Hydroxyurea
Disrupts purine and pyrimidine synthesis - inhibits ribonucleotide reductase
Adenosine deaminase deficiency
ADA is required for degradation of adenosine and desoxyadenosineConverts adenosine to inosine (which can be turned into hypoxanthine and used in the purine salvage pathway via HGPRT) - In ADA deficiency, ↑ dATP → lymphotoxicity - One of the major causes of autosomal recessive SCID
Lesch-Nyhan syndrome
Defective purine salvage due to absent HGPRT, which converts hypoxanthine to IMP and guanine to GMP. (Guanine, Hypoxanthine → Xanthine → Uric acid → Urine) - Results in excess uric acid production and de novo purine synthesis - X-linked recessive Findings: intellectual disability, self-mutilation, aggression, hyperuricemia (orange "sand" in diaper), gout, dystonia) Treatment: allopurinol or febuxostat
Probenecid
Increasis uric acid secretion in the urine (Low-dose aspirin has the opposite effect --> decreases uric acid secretion --> promotes hyperuricemia)
DNA polymerases
DNA polymerase III: - Prokaryotes only- Elongates leading strand by adding deoxynucleotides to the 3' end (5' → 3' synthesis).- Elongates lagging strand until it reaches primer of preceding fragment.- 3' → 5' exonuclease activity "proofreads" each added nucleotide.- Drugs blocking DNA replication often have modified 3' OH, thereby preventing addition of the next nucleotide. DNA polymerase I:- Prokaryotic only- Degrades RNA primer; replaces it with DNA.- Same functions as DNA polymerase III, also excises RNA primer with 5' → 3' exonuclease.
DNA topoisomerases
Create a single- or double-stranded break in the helix to add or remove supercoils. In eukaryotes: irinotecan/topotecan inhibit topoisomerase (TOP) I, etoposide/teniposide inhibit TOP II. In prokaryotes: fluoroquinolones inhibit TOP II (DNA gyrase) and TOP IV.
Mutations in DNA
Severity of damage: silent << missense < nonsense < frameshift. Transition = Purine to purine (eg, A to G) or pyrimidine to pyrimidine (eg, C to T).Transversion = Purine to pyrimidine (eg, A to T) or pyrimidine to purine (eg, C to G). Silent: Nucleotide substitution but codes for same (synonymous) amino acid; often base change in 3rd position of codon (tRNA wobble). Missense: Nucleotide substitution resulting in changed amino acid (called conservative if new amino acid is similar to chemical structure.- Sickle cell disease (substitution of glutamic acid with valine). Nonsense: Nucleotide substitution resulting in early stop codon (UAA, UAG, UGA). Frameshift: Deletion of insertion of a number of nucleotides not divisible by 3, resulting in misreading of all nucleotides downstream. Protein may be shorter or longer.- Duchenne muscular dystrophy, Tay-Sachs disease Splice site: Mutation at a splice site → retained intron in the mRNA → protein with impaired or altered function.- Rare cause of cancers, dementia, epilepsy, some types of β-thalassemia.
Nucleotide excision repair
Single strand DNA repair - Specific endonucleases release the oligonucleotides containing damages bases.- DNA polymerase and ligase fill and reseal the gap.- Repairs bulky helix-distorting lesions, thymidine dimers.- Occurs in G1 phase of cell cycle. Defective in xeroderma pigmentosum (inability to repair DNA pyrimidine dimers caused by UV exposure.- Findings: dry skin, extreme light sensitivity, skin cancer.
RNA polymerases
Eukaryotes:RNA polymerase I makes rRNA (5.8S, 18S, 28S); present only in the nucleolus.RNA polymerase II makes mRNA, snRNA, hnRNA, miRNA.RNA polymerase III makes 5S rRNA, tRNA.- No proofreading function but can initiate chains.- α-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II. Causes severe hepatotoxicity if ingested.- Actinomycin D inhibits RNA polymerase in both prokaryotes and eukaryotes Prokaryotes: 1 RNA polymerase (α2ββ'σ) makes all 3 kinds of RNA.- σ is required for the initiation of transcription at a promotor- Rifampin inhibits RNA polymerase in prokaryotes.
