (via catenin). Function: ↑ cell-cell
binding (cadherin receptor ↓ regulated in cancer metastasis)
 Selectin: calcium dependent. Function: binds carbohydrates in cell-cell
interactions. Subtypes:
o L-selectin (on leukocytes): have Sialyl-Lewis glycoproteins as ligands
o P-selectin (on platelets and endothelial cells)
o E-selectin (on endothelial cells): binds leukocytes strongly, ↑
expression on surface during acute inflammatory
response (stimulated by TNF-α)
 Integrin: calcium-independent, transmembrane; binds fibronectin and laminin
extracellularly and actin intracellularly. Function: binds leukocytes
weakly (neutrophilic attachment in rolling via an integrin LFA-1, defective in
leukocyte adhesion deficiency (LAD) type I: presents with recurrent bacterial
infections and delayed loss of umbilicus postpartum) and bind to laminin in
ECM (integrin receptor ↑ regulated in cancer metastasis)
Types of cell junctions:
 Tight junctions: aka zonula occludens. Structure: zona occuldens (ZOs) 1, 2, 3;
claudin proteins; membrane spanning proteins; bind cellA actin intracellularly on
one end, bind cellB actin intracellularly on opposite end
o Location: apical end of epithelial cells
o Function: prevent diffusion, create cell membrane polarity
 Adherens junctions: aka zonula adherens. Structure: cadherin proteins (Ca2+-
dependent adherin = cadherin)
o Location: belt around cell, below tight junction
 Desmosomes: aka macula adherens. Structure: cadherin proteins; link between two
cells; attach to intermediate filaments keratin and desmoplakin proteins
o Location: distinct sites
o Function: rivets; gives strength to junction between cells
o Clinical importance: pemphigus vulgaris: pathophysiology: auto-IgG against
desmosomal proteins in keratinocytes; type II hypersensitivity. Presentation:
painful, flaccid vesicles form on skin and oral mucosae; located above basal
layer, because it is above the basal layer the blister is weak; positive Nikolsky
sign: outer epidermis separates with gentle rubbing; acantholysis (loss of
connection between cells); post-inflammatory hyperpigmentation. Treatment:
immunosuppression (corticosteroids)
 Hemidesmosomes: structure: composed of integrins; bind type IV collagen,
fibronectin, laminin of basal lamina extracellularly and intermediate filaments
intracellularly
o Function: like a desmosome but instead of attaching cell-to-cell it attaches cell-
to-basement membrane; "half of a desmosome"
o Clinical importance:
o Bullous pemphigoid: pathophysiology: auto-IgG against basement membrane
hemidesmosomes; type II hypersensitivity, can be drug-induced
o Presentation: wide distribution of skin blisters, unlike pemphigus vulgaris →
does not involve oral mucosae; vesicles below the epidermis, stronger vesicles;
negative Nikolsky sign; NO acantholysis. Treatment: immunosuppression
(corticosteroids)
o Cancer: metastasis involves timely ↓ regulation of hemidesmosomal proteins
 Gap junctions: structure: pores formed by 6 connexon proteins
o Function: allow direct passage of small molecules from one cell to another (e.g.
Ca2+, cAMP); role in electrical and metabolic signaling; no role in strength
Extracellular matrix:
 Component: collagen, elastin, and:
 Glycoprotein: structure: protein + carbohydrates (protein content > carbohydrate
content; opposite true for proteoglycans), function: attach cells to various
components of the ECM. Examples: entactin, tenascin, laminin
 Fibronectin: structure: insoluble monomers, functions: attach cells to various
components of the ECM; can circulate in plasma to assist with clotting; biofilm
formation via cell adhesion to basement menbranes
 Proteoglycans: structure: protein + glycosaminoglycan (GAGs). GAGs have large
amount of negative charges due to sulfation, examples of GAGs: chondroitin sulfate,
dermatan sulfate, keratan sulfate, heparan sulfate, hyaluronic acid. Function: high
degree of hydration; sponge-like nature resists compression; found in high
concentrations in cartilage
Collagen: Synthesis and structure:
 Inside fibroblasts:
o pre-pro-collagen α chain formation: RER-bound ribosomes synthesize; contains
hydrophobic translocation sequence; chain formed mainly of repeating tripeptide
o Gly-X-Y: X and Y are proline, lysine
o pro-collagen α chain formation: hydrophobic sequence cleaved
o hydroxylated pro-collagen α chain formation: X and Y position prolines and
lysines are hydroxylated to form hydroxylysine and hydroxyproline. these amino
acids are unique to collagen; hydroxylated as peptide chain passes into ER;
performed by prolyl and lysyl hydroxylase (lack of lysyl hydroxylase function
results in weak collagen chains); requires ascorbic acid (Vitamin C): lack of
vitamin C causes scurvy: presentation: swollen gums, bruising, anemia, poor
wound healing; can also be caused by defective lysyl hydroxylase gene: Ehlers-
Danlos syndrome: nine different types, lysyl hydroxylase gene deficiency is one of
many causes, presentation: hyperextensible skin, hyperflexible joints, weak vessel
walls (↑ risk for aneurysm)
o glycosylated pro-collagen α chain formation: hydroxylysines are glycosylated
o pro-collagen α chain trimer formation: three α chains associate, moved from RER
to Golgi, secreted out of the fibroblast
 Outside fibroblasts:
o collagen molecule (tropocollagen) formation: propeptides cleaved from ends and
becomes insoluble; presence of propeptide does not allow assembly
intracellularly
o collagen fibril formation: catalyzed by lysyl oxidase: covalently links α chains by
crosslinking hydroxylysines, copper required as cofactor (lack of copper results
from Menke’s disease, at low serum concentrations of copper this enzyme cannot
function and weak collagen is formed, cause: X-linked gene mutation in ATP7A:
ATP-dependent copper efflux protein aka Ehlers-Danlos syndrome type IX:
inability of enterocytes to release absorbed copper, copper at toxic levels in small
intestine and kidneys, copper in circulation and in brain at low levels,
presentation: presents like a copper deficiency: seizures, failure to thrive,
neurodegeneration, steel-colored and brittle hair
 o collagen fiber formation: fibrils aggregate to form final bundles of triple helix
quaternary protein structure.
Types of collagen:
 Type I: thick, rope-like bundles of collagen; strongest tensile form of collagen;
majority of collagen in the body (approx. 90%); found in locations where high
tensile strength is needed (bone, fascia, tendons, teeth (dentin), cornea, skin); type
III of early wound repair converted to type I in late wound repair; defective in
osteogenesis imperfecta (OI) type I: aka brittle bone disease; AD, in most cases,
presentation: multiple fractures with minimal force (first fractures may occur
during delivery), blue sclerae (due to the translucency of the connective tissue over
the choroid due to lack of collagen), deafness (50%; abnormal middle ear bones),
dental abnormalities; defective in various forms of Ehlers-Danlos syndrome
 Type II: spongy collagen to absorb shock; found in tissues where there are
compression forces; cartilage (including hyaline), vitreous body of the eye, nucleus
pulposus of vertebral disc
 Type III: web-like fibers where forces pull from many directions; aka reticulin; found
in tissues where strength is needed (but not compression or tensile): skin, blood
vessels, uterus, fetal tissue; granulation tissue (type III of early wound repair
converted to type I in late wound repair); defective in Ehlers-Danlos type IV: faulty
collagen synthesis associated with: joint dislocation, berry aneurysms, organ
rupture
 Type IV: basement membrane; especially kidney, ears, eyes, skin; organizes/solidifies
cellular structure; defective in Alport's syndrome: effects the tissues where type IV
is most prominent (kidney → progressive hereditary nephritis, ears → deafness,
eyes → ocular disturbances), majority of cases are X-linked dominant; one of the
causes of epidermolysis bullosa: weak union of dermis and epidermis of the skin,
easily formed blisters; Goodpasture's syndrome involves an auto-antibody against
collagen type IV in pulmonary and glomerular capillaries; presents with hemoptysis
and glomerular disease
Elastin:
 Structure: rich in proline, lysine, glycine; unlike collagen, can exist in
nonglycosylated forms; fibrillin protein binds to tropoelastin to form elastic fibers:
Marfan's disease is caused by a defect in fibrillin, presentation: long extremities
including fingers, scoliosis, myopia and lens dislocations (upward like a martian
leaving for outer space as opposed to a downward dislocation in homocystinuria);
mitral valve prolapse (↑ for aortic aneurism)
 Function: is an elastic protein; found in tissues where stretch is needed (lungs, dermis
of the skin, large arteries, elastic ligaments, vocal cords, ligamenta flava of
vertebrae); desmosine interchain cross linking between lysine residues gives the
protein its elastic stretch
 Degraded by elastase, α1-antitrypsin normally inhibits elastase (class of protease
inhibitors synthesized in the liver)
 Excess elastase activity caused by α1-antitrypsin (AAT) deficiency (absent α1-globulin
peak in serum protein electrophoresis), autosomal codominant inheritance,
presentation: panacinar emphysema (worsened by smoking, early onset), smoking
without AAT deficiency usually causes centriacinar emphysema, cirrhosis
Marfan syndrome: Almost exclusively AD; mutation in FBN gene (encodes fibrillin-1 gene
on chromosome 15). Patients are susceptible to aortic disease, MVP, lens dislocation,
scoliosis, pectus deformity, arachnodactyly
A1-antitrypsin deficiency: Autosomal codominant inheritance; α1-antitrypsin inhibits
elastase; can lead to panacinar emphysema and hepatic disease
Cell cycle: stages:
 Interphase (G1): purpose: cellular growth to prepare for DNA replication (synthesis
of replication proteins, cyclin D), thymidine dimer repair. Variable duration, can exit
G1 and enter G0, chemotherapeutic agents that target G0 (cisplatin, nitrosoureas,
antitumor antibiotics, alkylating agents)
 S: purpose: DNA replication (cell is 2N before S, cell is 4N after S), DNA
proofreading. Constant duration, chemotherapeutic agents that target this
stage (etoposide, 6-mercaptopurine, 6-thioguanine, methotrexate, cytarabine,
hydroxyurea)
 G2: purpose: cellular growth to prepare for cell division, mismatch repair. Variable
duration, chemotherapeutic agents that target this stage (bleomycin)
 Mitosis (M): steps: prophase, metaphase, anaphase, telophase. Chemotherapeutic
agents that target this stage (vinblastine, vincristine, paclitaxel; all drugs that affect
microtubules)
Regulation: control transitions between phases of cell cycle.
 G1 checkpoint: at transition from G1 to S; most important checkpoint
 Cyclins/cyclin-dependent kinases: Cyclin D: produced during G1, complex with Cdk4
(cyclin dependent kinase) at sufficient concentration; signal to enter S phase
 Tumor suppressors: requires mutation in both copies before neoplasia occurs as
opposed to oncogenes which require just one mutation
 Rb: prevents cell from entering S phase; binds E2F to block its function as a
transcription factor; named for retinoblastoma
 P53: prevents cell from entering S phase; leads to inhibition of kinase activity of
Cdk4 via p21; can induce apoptosis if cell damage is severe via the BAX gene
(proapoptotic; inhibits BCL2 antiapoptosis gene; stimulates release of cytochrome c
from mitochondria)
Cell types:
 Permanent: enter G0 and cannot leave e.g. neurons, skeletal, cardiac muscle, RBCs
 Stable (quiescent): enter G0 and can leave when given appropriate stimulus e.g.
hepatocytes, lymphocytes labile; never go to G0; constant division with a condensed
G1 e.g. bone marrow, skin, gut epithelium
Signal transduction:
 Tyrosine kinase signaling: Tyrosine kinase structure: dimeric transmembrane protein,
intrinsic kinase activity, phosphorylated receptor can bind protein called insulin
receptor substrate (IRS; is a scaffold protein)
 Pathway: hormone binds to receptor, receptor is dimerized, auto cross-
phosphorylation of intracellular domain of the dimerized receptor, IRS
binds phosphorylated domain, IRS is phosphorylated on SH2-domains. Several
enzymes bind phosphorylated SH2-domians:
 Phosphatases: pathway signaled for by insulin
 Kinases: phosphoinositol-3 (PI-3) kinase
 G proteins: ras: oncogene present in many cancer; constitutive activation leads to
constant growth signal. Examples: insulin, PDGF, EGF receptors
G protein signaling: G-protein structure: trimeric enzyme (α, β, γ), α-subunit has intrinsic
GTPase activity (acts as timer for shutting off the active form)
 Pathway: hormone binds to receptor:G-protein complex on cell membrane, GDP
bound to inactive α-subunit is exchanged for GTP, α-subunit now active (can
be inhibitory (Gi) or stimulatory (Gs, Gq)), is released from β, γ subunits; can act as 2
different effector mechanisms:
 cAMP pathway: Gs activates adenyl cyclase: ATP → cAMP, cAMP can activate protein
kinase A, ADP-ribosylated by cholera and E. coli toxins (activates Gs, ↑↑↑ [cAMP]).
Gi inhibits adenyl cyclase: ADP-ribosylated by pertussis toxin (inhibits Gi,
↑↑↑ [cAMP])
 PIP2 pathway: Gq activates phospholipase C: PIP2 → IP3 + DAG, DAG can activate
protein kinase C, IP3 can release Ca2+ from the ER (Ca2+ then activates a number of
enzymes including protein kinase C)
 GTP eventually hydrolyzed to GDP and α-subunit becomes inactive. Examples: cAMP =
glucagon, epinephrine (β, α2); PIP2 = vasopressin, epinephrine (α1)
cGMP signaling: Pathway: hormone binds to receptor on cell membrane (there is also a
soluble receptor in the cytoplasm), receptor has intrinsic guanylate cyclase activity (GTP
→ cGMP; cGMP activate protein kinase G, protein kinase G mediates smooth muscle
relaxation/vasodilation, no G protein required). Examples: ANF → cell membrane
receptor; NO → diffuses across cell membrane and binds soluble receptor (mechanism
for pharmacologic nitrates (nitroprusside, nitroglycerine))
Cellular energy production:
Dietary energy content: Carbohydrate and protein (4 kcal/gm), fat (9 kcal/gm),
alcohol (7 kcal/gm)
Universal electron acceptors:
 Nicotinamides: NAD+; function: catabolic processes (transport reducing equivalents
as NADH)
 NADPH: function: regenerate glutathione (catalyzed by glutathione reductase;
newly reduced glutathione can convert H2O2 to H2O catalyzed by glutathione
peroxidase)
 , biosynthesis (fatty acids, cholesterol, nucleotides), respiratory burst, P-450
 Production: see HMP Shunt topic
 Flavin nucleotides: FAD+: function similar to NAD+ (does not carry as much energy;
generates 2 ATP in ETC while NADH generates 3). Production: succinate
dehydrogenase (citric acid cycle)
Glycolysis:
Function: generation of ATP from glucose via substrate-level phosphorylation (as
opposed to oxidative phosphorylation). Used by all cells:
 With O2: pyruvate enters citric acid cycle (after being created in glycolysis),
NAD+ regenerated via oxidative phosphorylation, produces 4 ATP per glucose
molecule. Without O2: pyruvate cannot enter citric acid cycle after glycolysis,
NAD+ must be regenerated via conversion of pyruvate to lactate catalyzed by
lactate dehydrogenase, produces 2 ATP per glucose molecule.
 Only source of energy for RBCs (generation of intermediates for other
pathways: 1,3-BPG (intermediate of glycolysis, can be converted to 2,3-DPG,
modifies the hemoglobin-O2 binding curve, binds HbA and ↓ binding affinity of
O2: a compensatory mechanism for ↓pO2)
Pathway: in cytoplasm; irreversible; net reaction: glucose + 2Pi + 2 ADP + 2 NAD+ → 2
pyruvate + 2 ATP + 2 NADH + 2H+ + 2 H2O
Important enzymes:
 Hexokinase: converts glucose into glucose-6-phosphate allowing "trapping" inside
cell. Distribution: widely present in most body tissues, allows trapping of glucose at
all blood glucose levels. Kinetics: high affinity → low Km, low capacity → low Vmax.
Regulation: feedback inhibited by glucose-6-phosphate
 Glucokinase (hexokinase IV): distribution: liver does not not normally use glucose a
fuel so the only function is storing excess glucose following a meal; β cells of
pancreas uses as a means to measure blood glucose and release insulin accordingly
mutated in the monogenenic, autosomal dominant form of diabetes called Maturity
Onset Diabetes of the Young type 2 (MODY2). Kinetics: low affinity → high Km, high
capacity → high Vmax. Regulation: induced by insulin (to store glucose in liver after a
meal), no direct feedback inhibition
 Phosphofructokinase-1: rate-limiting step; inhibited by ATP, citrate, stimulated by
AMP, fructose-2,6-bisphosphate. Fructose 2,6-bisphosphate synthesized by
phosphofructokinase-2
 Pyruvate kinase: catalyzes substrate-level phosphorylation; inhibited by ATP,
alanine, activated by fructose-1,6-bisphosphate
 Hormonal regulation: fasting state: ↑ glucagon → ↑ cAMP → ↑ protein kinase A → ↑
FBPase-2, ↓ PFK-2; fed state: ↑ insulin → ↓ cAMP → ↓ protein kinase A → ↓ FBPase-2,
↑ PFK-2
Disorders of glycolysis:
 Pyruvate kinase deficiency: AR (most commonly); pathophysiology: ↓ ATP
generation (inability to maintain Na+/K+ ATPase leads to RBC swelling, RBC lysis)
and back up of glycolysis (↑ 2,3-BPG and other glycolytic intermediates).
Presentation: chronic hemolysis, ↓ O2 affinity for HbA, due to ↑ 2,3-BPG; no Heinz
bodies unlike glucose 6-phosphate dehydrogenase deficiency.
 Hexose monophosphate shunt (HMP): function: generate NADPH required for FA
 synthesis, steroid synthesis, reduction of oxidizing agents (H2O2 see figure); provide
ribose 5-phosphate required for nucleotide synthesis
 Pathway: occurs in cytoplasm of all cells, no ATP consumed or generated. 2 phases:
o Oxidative: produces NADPH; glucose 6-phosphate (G6P) → 6-phosphogluconate
catalyzed by glucose 6-phosphate dehydrogenase (G6PDH): rate limiting step,
activated by NADP+, insulin, inhibited by NADPH; irreversible
o Nonoxidative: exchanging intermediate substrates between glycolysis and HMP
shunt catalyzed by transketolase, requires thiamine, reversible
Clinical relevance:
 Glucose-6-phosphate dehydrogenase (G6PDH) deficiency: pathophysiology: ↓ NADPH
production (cells (specifically RBCs) lose protection against oxidizing agents);
cannot regenerate glutathione; XR; most common human enzyme deficiency, ↑
prevalence among blacks, ↑ malarial resistance (by shortening the circulation life of
RBCs. Plasmodium does not have enough time for life span, plasmodium does not
have defense against free radicals, ↑ in free radicals kills parasite).
 Presentation: episodic hemolytic anemia (intravascular hemolysis, normocytic, 2-3
days post precipitating stress: foods (fava beans, common in Mediterranean foods,
presentation: pallor, hemoglobinuria 24-48 post ingestion), drugs (sulfonamides,
primaquine, antituberculosis drugs), infection (free radicals generated by the
immune system)), Heinz bodies (oxidized hemoglobin that precipitates within
RBCs), bite cells (result from the phagocytic removal of Heinz bodies by
macrophages), back pain.
 Test: active hemolysis screen (Heinz body prep)
Pyruvate dehydrogenase complex:
 Function: catalyze conversion of pyruvate to acetyl-CoA; net reaction: pyruvate +
NAD+ + CoA → acetyl-CoA + CO2 + NADH; irreversible
 Structure: 3 enzymes that require 5 cofactors: thiamine pyrophosphate (B1), FAD (B2),
NAD (B3), CoA (B5), lipoic acid; similar to α-ketoglutarate dehydrogenase complex
 Regulation: activators of pyruvate dehydrogenase, ↓ in energy status of the cell
(↑ NAD+/NADH ratio, ↑ ADP), ↑ exercise, ↑ Ca2+, ↑ insulin; inhibited by acetyl-CoA
 Clinical relevance: pyruvate dehydrogenase deficiency: pathophysiology: X-linked
(variable penetrance in females) accumulation of pyruvate, alanine; lactic acidosis
(pyruvate shunted to lactate to regenerate NAD+). Presentation: neurological
defects. Treatment: ↑ ketogenic nutrients (lysine and leucine)
 Arsenic poisoning: inhibits pyruvate dehydrogenase (via inhibition of lipoic acid);
presentation: vomiting, rice water stools, garlic breath
Citric acid cycle: Krebs cycle, tricarboxylic acid (TCA) cycle
 Function: generate high amounts of NADH/FADH2 that act as fuel for ATP synthesis in
the electron transport chain
 Pathway: occurs in the mitochondria, requires O2 to function, net equation: acetyl-CoA
+ 3 NAD+ + FAD + GDP + Pi → 2CO2 + 3 NADH + FADH2 + GTP + CoA. Will
theoretically yield 12 ATP (if ETC were 100% efficient), both carbons of acetyl-CoA
leave as CO2 in two of the reactions, conversion of isocitrate to alpha ketoglutarate,
conversion of alpha ketoglutarate to succinyl-CoA.
Important enzymes:
 isocitrate dehydrogenase (stimulated by ↓ energy, ↑ ADP; inhibited by ↑ energy, ATP,
NADH)
 α-ketoglutarate dehydrogenase complex (requires the same cofactors as the
pyruvate dehydrogenase complex: B1, B2, B3, B5, lipoic acid, in
alcoholics, B1 deficiency leads to Wernicke's encephalopathy (triad of ataxia,
confusion, ophthalmoplegia); inhibited by ↑ energy (↑ ATP, NADH), inhibited by ↑
intermediates in the cycle (↑ succinyl-CoA)
 succinyl-CoA synthetase: generates GTP
 succinate dehydrogenase: a member of both the citric acid cycle and the electron
transport chain
 important intermediates:
 citrate: functions to shuttle acetyl-CoA out of mitochondria for fatty acid synthesis,
citrate shuttle
 succinyl-CoA: building block for heme synthesis
 fumarate: enters from the urea cycle
 malate: functions as a gluconeogenic substrate
 Regulation: energy status control (NO hormonal control)
ETC: function: couple energy stored in electron acceptors (NADH, FADH2) to ATP
synthesis; process called oxidative phosphorylation: 3 ATPs per NADH (NADH enters
mitochondria from production in cytosol via malate-aspartate/glycerol-3-phosphate
shuttle), 2 ATPs per FADH2 (lower energy content than NADH)
 Pathway: located in inner mitochondrial membrane; series of carrier enzymes; NADH
and FADH2 create a proton gradient across the inner membrane, pass electrons in a
stepwise fashion, oxygen is the final electron acceptor, flow of proton back down
concentration gradient drives F0F1 ATP synthase complex (net production of ATP)
 Clinical importance: electron transport inhibitors disrupt membrane bound carrier
enzymes, result: ↓ proton gradient, ↓ ATP synthesis, ↓ O2 consumption, ↑
intracellular NADH/NAD+ ratio
 CO: source: combustion (smoking, fires, car exhaust, grills), paint strippers.
Presentation: ↓ O2 sat, cherry-red lips and cheeks, headache/nausea, tachypnea,
tachycardia. Treatment: 100% O2
 CN: source: nitroprusside administration byproduct give with thiosulfate to
consume produced CN, combustion of polyurethane (burning furniture, mattresses),
mining (gold), metal extraction. Presentation: seizures, tachypnea, tachycardia,
headache, flushing. Treatment: sodium thiosulfate (forms thiocyanate, less-toxic
metabolite, renally excreted), nitrites (convert hemoglobin to methemoglobin
(ferrous to ferric), does not allow cyanide transport to mitochondria, must be given
shortly after exposure)
Note: victims of house fires may have both CO and CN poisoning
 ATPase inhibitors: directly inhibit mitochondrial ATP synthase. Result: ↑ proton
gradient; no ATP is produced because electron transport stops e.g. oligomycin
 Uncoupling agents: "uncouples" ATP production from the proton gradient, ↑
permeability of membrane. Result: ↓ proton gradient, ↑ O2, NADH consumption, ATP
synthesis stops, but electron transport continues produces heat. Examples: 2,4-DNP,
aspirin/salicylates (fevers often occur after aspirin overdose), thermogenin in
brown fat (UCP protein, generates heat for newborns)
 CO (carbon monoxide) and CN (cyanide): site of inhibition: cytochrome a/a3
 Antimycin: SOI: sytochrome b/c1
 Doxorubicin: SOI: CoQ
 Retenone (pesticide): SOI: NADH dehydrogenase
Glucose transport:
 Sodium/glucose cotransporter (SGLT): function: transport glucose actively across
lumen against concentration gradient (energy provided by transport of sodium
down its concentration gradient). Location: small intestine (SGLT1; 2:1 Na+:Glu),
proximal tubule of nephron (SGLT2; 1:1 Na+:Glu)
 GLUT-1: function: basal glucose uptake (high affinity: transporters saturated at
normal blood glucose levels, ensures glucose entry to cells). Location: wide
distribution in tissues in the body (brain, erythrocytes, endothelial cells, cornea etc.)
 GLUT-2: function: low affinity glucose uptake (in the fasting state glucose does not
enter cells, mediates glucose surplus storage in liver when blood glucose levels rise,
facilitates insulin release in β-cells). Location: hepatocytes, pancreatic β-cells,
kidney, small intestines
 GLUT-3: function: high affinity glucose uptake (glucose preferentially accessed by
neurons in low-glucose states). Location: brain, neurons
 GLUT-4: function: insulin-controlled uptake of glucose, basal level of glucose intake
without insulin (presence of insulin ↑ translocation of transporters to the cell
membrane, ↑↑↑ glucose uptake, also stimulated by exercise). Location: adipocytes,
myocytes, cardiomyocytes
Gluconeogenesis:
 Function: de novo glucose synthesis: effectively glycolysis in reverse, can maintain
blood glucose when glycogen stores are exhausted, must supply brain and RBCs
which utilize glucose for energy; is NOT a source of energy for the liver (hepatocytes
use β-oxidation to supply the energy needed for gluconeogenesis). Potential
substrates: all amino acids (except for leucine and lysine), lactate (produced in
anaerobic glycolysis), glycerol-3-phosphate (produced in fat catabolism), propionyl-
CoA, produced in odd-carbon fatty acid catabolism
 Pathway: location: hepatocytes (primary), kidney, enterocytes, NOT muscle (no
glucose-6-phosphatase, cannot release free glucose)
 Enzymes: involves both mitochondrial and cytosolic enzymes, several steps of
glycolysis are reversible, the non-reversible steps must be bypassed with special
gluconeogenic enzymes:
o Pyruvate carboxylase: pyruvate → oxaloacetate; requires biotin and ATP;
activated by acetyl-CoA; oxaloacetate must be converted to malate to exit the
mitochondria via the malate-aspartate shuttle in mitochondria
o PEP carboxykinase (PEPCK): oxaloacetate → phosphoenolpyruvate (PEP);
requires GTP; activated by glucagon and cortisol in both cytosol and
mitochondria
o Fructose-1,6-bisphosphatase: fructose-1,6-bisphosphate → fructose-6-P;
important control point of gluconeogenesis; activated by ATP, inhibited by AMP
and fructose-2,6-bisphosphate in cytosol
o Glucose-6-phosphatase (G6P): glucose-6-P → glucose in ER of hepatocytes.
Clinical relevance: von Gierke disease = G6P deficiency
o Other enzymes: lactate dehydrogenase: lactate → pyruvate, requires free NAD+
 Regulation: stimulation (glucagon, acetyl CoA, citrate), inhibition (high NADH/NAD+
ratio; alcohol may cause elevated NADH/NAD+ ratio leading to hypoglycemia)
Glycogen: Structure: polymer of glucose: straight chain with α-1,4-bond, branches with
α-1,6-bond. Glycogen granule: core: glycogenin
 Function: energy reserves that can provide glucose during a fast or ↑ energy
demand (supplies exhausted in < 24 hrs), stored mainly in the liver and muscle.
Muscle does not have glucose-6-phosphatase so it cannot release free glucose;
stores for its own consumption, liver does have glucose-6-phosphatase so it can
release free glucose and can use supplies to maintain blood glucose levels.
 Glycogenesis: Glycogen synthesis
 Pathway: glucose-6-phosphate converted to glucose-1-phosphate, UDP group added
to form UDP-glucose, UDP-glucose added to polymer in an α-1,4 linkage catalyzed
by glycogen synthase (rate limiting step of glycogen synthesis), polymer rearranged
to create α-1,6 linked branches (catalyzed by branching enzyme, deficiency =
Anderson disease)
 Regulation: glycogen synthase (in liver: activated by insulin, inhibited by glucagon,
epinephrine; in muscle: activated by insulin, inhibited by epinephrine)
Glycogenolysis: Glycogen catabolism
 Pathway: glucose-glucose bond broken by addition of a phosphate catalyzed by
glycogen phosphorylase (rate limiting step of glycogenolysis; hepatic deficiency =
Hers disease (type VI); muscle deficiency = McArdle disease (type V)), glucose-1-
phosphate freed (converted to glucose-6-phosphate; debranching enzymes removes
α-1,6 linked branches; deficiency = Cori's disease (type III)), liver converts glucose-
6-phosphate to glucose catalyzed by glucose-6-phosphatase (deficiency = von
Gierke disease (type I)), muscle puts glucose-6-phosphate into glycolysis
 Regulation: glycogen phosphorylase in liver (activated by epinephrine/glucagon via
cAMP/protein kinase A, inhibited by insulin; remember: exact opposite of glycogen
synthase: hepatic glycogen regulatory processes both turn on forward direction and
turn off reverse). In skeletal muscle: activated by epinephrine, AMP, Ca2+, inhibited
by insulin, ATP), remember: since muscular glycogen can only supply itself, it is
regulated by its own energy supply (AMP/ATP ratio); while liver must supply
energy to many other tissues, it functions independently of AMP/ATP ratio in
hepatocytes
Glycogen storage disease (glycogenolyses): all disorders have abnormal glycogen
metabolism, leads to an accumulation of glycogen within cells, organ dysfunction.
Remember: disorders numbered in order of pathway from end (glucose release) to
beginning (breakdown of glycogen polymer)
 Glucose release: Type I: von Gierke: lacks glucose-6-phosphatase. Presentation: liver
cannot release stored glucose: hepatomegaly, severe hypoglycemia. Body must rely
on fat/protein catabolism for energy: hyperlipidemia, hyperuricemia, lactic acidosis,
normal glycogen structure. Tests: stimulation test with glucagon, fructose, galactose:
does not ↑ serum glucose
 Lysosomal pathway: Type II: Pompe "trashes the Pump (heart)": lacks lysosomal α1,4-
glucosidase, degrades glycogen-resembling material in endosomes. Presentation:
buildup of glycogen in cardiac muscle; electron dense granules inside lysosomes,
cardiomegaly, hypertrophic cardiomyopathy
 Branching/debranching:
o Type III: Cori: lacks debranching enzyme. Remember: Cori = can't Catabolize
branches, 6-pack core - alpha 1,6 glucosidase defective. Presentation: liver cannot
break down glycogen past a branch point: hepatomegaly, hypoglycemia, abnormal
glycogen structure (short outer glycogen chains)
o Type IV: Anderson: lacks branching enzyme; remember: Anderson = can't Add
branches. Presentation: liver cannot form branched glycogen granules: hypotonia,
cirrhosis
 Phosphorylase:
o Type V: McArdles: lacks muscle phosphorylase; remember: McArdles = Muscle,
can't breakdown glycogen to glucose-1-phosphate. Presentation: muscle
weakness/cramps upon exertion, myoglobinuria, normal glycogen structure
o Type VI: Hers: lacks hepatic phosphorylase; remember: Hers = Hepatic.