RNA processing (eukaryotes)
Initial transcript is called heterogenous nuclear RNA (hnRNA). hnRNA is then modified and becomes mRNA. The following processes occur in the nucleus:- Capping of the 5' end (addition of the 7-methylguanosine cap)- Polyadenylation of 3' end (±200 A's)- Splicing out of introns→ Capped, tailed, and spliced transcript is called mRNA. - mRNA is transported out of the nucleus into the cytosol, where it is translated.- mRNA quality control occurs at cytoplasmic processing bodies (P-bodies), which contain exonucleases, decapping enzymes, and microRNAs.- mRNAs may be degraded or stored in P-bodies for future translation- Poly-A polymerase does not require a template- AAUAA = polyadenylation signal
Splicing of pre-mRNA
1. Primary transcript combines with small nuclear ribonucleoproteins (snRNPs) and other proteins to form spliceosome.2. Cleavage at 5' splice site; lariat-shaped (loop) intermediate is generated.3. Cleavage at 3' splice site; lariat is released to precisely remove intron and join 2 exons. Antibodies to spliceosomal snRNPs (anti-Smith antibodies) are highly specific for SLE.
Protein synthesis
Initiation:Eukaryotic initiation factors (eIFs) identify either the 5' cap or an internal ribosome entry site (IRES). IRES can be located at many places in an mRNA (most often 5' UTR). The eIFs then help assemble the 40S ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal 60S subunit assemble with the complex. Requires GTP. Elongation:1. Aminoacyl-tRNA binds to A site (except for initiator methionine), requires an elongation factor and GTP.2. rRNA ("ribozyme") catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site.3. Ribosome advances 3 nucleotides toward 3' end of mRNA, moving peptidyl tRNA to P site (translocation). Termination:Release factor recognizes stop codon and halts translation → completed polypeptide is released from ribosome. Requires GTP.
Tumor suppressors
p53 induces p21, which inhibits CDKs --> hypophosphorylation (activation) of Rb Hypophosphorylated Rb binds to and inactivates transcription factor E2F --> inhibition of G1-S progression. Mutations in these genes result in unrestrained cell division (eg, Li-Fraumeni syndrome)
I-cell disease
Inclusion cell disease/mucolipidosis type II Inherited lysosomal storage disorder; defect in N-acetylglucosaminyl-1-phosphotransferase → failure of the Golgi to phosphorylate mannose residues (ie, ↓ mannose-6-phosphate) on glycoproteins → proteins are secreted extracellularly rather than delivered to lysosomes. Presentation: - Coarse facial features- Gingival hyperplasia- Clouded corneas- Restricted joint movement, kyphoscoliosis- Claw hand deformities- High levels of lysosomal enzymes Often fatal in childhood.
Peroxisome
Membrane-enclosed organelle involved in:- β-oxidation of very-long-chain fatty acids (VLCFA)- α-oxidation (strictly peroxisomal process)- Catabolism of branched-chain fatty acids, amino acids, and ethanol- Synthesis of cholesterol, bile acids, plasmalogens (important membrane phospholipid, especially in white matter of brain) Peroxismal disorders commonly lead to neurologic disease due to deficits in synthesis plasmalogens, important phospholipids in myelin. Peroxismal diseases include Zellweger syndrome, Refsum disease, and adrenoleukodystrophy.
Drugs that act on microtubules
- Mebendazole (antihelminthic) - Griseofulvin (antifungal) - Colchicine (antigout) - Vincristine/Vinblastine (anticancer) - Paclitaxel (anticancer)
Kartagener syndrome (1° ciliary dyskinesia)
Immotile cilia due to a dynein arm defect. - Autosomal recessive - Results in ↓ male and female fertility due to immotile sperm and dysfunctional fallopian tube cilia. - ↑ risk of ectopic pregnancy - Can cause bronchiectasis, recurrent sinusitis, cronic ear infections, conductive hearing loss, and situs inversus (eg, dextrocardia on CXR).
Sodium-potassium pump
Na+-K+ ATPase is located in the plasma membrane with ATP site on cytosolic side. For each ATP consumed, 3Na+ go out of the cell (pump phosphorylated) and 2K+ come into the cell (pump dephosphorylated).Plasma membrane is an asymmetric lipid bilayer containing cholesterol, phospholipids, sphingolipids, glycolipids, and proteins. Ouabain (a cardiac glycoside) inhibits by binding to K+ site. Cardiac glycosides (digoxin and digitoxin) directly inhibit the Na+-K+ ATPase, which leads to indirect inhibition of Na+/Ca2+ exchange → ↑ [Ca2+]i → ↑ cardiac contractility.
Collagen
Most abundant protein in the human body.Extensively modified by posttranslational modification.Organizes and strengthens extracellular matrix. Type I: Most common (90%) – bone (made by osteoblasts), skin, tendon, ligaments, dentin, fascia, cornea, late wound repair & scar tissue.- ↓ production in osteogenesis imperfecta type I Type II: Cartilage (including hyaline), vitreous body, nucleus pulposus. Type III: Reticulin – skin, blood vessels, uterus, fetal tissue, granulation tissue.- Deficient in the vascular type of Ehlers-Danlos syndrome Type IV: Basement membrane, basal lamina, lens.- Defective in Alport syndrome, targeted by autoantibodies in Goodpasture syndrome
Collagen synthesis and structure
1. Synthesis – translation of collagen α chains (preprocollagen) – usually Gly-X-Y (X and Y are proline or lysine). Glycine content best reflects collagen synthesis. 2. Hydroxylation – hydroxylation of specific proline and lysine residues. Requires vitamin C.- Deficiency → scurvy 3. Glycosylation – glycosylation of pro-α-chain hydroxylysine residues and formation of procollagen via hydrogen and disulfide bonds (triple helix of 3 collagen α chains).- Problems forming triple helix → osteogenesis imperfecta. 4. Exocytosis – exocytosis of procollagen into extracellular space. 5. Proteolytic processing – cleavage of disulfide-rich terminal regions of procollagen → insoluble tropocollagen. - Problems with cleavage → Ehlers-Danlos syndrome. 6. Cross-linking – reinforcement of many staggered tropocollagen molecules by covalent lysine-hydroxylysiene cross-linkage (by copper-containing lysyl oxidase) to make collagen fibrils.- Problems with cross-linking → Ehlers-Danlos syndrome, Menkes disease.
Osteogenesis imperfecta
Genetic bone disorder (brittle bone disease) caused by a variety of gene defects (most commonly COL1A1 and COL1A2). - Most common form is autosomal dominant with ↓ production of otherwise normal type I collagen. Manifestations include:- Multiple fractures with minimal trauma; may occur during the birth process- Blue sclerae due to the translucent connective tissue over choroidal veins- Hearing loss (abnormal ossicles)- Tooth abnormalities, including opalescent teeth that wear easily due to lack of dentin (dentinogenesis imperfecta) Treat with biphosphonates to ↓ fracture risk.
Ehlers-Danlos syndrome
Faulty collagen synthesis causing hyperextensible skin, tendency to bleed (easy bruising), and hypermobile joints.- Multiple types.- Inheritance and severity vary.- Can be autosomal dominant or recessive. - May be associated with joint dislocation, berry and aortic aneurysms, organ rupture. - Hypermobility type (joint instability): most common type.- Classical type (joint and skin symptoms): caused by a mutation in type V collagen (eg, COL5A1, COL5A2).- Vascular type (fragile tissues including vessels, muscles, and organs that are prone to rupture): deficient type III procollagen.
Menkes disease
X-linked recessive connective tissue disease caused by impaired copper absorption and transport due to defective Menkes protein (ATP7A).- Leads to ↓ activity of lysyl oxidase (copper is a necessary cofactor) → defective collagen (cross-linking). - Brittle, "kinky" hair, growth retardation, hypotonia, tortuosity of major arteries.- Low serum ceruloplasmin, decreased serum copper levels.
Elastin
Stretchy protein within skin, lungs, large arteries, elastic ligaments, vocal cords, ligamenta flava (connect vertebrae → relaxed and stretched conformations).- Rich in nonhydroxylated proline, glycine, and lysine residues (vs hydroxylated residues of collagen).- Tropoelastin with fibrillin scaffolding.- Cross-linking takes place extracellularly and gives elastin its elastic properties. - Broken down by elastase, which is normally inhibited by α1-antitrypsin.- α1-Antitrypsin deficiency results in unopposed elastase activity, which can cause emphysema. - Changes with aging: ↓ dermal collagen and elastin, ↓ synthesis of collagin fibrils; crosslinking remains normal.
Blotting procedures
Southern blot: DNA is electrophoresed Northern blot: RNA is electrophoresed Western blot: protein is electrophoresed- eg, confirmatory test for HIV after positive ELISA Southwestern blot: identifies DNA-binding proteins (eg, transcription factors) using labeled oligonucleotide probes
Zellweger syndrome
Disorder of peroxisome biogenesis due to mutated PEX genes. - Autosomal recessive - Hypotonia- Seizures- Hepatomegaly- Craniofacial abnormalities (eg, widened sutures, large anterior fontanelle)- Early death
Refsum disease
Peroxisome disorder. Disorder of α-oxidation → phytanic acid not metabolized to pristanic acid. - Autosomal recessive - Scaly skin- Ataxia- Cataracts/night blindness- Shortening of 4th toe- Epiphyseal dysplasia Treatment: Diet, plasmapheresis
Adrenoleukodystrophy
Peroxisome disorder. Disorder of β-oxidation → VLCFA buildup in adrenal glands, white matter of brain, testes. - X-linked recessive - Progressive disease that can lead to adrenal gland crisis, coma, and death.