Presentation: hepatomegaly, fasting hypoglycemia; can be mild due to
gluconeogenic compensation.
Sorbitol: A 69-year-old man presents to your clinic with a chief complaint of changes in
his vision. He has a past medical history of diabetes mellitus type II and medication non-
compliance. The patient states that after a very large meal in which he consumed a
family-sized bucket of chicken, a glass of maple syrup, and an entire wedding cake he
noticed that his vision became very blurry. In fact, he notes this happens every time he
eats a large meal (sorbitol accumulation in the lens & lens swelling).
 Pathway: alternative method of trapping glucose in the cell; tissues with sorbitol
dehydrogenase: liver, ovaries, seminal vesicles, lens (at low level of activity), tissues
without sorbitol dehydrogenase: Schwann cells, retina, kidney. Note: galactose can
also be converted to an aldose
 Clinical relevance: prolonged hyperglycemia (commonly caused by uncontrolled
diabetes mellitus); pathophysiology: glucose enters cells and is converted to sorbitol
in tissues without sorbitol dehydrogenase or low levels of activity (sorbitol trapped
in cell and is osmotically active). Presentation: pathology directly linked to which
tissues have aldose reductase but lack sorbitol dehydrogenase: peripheral
neuropathy, cataracts, retinopathy (all symptoms of chronic diabetes)
Fructose metabolism: source: sucrose in fruits and sweeteners (disaccharide of glucose
and fructose hydrolyzed by sucrase in brush border of small intestine).
 Pathway: liver is main site of metabolism, also in renal proximal tubule; DHAP and
glyceraldehyde enter into glycolysis downstream of regulation
 Clinical relevance:
 Fructokinase deficiency ; benign; presentation: fructosuria (does not have the osmotic
pathologies (e.g. cataracts) associated with galactokinase deficiencies (fructose is
not an aldose and therefore not substrate for aldose reductase).
 Fructose 1-P aldolase (aldolase B) deficiency: aka hereditary fructose intolerance; AR;
more severe than fructokinase deficiency because fructose 1-P acts as a phosphate
sink; presentation: fructosuria, lethargy, hypoglycemia, liver and proximal renal
tubule disorder (Hyperbilirubinemia, hyperuricemia (degradation of ADP due to
loss of Pi)); treatment: fructose (and sucrose)-free diet.
Galactose metabolism: A 2-week-old boy is brought to the emergency department after
progressive lethargy over the course of the previous week. His parents report extended
episodes of vomiting after feeding ever since birth though this has not previously been
extensively investigated. Physical exam shows the development of bilateral cataracts so
based on clinical suspicion a metabolic panel is conducted in order to evaluate for
disorders of galactose metabolism.
 Source: lactose in dairy products: disaccharide composed of glucose and galactose;
hydrolyzed by lactase in brush border of small intestine
 Metabolic pathway: trapped in cell by galactokinase; converted into glucose-1-
phosphate through UDP mediated epimerization, glucose-1-phosphate can then be
used: directly in glycogenesis, converted to glucose-6-phosphate and used in
glycolysis or released as free glucose in hepatocytes
 Pathophysiology:
o Galactokinase deficiency: presentation: galactosemia/galactosuria, cataracts in
childhood, excess galactose is converted to galactitol, catalyzed by aldose
reductase, galactitol is osmotically active. Treatment: galactose free diet
o Gal-1-P uridyl transferase deficiency: similar to galactokinase deficiency but
more severe, Gal-1-P acts as a phosphate sink; presentation: early cataracts,
more severe, with vomiting/diarrhea after milk ingestion, liver disease, lethargy,
mental retardation, hepatomegaly, Hyperbilirubinemia, jaundice.
Treatment: galactose free diet
Lactose intolerance: mechanism: lactase deficiency. Primary: hereditary; ↑ frequency in
african americans and Asians; age-dependent. Secondary: post-gastroenteritis.
 Presentation: bloating, cramps, osmotic diarrhea; symptoms due to fermentation of
indigestible lactose by intestinal bacteria, leads to production of H2, CH4, organic
acids (H2 used in diagnosis, detected on breath following oral lactose load)
 Treatment: eliminate dairy from diet, take lactase pills when consuming dairy
Fat Metabolism:
 Fatty acid metabolism: Structure: long chain of carbons with carboxyl group on one
end, can have a variable amount of double bonds (double bonds make a fat
unsaturated, naturally in a cis configuration: trans fats are unnatural and created via
hydrogenation of vegetable oils (↑ risk of atherosclerosis), double bonds ↓ melting
temperature: plant fat (e.g. olive oil) is unsaturated and liquid at room temperature,
animal fat (eg. butter) is saturated and solid at room temperature)
 Nomenclature:
o e.g. palmitic acid: C16:0; 16 carbons with no double bonds, numbered with
carboxyl carbon as 1
o e.g. linoleic acid: C18:2 (9,12); 18 carbons with 2 double bonds (one at the 9th
and one at the 12th carbon)
o omega system: count opposite to the numbered system (i.e. carboxyl carbon is
counted last), used to number unsaturated fats e.g. linoleic acid: omega 6 family,
double bond at position "12" is 6 in from the opposite side (18 carbons in total)
 Essential fatty acids (FA): cannot be synthesized. Examples:
o Linoleicacid: omega 6, can be used as a precursor for arachidonic acid (becomes
an essential fatty acid if linoleic acid is absent)
o linolenic acid: omega 3, ↓ risk of CV disease. Remember: omega 3 saves you from
triple bypass (found in cold water fish, nuts)
Fatty acid synthesis: FA synthesis:
o pyruvate (carbohydrate) → acetyl-CoA: activated by insulin, functions to store
excess carbs as fat, occurs in the mitochondria via pyruvate dehydrogenase
o acetyl-CoA + oxaloacetate → citrate: shuttled out of mitochondria into
cytoplasm (citrate shuttle), split back to acetyl-CoA and oxaloacetate
o acetyl-CoA + CO2→ malonyl-CoA: catalyzed by acetyl-CoA carboxylase, biotin
required, activated by insulin
o malonyl-CoA → CO2 + 2 carbons on fatty chain: catalyzed by FA synthase
 requires NADPH
 humans make palmitic acid (16:0) as stored fat: only de novo fat possible
 for 1 palmitic acid requires: 8 acetyl-CoA, 7 ATP, 14 NADPH
Fatty acid catabolism: Break down via β-oxidation: occurs in hepatocytes, myocytes,
adipocytes (neurons cannot use fat as energy: FAs do not cross BBB). Pathway location
differs based on length of Fas: short/medium (2-12 carbons) diffuse in mitochondria,
long (14-20 carbons) utilizes carnitine shuttle:
▪ carnitine added to FA in the intermembrane space of the mitochondria catalyzed
by carnitine acyltransferase (CAT) -1, inhibited by malonyl-CoA so as to prevent
newly synthesized FAs from being degraded
▪ carnitine: FA transported into the matrix catalyzed by the carnitine transporter
▪ carnitine exchanged for CoA catalyzed by carnitine acyltransferase (CAT)-2
▪ clinical importance: myopathic CAT deficiency: presentation: myoglobinuria
(muscle aches/weakness), ↑ TG content in muscles (unable to use as energy),
provoked by prolonged use of muscle; very long (>20 carbons) oxidized in
peroxisome
β-oxidation pathway: occurs in the mitochondrial matrix, reverses FA synthesis:
Removing an acetyl-CoA and producing NADH and FADH2 catalyzed by fatty acyl-CoA
dehydrogenase. Two types: long-chain acyl-CoA dehydrogenase (LCAD), medium-chain
acyl-CoA dehydrogenase (MCAD); blocked by ackee fruit toxin.
 Creates most of the energy used by the liver: acetyl-CoA created in liver does not enter
the citric acid cycle
 Clinical importance: MCAD deficiency: presentation: non-ketotic hypoglycemia, C8-
C10 acyl carnitines in the blood (liver unable to break FAs down further than C8-
C10), no ketone bodies (liver unable to produce ketones from β-oxidation), fasting
hypoglycemia (liver unable to produce enough energy from β-oxidation to supply
gluconeogenesis); symptoms often precipitated by infection or stress. Treatment:
low fat diet with frequent meals of high carbs.
Propionic acid pathway: Production: β-oxidation of odd-numbered fatty acids (2 carbon
acetyl-CoA groups removed from chain until there are 5 carbons remaining, 5 carbon
chain split into 1 acetyl-CoA (2C) + 1 propionyl-CoA (3C))
 Pathway: propionyl-CoA → methylmalonyl-CoA catalyzed by propionyl-CoA
carboxylase (requires biotin (B7)), methylmalonyl-CoA → succinyl-CoA catalyzed by
methylmalonyl-CoA (requires B12; deficiency of B12 results in a blockage of this step,
result is ↑↑↑ methylmalonate, can cause irreversible neuropathy due to pathologic
synthesis of myelin with methylmalonate, a means to determine whether a patient
with megaloblastic anemia is deficient in folate or B12: methylmalonic aciduria not
seen in folate deficiencies), succinyl-CoA → citric acid cycle (can be converted to
malate: gluconeogenic: exception to rule that fatty acids can be converted to
glucose).
Ketone bodies:
Structure: two types: acetoacetate, β-hydroxybutyrate:
 β-hydroxybutyrate + NAD+ → acetoacetate + NADH
 ↑ NADH:NAD+ ratio results in ↑ β-hydroxybutyrate:acetoacetate ratio
o 1 ketone body = 2 acetyl-CoA
Function
o produced by the liver: brain can use ketones if glucose supplies fall , >1 week of
fasting
o can provide energy to body in prolonged energy needs: prolonged starvation,
glycogen and gluconeogenic substrates are exhausted
o can provide energy if citric acid cycle unable to function
 diabetic ketoacidosis: cycle component (oxaloacetate) consumed for
gluconeogenesis
 alcoholism: ethanol dehydrogenase consumes NAD+ (converts to NADH)
 ↑ NADH:NAD+ ratio in liver favors use of oxaloacetate for ketogenesis
rather than gluconeogenesis.
o RBCs cannot use ketones as they lack mitochondria
Synthesis: occurs in hepatocyte mitochondria (liver cannot use ketones as energy, lacks
β-ketoacyl-CoA transferase (thiophorase) which converts acetoacetate to acetoacetyl)
o under normal conditions acetoacetate = β-hydroxybutyrate
o HMG CoA synthase is rate limiting enzyme
Clinical relevance:
Ketoacidosis: pathogenesis: ↑ ketone levels caused by poorly controlled type I diabetes
mellitus (liver ketone production exceeds ketone consumption in periphery), possible in
type II diabetes mellitus but rare, alcoholism (chronic hypoglycemia results in ↑ ketone
production). Presentation: β-hydroxybutyrate > acetoacetate (due to ↑ NADH:NAD+
ratio), acetone gives breath a fruity odor, polyuria, ↑ thirst.
Tests: ↓ plasma HCO3, hypokalemia (individuals are initially hyperkalemic (lack of insulin
+ acidosis) because K leaves the cells, overall though the total body K is depleted, replete
K in these patients once the hyperkalemia begins to correct), nitroprusside urine test for
ketones may not be strongly + (does not detect β-hydroxybutyrate, state favored by ↑
NADH:NAD+ ratio, should use a test specific for β-hydroxybutyrate)
triglycerides:
 Function: storage form of fatty acids in adipose tissue
 Synthesis: 3 fatty acids + glycerol-3-phosphate → triglyceride. Glycerol-3-phosphate
provided by: reduction of DHAP from glycolysis (by adipocytes and hepatocytes),
phosphorylation of free glycerol by glycerol kinase (only hepatocytes)
 Catabolism :
o triglyceride hydrolyzed into 3 fatty acids + glycerol, catalyzed by hormone
sensitive lipase, ↑ regulated by epinephrine, cortisol , ↓ regulated by insulin
o glycerol is a gluconeogenic substrate in the liver: converted to DHAP
o fatty acids undergo β-oxidation in liver and tissues: can travel in serum
complexed with albumin
Cholesterol: Function: component of cell membrane, precursor for hormone synthesis
(steroids, vitamin D), precursor for bile acid synthesis (means of excretion; >95% of bile
acids are reabsorbed; ↑ in bile acid production used as a means to treat
hypercholesterolemia), cholestyramine (resin that binds bile acids in the GI tract and
prevents reabsorption, increases serum cholesterol usage in bile acid production, ↑
regulates LDL receptor)
 Sources: dietary intake (circulating serum LDL: uptake via endocytosis of LDL:LDL
receptor complex; circulating serum HDL: transfer of cholesterol from HDL to
hepatocyte via scavenger receptor (SR-B1), high levels of this receptor in
hepatocytes and steroid producing tissues), de novo synthesis (occurs in
hepatocytes; HMG-CoA reductase is rate limiting enzyme, inhibited by statins,
glucagon, cholesterol, activated by insulin)
 Regulation: ↑ in intrahepatic [cholesterol] ↓ expression of: HMG-CoA reductase (↓ de
novo cholesterol synthesis), LDL-receptor (↓ in cholesterol-containing LDL uptake
from serum), scavenger receptor (SR-B1) (↓ cholesterol uptake from HDL)
 Transport: cholesterol is fat-soluble: ↑ transport by synthesis of a cholesteryl ester
(dissolves into center of HDL, catalyzed by lecithin-cholesterol acyltransferase
(LCAT): enzyme in serum activated by apoA-1 of HDL)
 Pathology:
o familial hypercholesterolemia: defective LDL receptor; AD; presentation:
cholesterol deposition in skin, xanthelasma (in eyelid), tendon xanthomas (in skin
above tendons), ↑ risk for coronary heart disease
o atherosclerosis: risk factors (causes of endothelial cell damage): smoking, ↑ LDL
in serum, homocystinemia, diabetes; protective factors: antioxidants (vitamin E,
protects LDL from oxidation); process: injury to endothelial cells of blood vessels,
an inflammatory state is induced (T-cell activation, similar to a granuloma),
cholesterol in the blood deposits in blood vessels, LDL oxidized and phagocytosed
by macrophages (can become full of cholesterol: foam cells), fatty streak formed,
fatty streak enlarges, fibrous cap formed (smooth muscle and endothelial cells
migrate over the fatty streak, underlying tissue forms necrotic core), cap at risk of
rupturing (exposes underlying endothelium and causes thrombosis)
Apolipoprotein: A 50-year-old woman presents to her primary care physician for a
regular checkup. She states that for the past four months she has not experienced
menses. Her primary care physician suspects that she has gone through menopause. On
routine lab work, her LDL has increased as compared to one year ago, and her HDL has
decreased drastically:
Mechanisms for fatty acid and cholesterol transport: different types that vary by:
Density: HDL (high density), LDL (low density), IDL (intermediate density), VLDL (very
low density), chylomicrons; arranged from most to least dense. types of apolipoprotein:
HDL: Function: transfer cholesterol (tissues → liver), good cholesterol. Structure : apoA-1
(activates lecithin cholesterol acyltransferase (LCAT)), apoE/apoC-II (apolipoproteins
donated to chylomicrons and VLDL)
LDL: Function: transfer cholesterol from liver → tissues (most recycles back to be
absorbed by the liver); bad cholesterol. Structure: apoB-100; mediates endocytosis of
LDL by binding to apoB-100 (LDL) receptor on liver and tissues
IDL: Function: VLDL + TGs → IDL; picks up cholesterol esters from HDL, catalyzed
by cholesterol ester transfer protein (CETP), IDL + cholesterol from HDL → LDL.