Cytoskeletal elements
A network of protein fibers within the cytoplasm that supports cell structure, cell and organelle movement, and cell division. Microfilaments: Muscle contraction, cytokinesisEg, actin, microvilli Intermediate filaments: Maintain cell structureEg, vimentin, desmin, cytokeratin, lamins, glial fibrillary acidic protein (GFAP), neurofilaments Microtubules: Movement, cell divisionEg, cilia, flagella, mitotic spindle, axonal trafficking, centrioles
Telomerase
Eukaryotes only. A reverse transcriptase (RNA-dependent DNA polymerase) that adds DNA (TTAGGG) to 3' ends of chromosomes to avoid loss of genetic material with every duplication. Often dysregulated in cancer cells, allowing unlimited replication.
Nucleolus
- Transcription of rRNA - Maturation and assembly of ribosomal subunits
Chromatin structure
DNA exists in the condensed, chromatin form to fit into the nucleus.DNA loops twice around a histone octamer to form a nucleosome. A nucleosome consists of 2x (H2A, H2B, H3, H4) H1 binds to the nucleosome and to "linker DNA," thereby stabilizing the chromatin fiber. Phosphate groups give DNA a Θ charge. Lysine and arginine give histones a ⊕ charge. In mitosis, DNA condenses to form chromosomes. DNA and histone synthesis occurs during the S phase. Mitochondria have their own DNA, which is circular and does not utilize histones.
Heterochromatin
Condensed, appears darker on EM. Transcriptionally inactive, sterically inaccessible. ↑ methylation, ↓ acetylation. Barr bodies (inactive X chromosomes) may be visible on the periphery of nucleus.
Euchromatin
Less condensed, appears lighter on EM. Transcriptionally active, sterically inaccessible.
DNA methylation
Changes the expression of a DNA segment without changing the sequence. Involved with genetic imprinting, X-chromosome inactivation, repression of transposable elements, aging, and carcinogenesis. DNA is methylated in imprinting.Methylation within gene promotor (CpG islands) typically represses gene transcription.
Histone methylation/acetylation
Histone methylation: Usually causes reversible transcriptional suppression, but can also cause activation depending on the location of methyl groups. Histone acetylation: Relaxes DNA coiling, allowing for transcription. Methylation makes DNA mute.Acetylation makes DNA active.
Nucleotides
Nucleoside = base + (deoxy)riboseNucleotide = base + (deoxy)ribose + phosphate; linked by 3'-5' phosphodiester bond. 5' end of incoming nucleotide bears the triphosphate (energy source for the bond).Triphosphate bond is target of 3' hydroxyl attack. Purines: A, GPyrimidines: C, T, U Deamination of cytosine forms uracil.Methylation of uracil makes thymine.Deamination of 5-methylcytosine forms thymine. Deamination of adenine forms hypoxanthine.Deamination of guanine forms xanthine. Amino acids necessary for purine synthesis: glycine, aspartate, glutamine.Pyrimidine out of carbamoyl phosphate, aspartate.
Origin of replication
Particular consensus sequence of base pairs in genome where DNA replications begins. - Single in prokaryotes- Multiple in eukaryotes AT-rich sequences (such as TATA box regions) are found in promotors and origins of replication.
Primase
Makes an RNA primer on which DNA polymerase III can initiate replication.
DNA ligase
Catalyzes the formation of a phosphodiester bond within a strand of double-stranded DNA. Joins Okazaki fragments.
Base excision repair
Single strand DNA repair - Important repair of spontaneous/toxic deamination. - Base-specific glycosylase removes altered base and creates AP (apurinic/apyrimidinic) site.- One or more nucleotides are removed by AP-endonuclease, which cleaves the 5' end. - Lyase cleaves the 3' end. - DNA polymerase-β fills the gap and DNA ligase seals it.- Occurs throughout cell cycle
Mismatch repair
Single strand DNA repair - Fixes DNA copy errors - Newly synthesized strand is recognized, mismatched nucleotides are removed, and the gap is filled and resealed.- Occurs predominantly in S phase of cell cycle. Defective in Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC).