Structure: apoE (recieves from HDL, mediates uptake by the liver, remember: apoE
empties to liver), apoB-100 (mediates endocytosis of LDL by binding to apoB-100 (LDL)
receptor on liver and tissues).
VLDL: Function: transports TGs from liver → tissues. Structure: apoB-100, form
packaged and secreted from liver; apoC-II activates lipoprotein lipase (catalyzes
hydrolysis of triglycerides into individual FAs for absorbtion; ↑ regulated by insulin);
apoE mediates uptake by the liver
Chylomicrons: Function: transport cholesterol, TGs, FAs, and fat soluble vitamins
(intestine → tissues), released from intestinal lumen cells into lymphatics. Structure:
apo-48 (form packaged and secreted by intestine), apoC-II (activates lipoprotein lipase,
added from HDL), apoE (mediates uptake by the liver, added from HDL)
lipoprotein disorder: A 12-year-old boy presents to the emergency department with
chest pain after gym class. His parents note that he has been having increased episodes
of difficulty catching his breath after exertion and has had previous episodes of chest
pain on exertion. Upon physical exam yellow deposits are found on his heels and on his
eyebrows. Based on clinical suspicion a LDL level is obtained and is found to be 480
mg/dL. He is referred to a geneticist for evaluation of deficiencies in LDL receptors and is
prescribed a statin.
Hyperlipoproteinemia:
 Type I hyperlipoproteinemia (hyperchylomicronemia): pathophysiology: deficiency in
lipoprotein lipase or apoC-II results in ↑↑↑↑ TGs, chylomicrons. Presentation:
eruptive xanthomas, steatosis, abdominal pain post-fat ingestion, retinitis
pigmentosa
 Type II hyperlipoproteinemia: pathophysiology: deficiency in LDL receptors
 type IIa (familial hypercholesterolemia) results in ↑↑↑↑ LDL (>260 mg/dL), type IIb
(familial combined hyperlipidemia) results in ↑↑↑↑ LDL, TGs, cholesterol
 Presentation: deposition of cholesterol in normal tissue (xanthomas, xanthelasma),
↑↑ risk for coronary heart disease
 Type III hyperlipoproteinemia (familial dysbetalipoproteinemia): pathophysiology:
deficiency in apolipoprotein E. Remember: III lacks E; results in ↑↑↑ in remnants
(chylomicron/ IDL), apolipoprotein normally clears remnants (empties)
 Presentation: similar to type II hyperlipoproteinemia, ↑↑ risk for coronary heart
disease
 Type IV hyperlipoproteinemia (familial hypertriglyceridemia): pathophysiology: ↓
removal or ↑ production of VLDL results in ↑↑↑ in VLDL. Presentation: pancreatitis
 Type V hyperlipoproteinemia: type I + type IV; ↓ lipoprotein lipase + ↑ VLDL.
Remember: 1+4=5
 Acquired hypercholesterolemia: oral contraceptive, obstructive jaundice
 Acquired hypertriglyceridemia: alcoholism, renal failure, diabetes mellitus
 Treatment for hyperlipoproteinemias: dietary modifications (type I), statins (type II -
IV), niacin (type II, IV, V), fibrates (type IIa, IV, V), bile acid sequestrants (type IIa)
Hypolipoproteinemia: Abetalipoproteinemia: AR; pathophysiology: deficiency in
apolipoprotein B-48 and B-100 (remember: A (without) beta (B)), ↓ chylomicrons (B-
48), VLDL/LDL (B-100). Presentation: malabsorption of fat (can enter enterocytes but
cannot exit because it cannot be packaged for release in lipoproteins, leads to histological
appearance of fat droplets inside enterocytes), ↓ vitamin E absorption, ataxia, hemolytic
anemia with acanthocytes
High yield:  fatty acid synthesis
▪ Remember that type I, IIa, and IV are the most common types of  glucose uptake in fat, muscle, liver
hyperlipoproteinemia  protein anabolism
 I and IV present with pancreatitis, IIa presents with early symptoms of ACS  insulin-resistant tissues
 Treat all types other than I with statins, niacin, and fibrates  brain
 RBCs
Metabolism overview: Rate limiting enzymes:  Post-absorptive state
Process Enzyme o between meals
Glycolysis Phosphofructokinase-1 (PFK-1) o epinephrine/glucagon in control
 glucagon-sensitive tissues
Gluconeogenesis Fructose-1,6-bisphosphatase  adipocytes
TCA cycle Isocitrate dehydrogenase  hepatocytes
Glycogen synthesis Glycogen synthase  pathways favored by glucagon
Glycogenolysis Glycogen phosphorylase  glycogenolysis
 fatty acid catabolism
HMP shunt Glucose-6-phosphate dehydrogenase (G6PD)
 protein catabolism
De novo pyrimidine  gluconeogenesis
Carbamoyl phosphate synthetase II
synthesis  glucagon-resistant tissues
De novo purine synthesis Glutamine-PRPP amidotransferase  brain
Urea cycle Carbamoyl phosphate synthetase I  RBCs
Fatty acid synthesis Acetyl-CoA carboxylase (ACC)  Starvation
o days 1-3
Fatty acid oxidation Carnitine acyltransferase I  main source of energy for body
Ketogenesis HMG-CoA synthetase  catabolism of triglycerides in fat stores
Cholesterol synthesis HMG-CoA reductase  main source of energy for brain
 glucose from gluconeogenic conversion of lactate and alanine
Metabolism of exercise and starvation state: o > 3 days
 100 meter sprint  main source of energy for body
o energy source used  catabolism of triglycerides in fat stores
 pre-synthesized ATP  main source of energy for brain
 creatine phosphate  mostly glucose with some ketone bodies
 anaerobic glycolysis o > several weeks
 100 meters to 1 kilometer  main source of energy
o energy source used  degradation of muscle and organs
 oxidative phosphorylation  after adipose tissue store have been exhausted
 main source of energy for brain
 1 kilometer to a marathon
o energy source used  2/3 ketone bodies
 glycogen  1/3 glucose
note: RBCs are always dependent on glucose
 β-oxidation of fatty acids
Pyruvate metabolism: A 23-year-old man is running a marathon. In the last mile of the
 Absorptive state race he experiences an intense burning in his muscles. Once he completes the race and
o immediately after eating rests, the burning slowly subsides. He asks his physician the next day why this may have
o insulin in control occurred and what he can do to mitigate it. His physician explains that a training
 insulin-sensitive tissues regimen can increase his ability to perform aerobic metabolism, and thus decrease the
 adipocytes conversion of pyruvate to lactate (via lactate dehydrogenase) which caused the burning
 myocytes in his muscles that he experienced.
 hepatocytes Pathways
 pathways favored by insulin  pyruvate → lactate
 glycogenesis o catalyzed by
  lactate dehydrogenase (LDH)  hypoxia alone cannot explain this effect
o reversible  generates high amounts of biosynthetic substrates for growth
o generated in anaerobic glycolysis  unique protein expression profile
 allows conversion of NADH → NAD+  Metabolic changes
o in the liver, LDH converts lactate to pyruvate o transport of glucose into cells
 for gluconeogenesis or for metabolism to acetyl-CoA  ↑↑ expression of GLUT1
 Cori cycle  allows for ↑↑ glucose uptake
 shifts energy generation from periphery to the liver  ↑↑ expression of hexokinase 2
 pyruvate → acetyl-CoA  promotes glycolysis
o catalyzed by  inhibits apoptosis
 pyruvate dehydrogenase (PDH)  ↑↑ affinity for ATP
o irreversible  ↓ inhibition by G6P
o acetyl-CoA enters the citric cycle o glycolysis
 pyruvate → oxaloacetate  ↑↑ expression of fetal pyruvate kinase
o catalyzed by  favors the anaerobic pathway
 pyruvate carboxylase (PC)  ↑↑ shunting into pentose phosphate shunt
o irreversible  NADPH needed for biosynthesis
o oxaloacetate can o fatty acid metabolism
 replenish the citric acid cycle  ↓↓ β-oxidation
 substrate for gluconeogenesis  ↑↑ lipid biosynthesis
 pyruvate → alanine o amino acid metabolism
o catalyzed by  ↑↑ utilization of glutamine
 alanine transaminase (ALT)  replenish intermediates in the TCA cycle
o reversible  e.g. oxaloacetate
o alanine carries amino groups to the liver from muscle o cell growth
o in the liver, ALT converts alanine to pyruvate  ↑↑ expression of Akt
 for gluconeogenesis  controls proliferation of cells
 regulates growth downstream of insulin
Ethanol metabolism: Function: elimate ethanol (EtOH): CNS depressant/toxin  Clinical importance
Kinetics: NAD+ is limiting reagent, alcohol dehydrogenase operates via zero-order o cancer therapy
kinetics, inhibitors (fomepizole inhibits alcohol dehydrogenase, antidote for suspected  inhibitors of pyruvate kinase
ethylene glycol or methanol poisoning; disulfiram (Antabuse) inhibits acetaldehyde  forcing the cancer cell to use the TCA cycle
dehydrogenase, acetaldehyde accumulates, leads to hangover symptoms, prescribed to
 inhibitors of fetal pyruvate kinase
help recovering alcoholics)
 ↑ amount of adult pyruvate kinase
Clinical relevance: ethanol hypoglycemia: pathophysiology: ↑ in EtOH metabolism → ↑
 ↑ use of the TCA cycle
NADH/NAD+ ratio, NADH/NAD+ ratio changes energy generating kinetics, lactate
DNA:
favored over pyruvate (lactate + NAD+ → pyruvate + NADH; no free NAD+ for required
Nucleus:
conversion), glycerol-3-phosphate favored over DHAP (G3P + NAD+ → DHAP + NADH),
Nuclear envelope: membrane bilayer structure, continuous with ER, contains nuclear
malate favored over oxaloacetate (malate + NAD+ → OAA + NADH). Presentation: lactic
pores: controls transport between cytoplasm and nucleus, more active cells have more
acidosis (↑ lactate), fatty liver (↑ G3P results in ↑ synthesis of TGs), ethanol + extreme
nuclear pores, small molecules freely traffic, large molecules require active transport
physical exertion (severe hypoglycemia, EtOH inhibits the Cori cycle by consumption of
(mediated by importins and exportins, nuclear localization signal (NLS) provides signal
free NAD+: conversion of lactate to glucose in anaerobic metabolism, lactic acidosis).
for nuclear access)
Nuclear lamina: on inner face of nuclear envelope, fibrous network of proteins, role:
Warburg hypothesis:
attach to chromatin, participate in construction and deconstruction of nucleus during cell
 Principle idea division (phosphorylation by lamin kinase in prophase results in blebbing of nuclear
o cancer cells generate energy via a unique mechanism envelope)
 anaerobic glycolysis
 low energy generating pathway Nucleolus: location of rRNA synthesis, site of ribosomal assembly
 cancer cells need high energy to facilitate their rapid division
Nucleic acid structure:  Clinical importance
 Nitrogenous bases o anticancer drugs
o purines  can intercalate DNA
 structure  daunorubicin, doxorubicin
 2 rings  can bind DNA
 examples  cisplatin
 adenine (A), guanine (G) Chromatin structure:
 found in DNA and RNA Composition:
 xanthine, hypoxanthine, uric acid DNA: - charge
 not found in either DNA or RNA Histone proteins: + charge from Lys and Arg residues; H2A, H2B, H3, H4 (core proteins);
o pyrimidines H1 (linking protein); post-translational modification of histone tails, forms a code,
 structure interpreted by effector proteins to regulate transcription downstream. Histone
 1 ring acetyltransferase (HAT): acetyl group added which blocks positive charge of histone
 examples protein and loosens interaction with DNA, ↑ transcription. Histone deacetylase (HDAC):
 cytosine (C) removes acetyl group which exposes positive charge and tightens interactions, ↓
 found in DNA and RNA transcription
 thymine (T) Electrostatic attraction of DNA with histone proteins
 found in DNA
Organization:
 uracil (U)
10 nm chromatin: DNA wraps around dimer of H2A:H2B:H3:H4; called nucleosome;
 found in RNA
sensitive to nuclease activity
 Nucleoside 30 nm chromatin: nucleosomes held together by H1; not sensitive to nuclease activity
o nitrogenous base + ribose 30 nm fiber loops: further condensation
 Nucelotide
o nitrogenous base + ribose + phosphate Euchromatic/heterochromatic: euchromatic = accessible to transcription,10 nm through
 3'-5' phosphodiester bond connects ribose and phosphate 30 nm fiber loops; heterochromatic = not accessible to transcription, any greater
 DNA condensation than 30 nm fiber loops, condensed to save room
o nucleotide interactions
 H-bonding De novo nucleotide synthesis:
 G with C
 3 H-bonds Phosphoribosyl pyrophosphate (PRPP): sugar building block formed in nucleotide
 stronger synthesis, added to nitrogenous base to form nucleoside, formed from ribose-5-
 A with T phosphate (product of pentose phosphate shunt: ATP + ribose-5-phosphate = PRPP +
 2 H-bonds AMP; catalyzed by PRPP synthetase). Once PRPP is made it can add to either a de novo or
 weaker salvaged base
 A with U in RNA
 melting or denaturing Pyrimidine: pathway diagram, important enzymes:
 H-bonds disrupted with changes in temperature, pH, chemical carbamoyl phosphate synthetase-2: rate limiting step; not the same carbamoyl
agents phosphate as in urea cycle
ribonucleotide reductase: inhibited by hydroxyurea; also reduces UDP, CDP, ADP, GDP;
 ↑ GC content = ↑ melting temperature
dADP and dATP negatively feedback and inhibit enzyme. Result = ↓ dTMP, dUDP, dCDP,
 DNA can form correct structure again if disrupting agent is
dADP, dGDP. Note: good to target thymidine synthesis because it is not involved in RNA
removed slowly (reanealing)
thymidylate synthase: inhibited by 5-fluorouracil (5-FU); result = ↓ dTMP
 key principle of Southern blotting and PCR
dihydrofolate reductase: inhibited by: methotrexate (MTX) in eukaryotes, trimethoprim
 see Biological lab techniques section (TMP) in prokaryotes (sulfamethoxazole (SMX) interferes with DHF synthesis in
o strands prokaryotes, co-trimoxazole = TMP + SMX), inhibited by pyrimethamine in protozoa.
 antiparallel Result = ↓ dTMP
 right handed double helix Deficiency: orotic aciduria: inability to convert orotic acid to UMP, defect in uridine
o Chargaff's rules monophosphate (UMP) synthase; AR. Presentation: ↑ orotic acid crystals in urine,
 A % = T/U % megaloblastic anemia (does not improve with administration of vitamin B12 or folic acid:
 G%=C%
not enough thymidine to sustain normal erythropoiesis), failure to thrive, no DNA gyrase (topoisomerase II) breaks the DNA to prevent coiling.
hyperammonemia (distinguishes between ornithine transcarbamylase (OTC) deficiency RNA primer removed by RNAase H in eukaryotes and filled by a DNA polymerase by DNA
with high [orotic acid] in urine with hyperammonemia). Treatment: oral uridine polymerase I in prokaryotes and can fill simultaneously.
administration, bypasses defect in de novo pyrimidine pathway DNA ligase seals the nick between fragments.

Purine: pathway diagram, insufficient capacity in most cells, important enzymes: PRPP Differences between prokaryotes eukaryotes: prokaryotes (single origin of replication),
amidotransferase: rate-limiting step, inhibited by AMP, GMP, IMP, indirectly inhibited by eukaryotes (multiple origins of replication)
allopurinol, 6-mercaptopurine. Note: base of inosine = hypoxanthine Clinical importance: antibiotics (quinolones, fluoroquinolones block bacterial
Nucleotide catabolism/salvage: topoisomerase; used to treat aerobic gram negatives in UTIs and gonorrhea; e.g. drugs
Purine: salvage pathway (see figure), clinical importance: ending in –floxacin), cancer chemotherapy (etoposide, teniposide block eukaryotic
Adenosine deaminase (ADA) deficiency: defective purine salvage, results in excess ATP topoisomerase)
and dATP, prevents DNA synthesis. ATP and dATP feedback negatively on ribonucleotide
reductase in the synthesis of purines and pyrimidines for DNA replication, ↓ lymphocyte Reverse transcription: Function: convert RNA to DNA, performed by reverse
count (major cause of SCID (severe combined immunodeficiency disease: lack of both T transcriptase, has high error rate and cannot proofread, three activities: RNA-dependent
and B cells); AR DNA polymerase, RNase, DNA-dependent DNA polymerase
Lesch-Nyhan disease: defective purine salvage; lacks hypoxanthine guanine Clinical relevance: present in retroviruses e.g. HIV, some HIV drugs inhibit reverse
phosphoribosyl pyrophosphate transferase (HGPRT) enzyme; XR, presentation: severe transcriptase: AZT (enters cells and modified by addition of ATP, functions as a
CNS symptoms (choreoathetosis, mental retardation), self-mutilation, hyperuricemia nucleotide analog, terminates replication). Also present in telomerase; used in RT-PCR.
(degradation of all purines since it cannot salvage)
Gout: pathophysiology: high urate levels due to: ↑ in cell breakdown (e.g. treatment of DNA repair:
large tumor masses with radiation or chemo), ↓ in renal excretion (most common cause). Damage tolerance:
Results in precipitation of monosodium urate crystals in joints, negative birefringence  Function
(yellow when parallel to slow ray; needle shaped). Presentation: recurrent acute o can allow DNA replication to continue despite presence of DNA
arthritis: pain in big toe first (podagra), chronic: tophi present (granulomatous damage (e.g. thymidine dimer)
deposition (multinucleated giant cells) of crystals in soft tissue; ↑ frequency in men >30  Process
y/o. Treatment: acute → colchicine or indomethacin, chronic due to ↓ in renal excretion o DNA polymerase stalls at dimer
→ probenecid, chronic due to ↑ in cell breakdown → allopurinol o sliding clamp releases regular DNA polymerase and binds the one of
two translesion polymerases
Pyrimidine: salvage (may be salvaged by pyrimidine salvage enzymes), degradation  error free
(completely broken down to ammonia)  recognizes that the dimer is normally a thymidine
Other causes of hyperuricemia: and the polymerase adds an adenosine opposite
↑ EtOH intake: can precipitate an acute gout attack and continues replication
↑ nucleic acid in diet: red meats, organ meats  error prone
Phosphate "trapping" diseases:
 polymerase adds any base opposite the lesion and
▪ e.g. glucose-6-phosphate deficiency (G6PD), galactose uridyltransferase
continues replication
deficiency
mismatch repair:
▪ caused by an inability to dephosphorylate common metabolites and therefore
leads to trapping of phosphate by these metabolites  Process
▪ lack of phosphate prevents synthesis of ATP, GTP, plus other nucleotide o repairs G/T or A/C pairing
phosphates. ADP, AMP and other hypophosphorylated bases are salvaged  sometimes misincorporated due to tautomerization of the
producing uric acid nucleotide
o involves MutS, MutH, MutL enzymes
DNA replication: o strand specific
Function: replicate cell genome in a manner that is highly accurate  recognizes which is the new strand because it is
Process: DNA melted to expose single strand to expose origin of replication, single unmethylated and the old strand is methylated
stranded binding proteins (SSBs) bind and stabilize melted DNA, RNA primer added in 5'  Deficiency
→ 3' direction by primase, DNA polymerase adds adds nucleotides in a 5' → 3': o hereditary nonpolyposis colorectal cancer
DNA polymerase III in prokaryotes, DNA polymerase α and δ in eukaryotes, can edit  aka Lynch syndrome
mistakes with a 3' → 5' exonuclease activity, adds continuously on the leading strand,  cause
adds discontinuous Okazaki fragments on the lagging strand because it must synthesize  hereditary absence of one copy of
in a 5' → 3' direction. enzyme hMLH1 or hMSH2
  second copy lost due to somatic mutation
 known as the two-hit model
 common to many DNA repair
deficiencies
 presentation
 microsatellite instability
 di-, tri-, tetranucleotide repeats that can
be amplified
 constant in number in normal
cells
 diagnostic in Lynch syndrome
 ↑↑ risk of colorectal cancer
 NOT preceded by benign polyps
due to the use of the opposite strand as a template)
Deficiency: Bloom syndrome: cause: lack of BLM helicase enzyme; presentation: short
stature, rash from sun exposure, café-au-lait spot, leukemias, lymphomas, carcinomas
BRCA-1 involved in: breast, prostate, ovarian cancer
BRCA-2 involved in: breast cancer
Non-homologous end joining:
Function: repair double-strand breaks (these breaks may be caused by ionizing radiation
or oxidative free radicals; mechanism of cancer radiation therapy), occurs when a sister
chromatid is not available to use as a template (prior to S phase of cell cycle)
Process: break recognized by MRN complex, additional enzymes (Artemis, XLF, Pol μ) cut
ends so they can bind, DNA ligase IV joins ends together
Deficiency: severe combined immunodeficiency disease (SCID) (one of many causes)

Base excision repair: Telomeres:

Function: specific endonucleases (glycosylasse) remove bases that have been modified  Structure
by several common mechanisms of damage e.g. deaminated cytosines (C → U) removed o regions of DNA at the ends of chromosomes that are non-coding and repetitive
by uracil glycosylase; can take place anytime during the cell cycle but occurs primarily in o added by telomerase enzyme
G1  Function
Process: glycosylase specific for the damaged nucleotide removed damaged base by o protect ends of DNA from degradation
breaking glycosidic bond, damaged base removed (sugar remains but base removed:  Clinical relevance
creates an apurinic/apyrimidinic (AP) site), gap filled by DNA polymerase, ligation of o each round of replication shortens the telomere because DNA polymerase requires
strand nick by DNA ligase III an RNA template and lagging strand cannot replicate all the way to the end of the
chromosome
Nucleotide excision repair: o telomere shortening is implicated in cell senescence
Function: removes thymidine dimers caused by UV-B light, removes damaged bases o cancer cell often have ↑ telomerase activity
caused by chemicals  makes unlimited replication potential possible
Process: maintenance repair: RNA:
• XPC recognises DNA lesion and recruits XPA  rRNA
• XPB-G binds DNA and removes a chunk spanning the damaged segment o ribosomal
• DNA polymerase fills the gap o rRNA + protein = ribosome
• DNA ligase seals the nick o most abundant of all RNA types
transcription-coupled repair o clinical importance
• RNA polymerase stalls at DNA lesion  shiga- and verotoxins bind to 28S rRNA
• CSB and XPG recognize stalled RNA polymerase  inhibit eukaryotic protein synthesis
• CSA joins complex and removes damaged site and allows transcription to continue  tRNA
Deficiency: xeroderma pigmentosum (XP): cause: lack any enzyme XPA – XPG. o transfer
Presentation: cannot repair UV damage: sunlight sensitivity, ↑↑↑ prevalence of skin o covalently attached to amino acids
cancer, corneal ulcers. Diagnosis: measurement of repair mechanisms in white blood o provides link between genetic code and a particular amino acid
cells. Treatment: avoidance of sunlight
 mRNA
Cockayne syndrome: cause: lack of CSA or CSB; AR; presentation: growth failure,
o messenger
photosensitivity, nervous system abnormalities; can affect any organ system
o carries DNA message from chromosome to translation by ribosome
 hnRNA or pre-mRNA
Homologous recombination: o heteronuclear
Function: repair double-strand breaks, requires a sister chromatid to use as a template o present only in eukaryotes
(therefore must occur after S phase of cell cycle) o mRNA transcript prior to splicing
Process: double-strand break recognized by MRN complex, BRCA and BLM enzymes  contains introns and exons
involved in end processing, Holliday junctions are formed (cross-shaped structures that  snRNA
mediate strand rejoining), junctions are resolved (may result in loss of heterozygosity, o small nuclear
 o present only in eukaryotes  α-amanitin
o functions in splicing of mRNA in the nucleus  poison in death cap mushrooms
 Ribozymes  actinomycin D
o present in eukaryotes and prokaryotes  chemotherapeutic
o have intrinsic enzymatic activity Gene organization:
 miRNA Enhancer: function: allow signal transduced to ↑ transcription, act by binding and
o micro bending DNA such that they can act on the promoter directly. Location: may be very far
o ↓ translation by binding to 3'-UTR of target mRNA from gene locus, may be within gene locus, orientation does not matter. Examples:
 double-stranded RNA signals for degradation glucocorticoid response elements (GREs) bind glucocorticoid, estrogen response
o uses Dicer and RISC enzymes in processing elements (EREs) bind estrogen, cAMP response element (CREs) bind cAMP
o often coded for in intron segments Silencers: act in a mechanism similar to enhancers but ↓ transcription
o RNAi is a synthetic lab technique that takes advantage of miRNA
pathway Upstream promoter elements: function: allow transcription factor induced transcription
RAN polymerase: ↑. Location: close to gene locus; examples: CCAAT box bind NF-1, TATA box
Prokaryotes:
 1 type of RNA polymerase (RNAp) 5'-UTR: untranslated region upstream of start codon
 Structure
Kozak consensus sequence: GCCGCCRCCAUGG, where R is A or G three bases upstream
o α2ββ' core subunits
from AUG start codon; plays a major role in initiation of translation, a mutation in the
 Initiation Kozak consensus sequence may cause β-thalassemia intermedia)
o σ (sigma) = initiation factor
o binds RNAp to DNA Start codon: codes for methionine (AUG), beginning of translated (coding) region
 Termination Coding region: between start codon and stop codon, following transcription contains
o ρ (rho) = termination factor introns and exons (eukaryotes)
 Clinical importance
o inhibited by Stop codon: UGA, UAG, UAA; terminates translation; 3'-UTR: untranslated region
 rifampin downstream of stop codon
 antibiotic
 actinomycin D Transcription: Process:
 antibiotic Initiation: RNAp binds the template DNA strand at the promoter site with the help of
eukaryotes: transcription factors
 3 types of RNA polymerase Elongation: RNAp synthesizes the mRNA strand in 5' → 3' direction (reads template
o RNAp 1 strand in 3' → 5' direction; eukaryotic RNAps transcribe introns as well as exons), RNAp
 synthesizes rRNA in nucleolus inhibited by alpha-amanitin (ingestion of the amanita phalloides mushroom)
 remember: rRNA is most abundant RNA; so it is #1 Termination:
o RNAp 2 prokaryotes: two mechanisms:
 synthesizes mRNA, snRNA in nucleus rho-dependent: rho protein binds the mRNA; causes RNAp to fall off
o RNAp 3 rho-independent: mRNA has a GC-rich segment that naturally forms a hairpin; causes
 synthesizes tRNA in nucleus RNAp to fall off
 remember: tee = three eukaryotes: transcription continues past the gene and eventually falls off
 makes eukaryotic 5s rRNA
Mono/polycistronic: monocistronic = mRNA codes for 1 protein (eukaryotes only have
 Structure monocistronic mRNA), polycistronic = mRNA codes for >1 protein (e.g. bacterial lac
o similar in structure to prokaryotes operon)
 Initiation Transcription regulation:
o TFIID = initiation factor Function: can turn transcription on/off, can ↑ or ↓ rate of transcription, can act in cis or
o binds RNAp to DNA trans:
 Termination cis = regulation near gene locus: DNA binding sequence; a mutated cis regulatory
o translation continues far past gene and RNAp eventually falls off element can result in gene that is constitutively on or off
 Clinical importance trans = regulation from gene locus: transcription factor protein; because it acts at a
o inhibited by distance a good allelic copy can compensate for a mutated copy

Mechanisms of control: modify RNAp binding stability, transcription factors:
Function: modify basal transcription levels, two types:
General: must bind to DNA and RNAp to begin baseline transcription of most every gene
e.g. TFIID binds TATA box and RNAp II
Specific: acts through enhancers and silencers; can regulate specific gene responses.
Structure: DNA binding domain (can be zinc fingers, helix-turn-helix, helix-loop-helix, or
leucine zippers), regulatory element binding domain (e.g. binds hormone, ion, other
transcription factors, etc).
Modify RNAp accessibility to DNA:
histone modifiers: histone acetylases (HATs) open DNA and ↑ transcription, histone
deacetylase (HDACs) close DNA and ↓ transcription
Imprinting: methylation effectively shuts a gene off; often irreversible, some genes
methylate a gene locus on paternal or maternal chromosome, allows only one allele to be
active e.g. Prader-Willi/Angelman syndrome
inactivation of a chromosome: condensation of # of X chromosomes - 1 to form Barr
bodies e.g. Turner's Syndrome (XO) the patient would have no Barr bodies as they only
have 1 X chromosome
increase number of gene copies: more sites for RNAp to bind; common in oncogenes
Embryonic gene regulation:
sonic hedgehog (SHH) gene: mutations causes holoprosencephaly (HPE); failure of
midline brain to separate into right and left
homeobox (HOX) genes: control proper timing of gene activation
paired box (PAX) genes: mutations cause Klein-Waardenburg syndrome; presentation:
neural crest abnormalities, deafness, variation in pigmentation (forelock of white hair,
patches of different colored skin), dystopia canthorum (broad nasal root)
Examples of gene regulation:
peroxisome proliferator-activated receptors (PPARs): controls fat metabolism: turned on
by endogenous ligands (fatty acids, prostaglandins), also turned on by exogenous ligands
(fibrates, thiazolidinediones); bind PPRE region in DNA
Clinical importance: fibrates given to hyperlipidemic patients to ↑ transcription of
lipoprotein lipase also used in treatment for Zellweger syndrome.
RNA processing: Only in eukaryotes; pre-processed transcript = hnRNA
Process:
Capping: 5' end (7-methylguanosine cap, occurs before transcription has finished,
functions to help in ribosomal binding, also protects against degradation), 3' end
(polyadenylated (poly-A) tail, added by poly-A-polymerase, functions to protect from
degradation, some viruses can steal host cell caps so that the viral mRNA gets translated)
Splicing: process of intron removal, mediated by the spliceosome (snRNPs) (lariat is
formed, adjacent exons are attached).
Alternative splicing: one hnRNA can be spliced differently to produce different protein
products
Location: occurs in nucleus after transcription; only processed RNA is transported out of
the nucleus
Protein synthesis: function: converts mRNA nucleotidemessage into a polypeptide.
Location: mRNA must move from nucleus to cytoplasm for translation. Example: 5' - UUC
- CCU - CAU - 3' mRNA codes for (N-term) - Phe - Pro - His - (C-term) peptide
Amino acids:
Structure: carbon attached to carboxyl group + amino group + "R" group. "R" group gives
each of the 20 amino acids its unique properties: only L-form amino acids (AA) are found
in proteins.
Properties:
hydrophobicity: allow for protein folding such that hydrophilic AAs face externally and
hydrophobic AAs face internally, hydrophobic AAs have aliphatic or aromatic R-groups,
hydrophilic AAs have oxygen or nitrogens in R-group.
pH: acidic AAs are negatively charged at body pH (Asp, Glu), basic AAs are positively
charged at body pH (Arg, His, Lys)
essential/non-essential: essential AAs must be part of the diet. Remember: PVT TIM
HALL (Phe/Val/Thr/Trp/Ile/Met/His/Arg/Lys/Leu)
catabolic product:
glucogenic AAs can become glucose (Met, Val, Arg, His)
ketogenic AAs can become ketone bodies (Leu, Lys)
Some AAs can become both ketone bodies or glucose (Ile, Phe, Thr, Trp)
AA derivatives:
Tyrosine: thyroid hormones (T3/T4), catecholamines, melanin:
Deficiency: albinism; cause: defective tyrosinase (inability to synthesize melanin from
tyrosine), defective tyrosine transporters (↓ amounts of tyrosine and thus melanin), lack
of migration of neural crest cells forming melanocytes; presentation: lack of melanin ↑
risk of skin cancer. Genetics: AR, locus heterogeneity can result in variable inheritance:
XR for ocular albinism
Phenylalanine: catecholamines
Tryptophan: NAD, NADP (niacin), serotonin
Histidine: histamine
Arginine: nitric oxide (NO)
Glutamate: GABA
Glycine: protoprophyrin → heme
Amino acid absorption:
Nitrogen balance:
Positive: amino acid (AA) intake > excretion; examples: growth, pregnancy
Negative: AA excretion > intake; examples: kwashiorkor, starvation, infection
AA absorption in intestine:
Proteins degraded by trypsin and pepsinenteropeptidase cleaves trypsinogen (inactive)
to trypsin (active)
Absorbed as AA's across the gut lumen with specific transporter for similar AA's; e.g.,
transporter for basic AA's, transporter for large neutral AA's.
Deficiencies: lack of absorption with pancreatitis due to ↓ in degradative enzyme
production. Hartnup disease: defect in large neutral AA transporter in the intestine and
renal proximal tubule cells, result is a tryptophan deficiency, presents similar to pellagra
AA absorption in kidney: AA's that are filtered from the glomerulus can be actively
reabsorbed in the proximal convoluted tubules with similar transporters as the gut;
deficiencies: cystinuria: genetic defect in transporter for cysteine, ornithine, lysine,
arginine; AR; presentation: cystine staghorn calculi: note: cystine = 2x cysteine attached
by a disulfide bridge, cystine kidney stones result from high concentrations in urine.
Treatment: alkalinization of the urine with acetazolamide.
Amino acid catabolism: Three possible fates: enter citric acid cycle, form ketone bodies,
substrates for gluconeogenesis
Urea cycle: function: degrade excess amino acids and safely remove nitrogen, (surplus
amino acids cannot be stored), produce urea.
Pathway: aspartate and carbamoyl phosphate provide nitrogens (carbamoyl phosphate
synthesized from NH4+ + HCO3- + 2 ATP via carbamoyl phosphate synthetase I, rate
determining step of pathway, requires N-acetylglutamate which regulates the cycle, only
produced when excess amino acids are present), nitrogen added from systemic pool via
alanine cycle, one turn of the cycle: aspartate + NH3 + CO2 + 3 ATP → urea (containing
2N)+ fumarate + 2 ADP + Pi + AMP + PPi + 3 H20, connected to citric acid cycle via
aspartate-argininosuccinate shunt (fumarate of urea cycle → malate of citric acid cycle,
oxaloacetate of citric acid cycle → aspartate of urea cycle).
Location: Cellularly: formation of carbamoyl phosphate occurs in the mitochondrial
matrix; addition of aspartate and removal of fumarate and urea occurs in the cytoplasm.
Systemic: liver and kidney
Deficiencies: common presentation: hyperammonemia + ↑ [glutamine]blood + ↓ blood urea
nitrogen (BUN), onset shortly after birth (< 1-3 day), hyperammonemia, intoxication
presents with cerebral edema, vomiting, hyperventilation, lethargy, blurring vision; α-
ketoglutarate consumed, stops TCA cycle
carbamoyl phosphate synthase I creates carbamoyl phosphate: AR inheritance pattern;
orotic aciduria absent
ornithine transcarbamoylase forms citrulline from carbamoyl phosphate; XR inheritance
pattern, most common urea cycle disorder. Orotic aciduria because excess carbamoyl
phosphate is shunted into the UMP synthetic pathway in which orotic acid is an
intermediate.
Treatment: low protein diet, benzoate or phenylbutyrate (chelate nitrogen by becoming
aminated)
Ammonia transport: function: safely move nitrogenous wastes from tissues to kidney
and intestine in the form of glutamine. Pathway: ammonia loaded via glutamine
synthetase (NH3 + glutamate → glutamine; occurs in nearly all tissues), ammonia
unloaded via glutaminase (glutamine → NH3 + glutamate, specific to kidneys and
intestine (and low concentration in liver), induced by acidosis).
Glucose-alanine cycle: function: transport pyruvate from muscle to liver for
gluconeogenesis. Pathway: involves reversible aminotransferase reactions
alanine aminotransferase (ALT): glutamate + pyruvate → α-ketoglutarate + alanine in
muscle; α-ketoglutarate + alanine → glutamate + pyruvate in liver, requires vitamin B6
aspartate aminotransferase (AST): glutamate + oxaloacetate → α-ketoglutarate +
aspartate in liver; relationship between amino acids and α-keto acids:
alanine - NH3 = pyruvate
aspartate - NH3 = oxaloacetate
glutamate - NH3 = α-ketoglutarate
Defects in specific amino acid catabolism: all are part of newborn screening program:
phenylketonuria (PKU): inability to break down phenylalanine, deficient in
phenylalanine hydroxylase, ↓ tetrahydrobiopterin cofactor. Presentation: ↑
phenylalanine, ↓ tyrosine, requires tyrosine supplementation, mental retardation,
microcephaly, musty/mousy odor to sweat and urine, restriction of phenylalanine in the
diet though cannot eliminate as it essential for protein synthesis, very strict adherence to
diet during pregnancy for a mother with PKU, avoid aspartame
maple syrup urine disease: inability to breakdown branched-chain amino acids (Val, Leu,
Ile), deficient in branched-chain ketoacid dehydrogenase. Presentation: infantile onset
(normal for first week, progressive onset of symptoms), lethargy, weight loss,
hyper/hypotonia, mental retardation, urine smells of maple syrup, death if dietary intake
of Val, Leu, Ile is not restricted
alkaptonuria: inability to breakdown homogentisic acid (breakdown product of tyrosine
and phenylalanine), deficient in homogentisate oxidase. Presentation: arthritis
(accumulates over years in the cartilage (ochronosis), onset prior to third decade, urine
that darkens upon sitting in air, dark coloration of the sclera
Hartnup's disease: deficiency of neutral amino acid transporter, leads to ↓ tryptophan
absorption. Presentation: pellagra, result of niacin deficiency (niacin produced from
tryptophan)
Homocystinuria: inability to breakdown homocystinuria (methionine degradation
pathway). Causes: cystathionine synthase deficiency, ↓ affinity of cystathionine synthase
for pyridoxal phosphate (B6), homocysteine methyltransferase deficiency, deficiency in
folate, B6 or B12 in the diet can produce a less severe form of homocystinuria.
Presentation: vessel damage (DVT, atherosclerosis, MI before 2nd decade of life), similar
to Marfan's: mental retardation, lens dislocations downward as opposed to upward in
Marfan syndrome, tall with long extremities, ↑ homocysteine in the urine.
Treatment varies by cause: cystathionine synthase deficiency (↓ intake of Met, ↑ intake of
Cys, B12 and folate), ↓ affinity of cystathionine synthase for pyridoxal phosphate (↑ intake
of B6)
propionyl-CoA carboxylase/methylmalonyl-CoA deficiency: inability to handle Val, Met,
Ile, Thr; part of propionic acid pathway. Presentation: ketoacidosis, propionyl-CoA
carboxylase deficiency has ↑ propionic acid, methyl citrate, hydroxypropionic acid,
methylmalonyl-CoA mutase deficiency has ↑ methylmalonic acid. Treat by restricting Val,
Met, Ile, Thr in the diet
tRNA: Structure: cloverleaf structure, 3' aminoacyl end = CCA, bound covalently to amino
acid (AA)
Charging: purpose: adds correct AA to correct tRNA: each AA has a specific tRNA, correct
AA is only checked when attachment occurs by aminoacyl-tRNA synthetase to the tRNA,
thus, if a AA is mischarged, it has no other correction mechanism and will insert for the
AA specified by the tRNA anticodon. Charged tRNA contains the needed energy for the
formation of the peptide bond. Process: tRNA + ATP + aa → aa-tRNA + AMP + PPi,
catalyzed by aminoacyl-tRNA synthetase, inhibited by tetracyclines
 2 GTP to GDP for elongation (2)
Wobble: first 2 nucleotide positions provide specificity for an individual amino acid e.g.,
mRNA triplets CUU, CUC, CUA, CUG all code for Leu. Genetic code is degenerate, all amino Pharmacological inhibition of prokaryotic protein synthesis:
acids except for one have more than one codon (explains silent mutations: mutation from  Bind 30S
CUU to CUC would not alter the protein) o aminoglycosides
 inhibit formation of the initiation complex
Protein synthesis:  promotes misreading of mRNA
Ribosome: eukaryotes: small subunit (40S) synthesized in the nucleus, large subunit  Bind 50S
(60S) synthesized in the nucleolus, total = 80S, S = sedimentation; prokaryotes: small o chloramphenicol
subunit (30S) + large subunit (50S) → 70S  inhibits peptidyl transferase
o macrolides
Catalytic sites :
 inhibits translocation
A (acceptor) site: accepts charged aa-tRNA
o clindamycin
P (peptidyl) site: holds the growing peptide chain and transfers to the aa-tRNA in the A
 inhibits translocation
site
E (exit) site: allows tRNA to exit the ribosome after releasing its amino acid to the aa-
Protein structure:
peptide chain
Peptide bond: formation: carboxyl group on one amino acid + amino group on another
amino acid, results in a loss of water, bond can be broken (hydrolyzed) with addition of
Forms: polysomes: single mRNA being translated simultaneously by several ribosomes,
water across the bond
can be: free (proteins for nucleus or mitochondria), membrane bound (found on rough
ER, proteins for secretion or membrane insertion)
Orders of protein shape:
Primary: amino acid sequence determined by covalent peptide bonds
Steps:
Secondary: stable folding of individual protein domains (a protein may have
Initiation:
combinations of different secondary structures), common forms (α-helix, β-pleated
initiation complex generated from:
sheet), determined by amino acid-amino acid interactions via hydrogen bonds
small ribosomal subunit
Tertiary: shape of protein as a whole which imparts functionality to a protein (shape
mRNA: 5'-cap (eukaryotes), Shine-Dalgarno sequence (prokaryotes) may be disrupted (denatured) with changes in solution), common forms (globular,
initiation aa-tRNA: met-tRNA (eukaryotes), fmet-tRNA (prokaryotes) fibular), determined by h-bonding, hydrophobicity, disulfide bridges, ionic bonds
initiation factors (IFs): help in assembly, released when mRNA joins the ribosomal Quaternary: combination of tertiary sub-units, examples: α + β subunits in hemoglobin
subunit
Once assembled, recruits the large ribosomal subunit: initiation aa-tRNA occupies the P- Proetein folding and degradation:
site (all other aa-tRNAs bind the A-site), the A-site is empty Folding:
 Overview
Elongation: aminoacyl-tRNA binds to A site (consumes 1 GTP), peptidyl-bond formation
o required for a protein to achieve a proper tertiary protein structure
(peptidyl transferase reaction catalyzed by ribosomal rRNA ("ribozyme") , transfers
o involves heat shock proteins (Hsp)
growing polypeptide to amino acid in the A site)
 essential for normal protein folding
Translocation: ribosome moves 3 nucleotides at a time in a 5' to 3' direction: moves  some function as chaperones and some function as
peptidyl RNA from A site to P site, moves empty tRNA from P site to E site for exit, A site chaperonins
now empty and ready to accept next aa-tRNA, catalyzed by elongation factor-2 (eEF-2) o the more mutated a protein, the more help it needs from chaperones
(inactivated via ADP-ribosylation by bacterial toxins (Pseudomonas and Diphtheria)), o if a protein is not folding properly, a chaperone may send it directly
consumes 1 GTP. Cycle is repeated for each amino acid addition to the polypeptide for degradation
Wobble phenomenon: The anticodon of certain tRNA molecules can bind to multiple o clinical relevance
codons. The first two nucleotides of the codon adequately specify which tRNA (and  cystic fibrosis
amino acid) binds the codon, thereby permitting multiple nucleotides in the third  pathogenesis
position to bind the same tRNA.  3 nucleotide deletion on chromosome 7
 ΔF508 mutation in chloride channel
Termination: stop codon reached in mRNA, release factor binds mRNA, peptide bond is (CFTR) ↓ stability of the protein and ↑
hydrolyzed and completed protein is released, ribosomal complex dissociates folding time

Overall energy requirement: 4 high energy bonds: ATP to AMP for tRNA charging (2)
  instead of insertion into the plasma  Defects in destruction of misfolded proteins
membrane the protein is degraded in the o inability to send degraded proteins to proteasome results in
Golgi apparatus accumulation in ER
 ↓ chloride conductance results in ↓ o examples
Na+ and Cl reabsorption in sweat glands  α1-antitrypsin (AAT) deficiency
 presentation  normally synthesized by hepatocytes and
 ↓ water content of mucus which results in exocytosed into circulation
a thick mucus that cannot be cleared  inhibit proteases
 respiratory infections  in AAT deficiency misfolded α1-antitrypsin
 nasal polyps accumulates in ER and damages hepatocytes
 malabsorption  PAS+ granules
 meconium ileus  many genetic variations
 biliary cirrhosis  MC are Z and S variants due to point
 Chaperones mutations
o types  co-dominant allelic expression
 Hsp70  presentation
 associates with directly with the ribosome  micronodular cirrhosis
 hides hydrophobic regions of protein to allow for  fibrosis
proper folding  test with PCR
 ATP hydrolysis required
 essential post-translational modification:
 Hsp90  Covalent alterations
 used for fewer proteins than Hsp70 o phosphorylation
 ATP hydrolysis required  kinase additions of phosphate groups
 essential o glycosylation
 role in folding mutant proteins in cancer  addition of oligosaccharide
 Chaperonins  signals for translocation into ER/Golgi
o group 1 o hydroxylation
 Hsp60  allows for ↑ in hydrogen bonding
 ring shaped  needed for collagen synthesis
 ATP hydrolysis required o γ-carboxylation
 called GroEL/GroES in prokaryotes  addition of carboxyl group
 peptide chain enters the cage and it is capped  allows for Ca2+ binding site
 once folded the cap is removed and the protein is  needed for several clotting factors
released o prenylation
o group 2  addition of farnesyl or geranylgeranyl lipid groups
 TRiC/CCT  signals for membrane insertion
 composed of 8 Hsp60s  Proteolysis
 similar function to GroEL/GroES o cleavage of N- or C- terminal propeptides
 required for folding of actin and tubulin  peptides beginning in "pro-"
o converts zymogens to mature enzymes
degradation:  examples
 Ubiquitination  enzymes ending in "-ogen"
o cell's mechanism to mark a protein for destruction
o mechanism types of DNA mutation:
 several copies of ubiquitin added to a misfolded/unneeded silence:
protein  Exchange of one base for another results in same amino acid
 polyubiquitinated protein enters the proteasome o often an alteration in 3rd position of codon
 protein hydrolyzed into peptide fragments  tRNA wobble
  No change in protein function
Missense: Exchange of one base for another results in changed amino acid:
transversion → exchanges a purine to a pyrimidine or a pyrimidine to a purine e.g., C → A
or G → T
transition → exchanges a purine for another purine or a pyrimidine to another
pyrimidine e.g., A → G or C → T
Variable change in protein function: if the new amino acid is similar to old (leu → ile) the
protein will most likely function the same, if the new amino acid is different (glu → val)
the protein folding/stability will likely be affected e.g., sickle-cell anemia (glu → val
mutation in β-globin gene)
Nonsense:
 Exchange of one base for another results in a stop codon
 Loss of function mutation as peptide is truncated
Frameshift:
• Deletion or addition of 1 or 2 bases resulting in misreading of all nucleotides
downstream
• Loss of function mutation as peptide is completely different
Large segment deletion: Unequal crossover at meiosis results in loss of large segment of
DNA
 Loss of function mutation
 e.g., α-thalassemia (deletion of α-globin gene)
change at splice site:
 Alteration in base sequence at mRNA splicing site results in altered splicing:
o can remove parts of exon
o can leave parts of intron
 Variable effect on protein function as number of spliced amino acids varies
 e.g., β-thalassemia
triple repeat expansion:
 Expansion of short nucleotide sequence results in longer polypeptide
o can be in coding or noncoding region
 Addition of amino acids affects protein structure/folding and affects function
 Disease display anticipation
o earlier disease onset in successive generations
 e.g., myotonic dystrophy, Huntington's disease, and Fragile X
In-frame mutations
Heme metabolism:
Heme synthesis: function: hemoglobin, cytochrome b4, P450
Location of synthesis: involves both the mitochondria and the cytosol, occurs in nearly
every cell, occurs in RBC progenitor cells (CANNOT occur in RBCs because they lack
mitochondria)
Pathway: ALA synthase is rate limiting enzyme (see above)
Deficiencies in heme synthesis: hemin (occurs when Fe3+ is incorporated instead of Fe2+),
porphyria (causes symptoms due toxic accumulation of pathway intermediates:
aminolevulinic acid (ALA) causes neurological symptoms, protoporphyrins cause
photosensitivity: conjugated structure what absorbs light energy and forms free
radicals), symptoms worsened by: sunlight, P450 inducing drugs (stimulate the heme
synthesis pathway to ↑ production, ex: barbiturates, alcohol). Treatment: limit exposure
to sun and P450 inducing substances, hemin (inhibits new heme production). Types:
porphyria cutanea tarda: deficiency in uroporphyrinogen decarboxylase; AD, late onset
(4th or 5th decade), symptoms often noticed with alcohol consumption. Presentation:
photosensitivity, hyperpigmentation (body's attempt to protect the skin), dark
red/brown colored urine
acute intermittent porphyria: deficiency in porphobilinogen deaminase; AD, late onset.
Presentation: NO photosensitivity, episodic psychological symptoms (paranoia, anxiety,
depression), vague abdominal pain (patients can present with a history of laparoscopies,
dark red/brown colored urine, ALA and porphobilinogen (PBG) present in urine during
symptoms)
poisoning:
lead: induced deficiency in ALA dehydratase and ferrochelatase, both enzymes are Zn2+
dependent metalloenzymes. Pb2+ replaces the Zn2+ at the active site. Presentation: ↓ in IQ
(microcytic anemia with coarse basophilic stippling), abdominal pain (↑ in ALA without ↑
in PBG, differentiates from porphyrias), lead lines in bone and teeth xrays,
nephrotoxicity (deposition in nuclei of proximal renal tubular cells)
hexachlorobenzene: induced deficiency in uroporphyrinogen decarboxylase;
presentation: hypertrichosis (↑ body hair coverage), found in (now banned in USA)
pesticides.
iron deficiency (iron incorporated in the final step), result is microcytic hypochromic
anemia
vitamin B6 deficiency (rate limiting enzyme (ALA synthase) requires), most commonly
due to isoniazid therapy
degradation of heme:
 Function
o rid body of hemoglobin removed from degraded RBCs
 Location of synthesis
o spleen
 site of RBC destruction
o liver
 site of bilirubin conjugation
o intestine
 conversion by normal gut flora
bilirubin:
 Properties
o insoluble
 must travel in blood bound to albumin
o conjugation
 direct (conjugated)
 glucuronate group added
  soluble Polygenic inheritance: multiple genes are responsible for inheritance of a disease e.g.)
 indirect (unconjugated) androgenic alopecia
 glucuronate group not yet added
 insoluble Heritability: can measure the relative effect of genetic vs. environmental factors on a
 Modified forms phenotype, calculated by phenotypic relationship between dizygotic (DZ) and
o urobilinogen monozygotic (MZ) twins, heritability = (CMZ-CDZ) / (1-CDZ), where C = concordance.
 gives urine yellow color Prevalence of disease in both twins entirely environmental disease should have CMZ =
o stercobilin CDZ, entirely genetic disease should have CMZ=1.0 and CDZ=0.5; siblings share 50% of their
 gives feces brown color genes.
 with a blocked bile duct no stercobilin in feces and
Mode of inheritance:
it is clay colored
AD: Genders affected: male and female at equal frequency; does not skip generations (if
genetics:
two parents without the AD disease have child with an AD disease): Possibility is
reduced penetrance (have mutant gene but phenotypically normal), de novo germline
Codominance: both allelic copies are expressed e.g.) blood groups (A, B, AB)
mutation
Variable expression: nature and degree of phenotype vary from 1 individual to another
An affected child must receive disease from an affected parent: a homozygote dominant
with the same mutation e.g.) 2 patients with neurofibromatosis may have varying
parent has a 100% of having an affected child, two heterotyzgote parents with the AD
disease severity
disease condition have a 75% chance of having a child with the disease phenotype.
Incomplete penetrance not all individuals with a mutant genotype have diseased
Pathology: defects in structural genes. Presentation timing: usually after puberty
phenotype, explanation for a dominant disease "skipping" a generation, penetrance can
Other notes: often pleiotropic (several organ systems affected by single genetic defect),
be calculated by ( # with symptoms) / (# with disease genotype), must be figured into
only one copy of the defective gene is required to express the disease phenotype.
recurrance calculations: if parents have a 50% chance of giving defective gene but the
Examples: von Willebrand disease (most common), Huntington's disease, osteogenesis
penetrance is 50%, 0.5 x 0.5 x100 = 25% of recurrence, observed in recessive and
imperfecta, achondroplasia, Marfan syndrome, neurofibromatosis type, acute
dominant diseases
intermittent porphyria
Pleiotropy: single mutation has diverse effects upon several organ systems e.g.) PKU
AR: genders effected: male and female; generations affected:1/4 of offspring affected
Anticipation: changes in disease presentation in succeeding generations, ↑ severity,
when both parents are carriers, usually 1 generation
earlier onset, caused by trinucleotide expansion, region of repeating triplets expands in
Pathology: defects in enzymes. Presentation timing:infancy to childhood
each generation e.g.) Huntington's disease, fragile X, myotonic dystrophy, Friedreich
Other notes: most often more severe than AD, must have 2 defective copies of the gene,
ataxia
chances greatly increased with consanguinity
Examples: cystic fibrosis - deficiency in the chloride channel CFTR, inborn errors of
Loss of heterozygosity: "two-hit model": individual inherits or develops a mutation in
metabolism (PKU, von Gierke's, Pompe's, glycogen storage diseases, sphingolipidoses
one copy of gene, disease occurs when the the complementary allele is lost e.g. tumor
(except Fabry's), and mucopolysaccharidoses (except Hunter's)), sickle cell anemia,
suppressor diseases (Li-Fraumeni, retinoblastoma)
thalassemias, albinism, ARPKD, hemochromatosis
Dominant negative mutation: mutant gene product antagonizes wild-type gene product,
X linked recessive: genders affected: males must receive defective gene from carrier
exerts a dominant effect e.g.) common in multimeric proteins where one mutant subunit
mother, carrier mother's sons have 50% of having disease, affected males give copy to all
can change function of entire enzyme
of their daughters
Generations affected: skips generations, male-to-male transmission not allowed, diseases
De novo mutation: genetic disease in an individual with no familial history, recurrence
passes through carrier daughters
risk for offspring of same parents is low
Pathology: defects in enzymatic genes, similar to AR diseases
Presentation timing: usually after puberty
Locus heterogeneity: different mutations can produce the same phenotype e.g.)
Other notes: only one defective copy necessary for disease in males because males are
marfanoid habitus caused by Marfan's syndrome, MEN 2B, homocystinuria
hemizygous for X chromosome, two defective copies necessary for disease in females
(can be affected with just one defective copy if normal X chromosome is inactivated to
Heteroplasmy: presence of both normal and mutated mitochondrial (mt)DNA in the
Barr body, called manifesting heterozygotes, phenotype usually milder than affected
same cell; results in variable expression in mitochondrial inherited disease
males)
Examples: hemophilia A and B, Menke's disease, Duchenne muscular dystrophy, Lesch-
Uniparental disomy: offspring receives both copies of a chromosome from 1 parent, no
Nyhan syndrome, Ornithine transcarbamoylase deficiency, red-green color blindness
copies from the other parent, causes disease if the chromosome is usually imprinted
X linked dominant: genders affected: male and female at equal frequency
  Generations affected o attachment of mitotic spindle fibers
o does not skip generations  allows chromosomes to be pulled to opposite poles during
 only possibility is reduced penetrance anaphase
o females of affected fathers are always affected o variations in position
 male-to-male transmission not seen  metacentric
o male or females of affected mothers can be affected  centromere in the middle
 Pathology  submetacentric
o defects in structural genes  centromere offset slightly towards one end
 Presentation timing  acrocentric
o usually after puberty  near complete displacement of centromere to one
 Examples end
o hypophosphatemic rickets  Nomenclature
o Fragile X syndrome o long = q
o Alport syndrome o short = p
Mitochondrial inheritance: genders affected: male and females at equal frequency  remember: p = petite
Generations affected: does not skip generations, only transmitted from affected female: o translocation = t
gives to all offspring, due to the fact that the sperm do not contribute mitochondria to the o deletion = del
zygote types of chromosomal alteration:
Pathology: defects in electron transport/oxidative phosphorylation process, presents as Nondisjunction: homologous chromatids do not separate properly during meiosis (stage
neuropathies/myopathies (neurons and muscle cells require high amounts of energy and of nondisjunction affects gamete production outcome, nondisjunction in meiosis I results
depend on mitochondria) in 2 gametes with x 2 and 2 gametes x 0, nondisjunction in meiosis II results in 2 normal
Presentation timing: usually after puberty gametes, 1 gamete x 2 and 1 gamete x 0; zygote receiving 3 copies = trisomy, zygote
Other notes: variable expression due to heteroplasmy, a small percentage of receiving 1 copy = monosomy), risk greatly ↑ with ↑ in maternal age, more common in
mitochondria within a cell are affected leading to variable severity oogenesis than spermatogenesis
Examples: myoclonic epilepsy with ragged red muscle fibers, Leber hereditary optic
neuropathy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like Translocation: exchange genetic info between nonhomologous chromosomes by
episodes) breakage and repair. Balanced: where exchanged fragment is still functional on another
chromosome, unbalanced: where exchanged fragment cannot function properly;
Inheritance algorithm: Does offspring with disease have a parent with disease? (Y/N) common in cancers. Types:
o if YES Robertsonian: balanced, always involve two acrocentric chromosomes (13, 14, 15, 21,
 dominant (does not skip generations) 22), results in loss of short arm and fusion of two long arms of different chromosomes,
 is there male-to-male transmission of disease? (Y/N) no clinical presentation because short arms of acrocentrics contain no vital info (is a
 if YES translocation carrier, problems with gametogenesis and therefore reproduction
 autosomal dominant (miscarriage, aneuploidy): depends on how chromosomes segregate during homologous
 if NO pair separation).
 do daughters of affected male have disease? (Y/N)
Reciprocal: exchange of DNA between two non-homologous chromosomes, as long as no
 if YES
DNA is lost the phenotype is normal for that generation is a translocation carrier
 X-linked dominant
 if NO Inversion: type of rearrangement where part of chromosome is inverted in orientation;
 mitochondrial types: pericentric (inverted chromosomal segment includes centromere, remember:
o if NO pericentric involves centromere), paracentric (inverted chromosomal segment does not
 recessive (can skip generations) include centromere)
 predominantly males with disease? (Y/N)
 if YES Ring chromosomes: causes: product of two breakage sites on the chromosome and the
 X-linked recessive segment lost circularizes; ends of chromosomes join circularizing entire chromosome
 if NO (usually lost during gametogenesis → monosomy)
 autosomal recessive
chromosomal structure: Centromere: Isochromosome: replication of one arm of a chromosome with loss of the other, p-q → p-
o holds sister chromatids together p'; lethal for autosomes, can be observed on sex chromosomes
  Repeat expansion
Deletions: loss of chromosome segment; types: terminal (end of chromosome), o CGG repeat on FMR1 gene
interstitial (within the chromosome)  Presentation
o mental retardation
Chromosomal disease: o autism
Down syndrome: most common chromosomal disorder, most common cause of o long face large jaw
congenital mental retardation; causes: o large ears
trisomy 21: nondisjunction (most common cause), occurs during anaphase of meiosis I, o large testicles (macroorchidism)
Robertsonian translocation, mosaicism (least common cause); presentation: appearance: friedrich’s ataxia:
short stature, hypotonia, unique facial structure (epicanthic folds, macroglossia, flat
 Inheritance pattern
profile, depressed nasal bridge), simian crease in palm, ↑ risk for congenital heart disease
o autosomal recessive
(combined ASD and VSD), AML (< 3 y/o), ALL (> 3 y/o) , Alzheimer's disease (by 5th
decade, due to amyloid precursor protein (APP) gene on 21), Hirschsprung's disease,  Repeat expansion
duodenal atresia, congenital heart anamolies (atrioventricular canal is most common,
o GAA repeat on chromosome 9
endocardial cushion defects also very common). Screening: + quad screen (↓ α-  results in
fetoprotein, ↓ estriol, ↑ inhibin A, ↑ β-hCG), remember: high (hCG, inhibin); deficit (estriol,  defect in frataxin (an iron binding protein) that
fetoprotein) + ultrasound shows high amount of fluid behind the neck (↑ nuchal leads to impaired mitochondrial function
translucency), can confirm diagnosis with amniocentesis or chorionic villus sampling  degeneration of various spinal cord tracts
 Presentation
Edwards' syndrome: most common trisomy resulting in live birth after Down syndrome, o neurological findings
cause: trisomy 18, nondisjunction. Presentation: mental retardation, unique appearance,  muscle weakness
rocker-bottom feet, micrognathia, low-set ears, clenched hands with overlapping fingers,  loss of deep tendon reflexes
prominent occiput, congenital heart disease (VSD), death < 1 y/o  loss of vibratory sensation and proprioception
 clumsy gait with falls, nystagmus
Patau's syndrome: cause: trisomy 13: nondisjunction. Presentation: mental retardation, o other findings
unique appearance: microphthalmia, microcephaly, cleft lip/palate, holoprosencephaly,  pes cavus
polydactyly, VSD, cystic kidneys, death < 1 y/o  diabetes mellitus
 hypertrophic cardiomyopathy
Cri-du-chat syndrome: cause: microdeletion of short arm of chromosome 5. huntington’s disease: inheritance pattern: autosomal dominant
Presentation: high-pitched crying/mewing (origin of name: French for cry-of-the-cat), Repeat expansion: CAG repeat on chromosome 4
microcephaly, moderate to severe mental retardation, epicanthal folds, VSD Presentation: neurologic findings:
• caudate atrophy with enlarged ventricles on head CT
Williams syndrome: cause: microdeletion of long arm of chromosome 7 (region lost • decreased GABA and Ach
includes elastin gene); presentation: distinctive "elfin" facies, mental retardation, • increased dopamine
hypercalcemia (↑ sensitivity to Vitamin D), unique behaviors, well-developed verbal • NMDA mediated excitotoxicity
skills, extreme friendliness with strangers, musical talent, supravalvular aortic stenosis • movement disorder
• aggression/pyschosis
22q11 microdeletions: cause: microdeletion at chromosome 22q11, abnormal • depression
embryological development of 3rd and 4th pharyngeal pouch. Variable pressenation: • dementia
CATCH-22 disease: cleft palate, abnormal facies, T-cell deficiency (due to thymic aplasia), occurs typically a young patient (age 20 - 50)
cardiac abnormalities, hypocalcemia (due to parathyroid aplasia, results in tetany). mnemonic: HUNT 4 an animal, put it in a CAGe

Specific presentation of 22q11 microdeletions: myotonic dystrophy type 1:

DiGeorge syndrome: defects in thymus, parathyroid, heart  Inheritance pattern
Velocardiofacial syndrome: defects in palate, face, heart; no abnormalities thymus, o autosomal dominant
parathyroid  Repeat expansion
Trinucleotide repeat expansion disease: o CTG repeat on DMPK gene
Fragile X syndrome:
 results in
 Inheritance pattern  abnormal expression of myotonin protein kinase
o X-linked dominant
 Presentation
 o myotonia gene). Presentation: severe cognitive disability, frequent seizures, ataxia, speech
o muscle wasting impairment, hyperactivity, inappropriate laughter ("happy puppet"); affects both male
o cataracts and females
o testicular atrophy Population genetics: Forces responsible for genetic variation:
o arrhythmia Mutation: de novo mutation rates constant among populations; intrinsic error rate in
o frontal balding DNA polymerase
o classically "can't release a doorknob/handshake" founder effect: if one member of a small community carries a triat, as the population
epigenetics: expands there will be a higher frequency of that trait in the new community than there is
 Changes in gene expression caused by mechanisms other than changes in actual in the general population. Ex.) Pennsylvania Amish and Ellis-van Creveld syndrome
DNA sequence
 Examples genetic drift: a dramatic change in allele frequency based on chance; small populations
o X-inactivation are more vulnerable to genetic drift
o imprinting
o histone modification natural selection: ↑ in allelic frequency that ↑ species fitness, ↓ in allelic frequency that ↓
x- inactivation: species fitness. some genes ↑ species fitness as heterozygote but ↓ species fitness as a
homozygote ex.) sickle cell trait lowers malarial infections, while sickle cell anemia is
 Overview
o normalizes the genetic amount of males and females (lyonization) detrimental
o inactivates # of X chromosomes - 1 in a Barr body
bottleneck: Even when fitness is equal for all phenotypes, a population bottleneck can
 triploid X will have 2 Barr bodies
result in disrupted allelic frequencies or loss of a genotype all together by chance
 Mechanism
o mediated by XIST gene gene flow: transfer of alleles from one population to another
o inactivation through methylation
o occurs at blastocyst stage in female embryos Hardy-Weinberg equilibrium: states that genotype and allele frequencies remain
o X copy chosen for inactivation is random constant through generations: disease prevalence equation: p2+ 2pq + q2 = 1, where p =
 after choosing every subsequent cell will have the same X frequency of allele A, where q = frequency of allele B, p2 = frequency of homozygous
copy inactivated individuals for allele A, q2 = frequency of homozygous individuals for allele B, 2pq =
 Clinical relevance frequency of heterozygotes
o mosaicism
 non-homogenous X inactivation requirements for validity: large population, random mating
 some cells express paternal X and some cells express
maternal X the genotypic frequencies of the population will remain stable from generation to
imprinting: describes differences in transcriptional activity based on whether the generation, assumptions: no mutation, no selection for any of the genotypes at the locus,
chromosome is of maternal or paternal origin, at a single locus: 1 allele is active, 1 allele no migration
is inactive: creates a hemizygous state
Mechanism: inactive ("imprinted") allele is methylated during gametogenesis, creates other notes: prevalence of an X-linked recessive disease in males = q; prevalence of an X-
transcriptional inactivity; is maternal/paternal specific (gene at one locus always linked recessive disease in females = q2, possible to assume in most cases that p = 1 as
methylated on a specific copy), all cells of an individual have same imprinting level. the wild-type allele is approximately 1
During gametogenesis of the individual the methylation state is erased, reset to be either
maternal or paternal depending on the sex gene mapping: linkage analysis:
Principles
Clinical importance:  linkage
Prader-Willi syndrome: cause: deletion of normally active paternal allele on 15q; o the rate of cosegregation of two syntenic alleles (alleles appearing on
remember: Prader = paternal deletion. Presentation: mental retardation, hyperphagia → the same chromosome) is inversely proportional to the distance
obesity, hypogonadism, neonatal hypotonia, behavior problems; affects both male and between the alleles
females  i.e. genes that are physically close on a chromosome will be
inherited together more often
Angelman's syndrome: cause: disruption of the maternally expressed and paternally  genes that are physically far apart will be inherited together
imprinted gene UBE3A, which encodes an E3 ubiquitin ligase, typically results from less often
deletion of the normally active maternal allele on chromosome 15q, same region of the
 linkage group
genome as Prader-Willi syndrome deletion but opposite chromosome (not the same
 o set of genes at different loci on the same chromosome that tend to act
as a single set of genes in meiosis rather than undergoing independent
assortment
 an exception is if crossing over occurs
 linkage analysis
o using a pedigree to determine the location of an allele based on the
rate of cosegregation with other alleles of known location (genetic
markers)
o can be used to track disease-causing mutation
 linkage analysis can localize a gene responsible for a trait
when the sequence is unknown
 centi-Morgan (cM):
o a unit used to measure genetic linkage
o a length of DNA over which the frequency of homologous
recombination is 1%
 1% chance that a marker on a chromosome will become
separated from a second marker on the same chromosome
due to crossing over
 two loci that are 1 centimorgan apart will experience
recombination between these two loci in 1% of meiosis.
 parental gamete
o the portion of chromosome looks the same as that of one of the
parents
o no recombination
 nonparental gamete
o homologous recombination has occurred in the region of interest
 linkage disequilibrium
o when alleles occur together more often than can be accounted for by
chance
 indication that 2 alleles are physically close together
Biochemical lab technique: PCR: polymerase chain reaction:
Function: can amplify a selected region of DNA
Process: solution prepared containing: DNA primers specific for selected DNA region,
DNA sample of interest, heat stable DNA polymerase, deoxyribonucleotides;
denaturation of dsDNA by heating, annealing of DNA primer specific for region of
interest and slowly cooling the solution, replication of DNA at the primer by heat stable
DNA polymerase, repetition of the process several times, gel electrophoresis used to
separate the various components of the solution
Clinical use: in all uses PCR functions to amplify the amount of DNA present in a sample;
high specificity bacterial/viral infection testing:
HIV: first step is ELISA (high sensitivity), PCR is used to determine viral load, test
examines the amount of viral DNA integrated into host cell DNA. Advantages over ELISA:
PCR becomes positive earlier in disease course (Positive ELISA result is dependent on
antibody formation), PCR does not require that the patient have a competent immune
system (ELISA requires the host to make antibodies), important specific cases when PCR
should always be used: (a newborn whose mother is HIV+ will have antibodies even if
not infected, so ELISA does not work) when earliest possible detection is required
Genetic identification: forensic/paternity testing; use of variable number tandem repeats
(VNTRs) or short tandem repeats (STRs): unique copies of non-coding regions of DNA
between individuals, since they exist on both chromosomes, individuals have two copies
at each locus, 1 paternal and 1 maternal; can only prove with certainty that the sample
DOES NOT belong to the test subject, cannot prove with 100% certainty that DNA
belongs to individual of interest because there is a small chance that someone shares the
same VNTR or STR
Direct mutation: if DNA region is known, PCR can amplify that region for sequencing
RT-PCR:
Function: used to measure the amount of RNA present in a sample
Process: reverse transcriptase is added to solution containing RNA, dNTPs, primers for
specific sequence of interest, and heat stable DNA polymerase: RNA is converted to DNA
and DNA sequence of interest is amplified, the amount of amplified PCR product after a
set number of PCR cycles is directly proportional to the concentration of RNA in the
original sample
Clinical use: HIV viral load: measures transcriptional activity of the virus by detecting the
amount of RNA present, gives a more detailed picture of the infection and treatment
results
Blotting:
Function: probe for specific substance in solution:
Southern blot - used to analyze DNA; normally used to examine the presence of a
particular DNA sequence, remember: Southern Dixieland
Northern blot - used to analyze RNA; normally used to examine gene expression
Western blot (immunoblot) - used to analyze protein
Process: run a gel electrophoresis to separate the components of the solution, bands are
transferred ("blotted") to a filter/membrane, radiolabeled or fluorescently labeled probe
is incubated with the membrane: Southern blot - 32P-DNA, Northern blot - 32P-DNA,
Western blot - enzyme-linked or 131I antibody. Specific probe binds with high specificity
to DNA/RNA segment or protein of interest, membrane is visualized under conditions to
illuminate the probe
Clinical use:
restriction length polymorphism (RFLP): can be used for genetic testing; based on the
principle that individuals have unique/heritable variations in RFLPs, loss or gain of a
restriction site changes the fragment lengths after digestion by an endonuclease,
variation provided in part by variable number tandem repeats (VNTRs). Southern blot
used to visualize results ex.) testing for sickle-cell anemia: wild type (wt) gene contains a
restriction site in the gene fragment, sickle cell (sc) gene lacks this restriction site, the
diagnostic results following a Southern blot: wt homozygote would have only medium
and small fragments, heterozygote would have large, medium and small fragments, sc
homozygote would have only large fragments
gene expression profiling: measure whether a particular region of DNA is being
expressed, Northern blot used to visualize results ex.) testing for fragile X syndrome: ↓
expression of FMR1 gene, trinucleotide repeat disorder
microarrays:  because mRNA has had the non-coding introns spliced out
Function: can probe for thousands of different mRNAs simultaneously it can express genes from the cloned host
Process: a chip has thousands of DNA sequences robotically attached on a grid, a solution  bacteria do not contain the enzymes to splice RNA
of degraded mRNA is added to the chip, thousands of probes are added, a computer  requires reverse transcriptase to convert mRNA into DNA
analyzes the degree of binding to various regions of the chip for the library
 similar mechanism of accessing the library as the genomic
Clinical use: analyze cancer cell gene expression; can determine probability of becoming library; however the probe is an 125I-antibody against the
malignant based on population comparison studies, can be used to guide therapy protein of interest
o gene therapy
Cloning:  allows introduction of a normal copy of a gene into a cell that
 Function was defective in that gene
o transfer production of a protein from one organism to another  uses a delivery vector to introduce the gene
o synthesize large quantities of a plasmid or its product  usually modified viruses
 Process  viral genome is replaced with plasmid
o insertion of a DNA segment of interest into a vector  virus inserts the gene into the host as part
 vector is usually a plasmid of its normal life cycle
 first: digestion of vector with restriction  problems
endonuclease  viruses can indiscriminately
 second: sealing of the segment into the vector insert copies of gene into
with DNA ligase undesirable regions
o insertion of the vector into a bacteria via transformation  oncogenesis
o selection of the bacteria via a mechanism to distinguish which cells  cells must be dividing for viral
received the vector and those that did not integration
 usually via an antibiotic resistance gene  adenovirus vectors do
 Applications not require active
o recombinant proteins division, but do not
 produced by introduction of a plasmid containing the actually insert into the
protein product of interest into bacteria genome
 bacterial colony grown  can be performed
 some plasmids contain regulatory sequences which  ex vivo
can turn on/off expression of the plasmid  cells removed from the body, modified,
 bacteria lysed and produced protein is purified and transplanted back into the body
 ex.) recombinant insulin, factor VIII, bacterial factors for  in vivo
vaccination  cells modified in the body without
o genome sequencing/libraries removal
 contains the entire genome of an organism spread between  has been used to treat SCID caused by deficiency in the IL-
bacterial colonies receptor γ chain
 the genome of an organism is degraded with a restriction biochemical model systems:
endonuclease at specific palindromic sites  Transgenic mice
 individual pieces are inserted into a bacterial colony and o construction
cloned individually  germline modification
 to access the library for amplification of a particular segment  cloned gene (transgene) introduced into a fertilized
a blot is taken of the library and radiolabeled segment of mouse ovum
interest is hybridized  constitutive
 the labeled colony (in which hybridization took place) can be  non-specific insertion into genome
harvested and grown  conditional
o cDNA libraries  specific (targeted) insertion into genome
 "expression libraries"  the altered phenotype with the added gene is observed
 contain all the expressed genes of an organism in a similar through the entire lifespan
fashion as a genomic library
  including embryonic development karyotyping:
 number of gene copies is variable Function: examine chromosomal structure
 altered phenotype passed to offspring as it is a germline Process: mitotic cells in metaphase selected from actively dividing cell population (white
mutation cells, bone marrow, placenta, amnion; when chromosomes are most condensed),
o function chromosomes are stained with or without G-banding (G-banding involves partial
 studying dominant disorders digestion with trypsin, leaves alternating dark and white bands), staining with Giemsa or
 due to the fact that the transgene will coexist with other stains which have affinity for DNA
normal gene product
 Knockout mice spectral karyotyping: 5 different fluorescent probes are added, special digital image
o construction processing colors each chromosomes differently; chromosomes are ordered, numbered
 similar to transgenic mice except instead of adding a foreign based on unique properties of all 23 chromosomes
gene, a normal gene is removed
Clinical use: can analyze whether a gross chromosomal abnormality exists ex.) trisomy,
 also a germline modification
monosomy; used commonly for prenatal diagnosis via chorionic or amniotic sampling.
o function
spectral karyotyping can also diagnose translocations, inversions, or deletions of specific
 useful for studying loss-of-function mutations in human
chromosomal segments if the change is large enough
diseases
 Chimeric mice FISH: fluorescence in situ hybridization:
o construction
 Function
 a genetic mix between two distinct cell lines
o detect the presence/location of a specific region of DNA in the genome
 fusion of two early embryogenic lines in embryogenesis
 two lines recognized as self due to absence of immune  Process
system
o a probe for a desired DNA sequence is created with a fluorescent tag
o function o probe is added to DNA sample and color is detected
 can reproduce to create offspring of various types in  Clinical use
subsequent generations o detect microdeletions, translocations, and aneuploidies
 non-transgene homozygous  similar role to karyotyping however the DNA does not have
 transgene homozygous to be condensed during mitosis to be visualized
 transgene heterozygous  ex.) a probe for chromosome 7 would only have one signal if
the patient had a monosomy as opposed to 2 in a normal
 Cre-lox system
sample
o construction
 can also be used to diagnose Prader-Willi syndrome (15q)
 a system that allows turning on/off of gene of interest with deletion
an antibiotic-controlled promoter
DNA sequencing:
o function
 to study a gene whose deletion is lethal to embryos  Function
o determining sequence for a desired DNA sample
 allows the embryo to develop to a certain stage and
can then turn off a certain gene  Process (Sanger/direct method)
 can also study the timing of when a gene is important in o DNA sample put in 4 different reaction mixtures
development  each contains all 4 deoxynucleotides, 1 different
dideoxyneucleotide (ddNTP), and a DNA polymerase
 RNAi
o dideoxynucleotides stop DNA polymerization at each base
o construction
 generates DNA chains of various lengths that encompass the
 dsRNA is synthesized complementary to the mRNA sequence
entire original sequence
of interest
o solutions are separated by gel electrophoresis
 transfected into cells
o sequence can be deduced by looking at each electrophoresis row and
 dsRNA separates and binds the complementary mRNA reading which ddNTP was added to that tube column
 dsRNA (where one strand is the host mRNA) is seen as a
viral product and degraded
 Clinical use
o function o most commonly used when no specific set of mutations is a common
cause of disease
 ↓ expression of a particular gene