Module 11 Flashcards
What is Cancer?
- ‘Cancer derives from the Greek for cram, karcinos
- Hippocrates coined the word ‘karcinos’ after observing that distended veins radiating from a breast tumor resembled the legs of a crab
- Abnormal mass of tissue resulting from excessive cell division
- May be benign or malignant
Tumor/Neoplasia
Benign vs Malignant
Benign: cells remain clustered together in a single mass
Malignant = Cancer
- Invade surrounding tissue
- Spread via bloodstream or lymphatics = metastasis
Hallmarks of Cancer
- Growth signal autonomy
- Evasion of growth inhibitory signals
- Avoiding immune destruction
- Unlimited replicative potential
- Tumor-promoting inflammation
- Invasion and metastasis
- Angiogenesis
- Genome instability and mutation
- Evasion of cell death
- Reprogramming energy metabolism
- Growth signal autonomy
Cancer cells can be distinguished from normal cells in cell culture conditions
- Normally, cells grow as a single layer, or monolayer, in a Petri dish due to a property called contact inhibition; contact with neighboring cells inhibits growth.
Cancer cells can be distinguished from normal cells in cell culture conditions (2)
- Transformed cells (cells that have become cancer cells) acquire the following phenotypes:
• they fail to exhibit contact inhibition and instead grow as piles of cells or “foci” against a monolayer of normal cells
• they can grow in conditions of low serum
• they adopt a round morphology rather than a fl at and extended one
• they are able to grow without attaching to a substrate (e.g. the surface of a Petri dish), exhibiting “anchorage independence.
Three distinct phenotypes of cancer cells
- Immortality – indefinite proliferative lifespan
- Transformation – loss of response to normal regulators of cell growth
- Metastasis – ability to break off from a tumor and invade tissues in another location in the body
Normal to Cancer Cell
- Change in phenotype
- Genetic – change in DNA sequence
- Many, but not all, cancer causing agents damage DNA
- Epigenetic – heritable change in gene expression
HOW PROTO-ONCOGENES BECOME ONCOGENES
• Point mutations/deletions in coding sequence —> structural and functional changes
• Point mutations and deletions in regulatory sequences —> over-expression
• Chromosomal translocations —> fusion proteins with novel functions
• Insertional mutagenesis caused by viral integration —> aberrant expression
Gene amplification—>increase in gene dose and protein production
Examples of Inherited Predisposition to Cancer
- RB - Retinoblastoma
- Ip53 - Li-Fraumeni syndrome (various tumors)
- p16/INK4A - Melanoma
- APC - Familial adenomatous polyposis/colon cancer
- NF1, NF2 - Neurofibromatosis 1 and 2
- BRCA1, BRCA2 - Breast and ovarian tumors
Examples of Inherited Predisposition to Cancer 2
- MEN1, RET - Multiple endocrine neoplasia 1 and 2
- MSH2, MLH1, MSH6 - Hereditary nonpolyposis colon cancer
- PTCH - Nevoid basal cell carcinoma syndrome
- PTEN - Cowden syndrome (epithelial cancers)
- LKB1 - Peutz-Jegher syndrome (epithelal cancers)
- VHL - Renal cell carcinomas
Development of cancer occurs in stages
Normal -> Hyperplasia -> Mild dysplasia -> Carcinoma in situ (severe dysplasia) -> Cancer (invasive)
G1 checkpoint signals
- Is the cell big enough?
- Is the DNA damaged?
- Is the environment favorable?
- Is the cell ready to divide again?
G2 checkpoint signals
- DNA replicated once and only once?
- Is the DNA damaged?
- Cell size and nutritional state?
- Is the cell ready to enter mitosis?
M phase checkpoint signals
- Chromosomes attached to opposite poles?
* Is the cell ready to exit mitosis?
Cell Cycle Regulation
• Cyclins – family of proteins whose concentration oscillates during the course of the cell cycle
• Cyclin-Dependent Kinases
- family of enzymes that control progression of the cell cycle
- Exerts its influence by phosphorylation
• Complex = Maturation Promoting Factor (MPF)
Cell Cycle Activation
- Response to mitogen
- Early-response genes
- Delayed-response genes
- Transcription within minutes
- Peaks at 30 min. then drops
- Encode transcription factors (c-Fos and c-Jun)
- c-Fos and c-Jun -> transcription of delayed-response genes
Early-Response Genes
- Stimulated by products of early-response genes
- Include transcription factors such as E2F, cyclins D and E, CDKs
Delayed-Response Genes
E2F Target Genes
- c-Myc, c-Fos
- Thymidine kinase, thymidine synthetase
- Dihydrofolate reductase
- DNA polymerase
- Cyclin E and A
- Dependent on activation of E2F transcription factors
- Allows passage from G1 -> S phase
Restriction Point
- mRNA for replication proteins
- synthesis of replication proteins
- replication of DNA
S Phase
Key points: Cancer
• Cyclin A: required for DNA synthesis • CDK1: required for entry into mitosis • Cyclin B - synthesized during the S phase - Transported from cytoplasm into nucleus just before nuclear membrane breaks down
- Distorts active binding site
- Inserts into ATP-binding site
Cyclin Kinase Inhibitors
Cdk Inhibitory Proteins
• p53
- Arrests cell cycle in response to DNA damage
- Stimulates production of p21 and p27
• p21 and p27
- Bind to Cdk-cyclin complexes, inhibiting them
- Greek word meaning “falling off”
- Programmed cell death
- The number of cells in multicellular organisms is tightly regulated
- Billions of cells die in the bone marrow and intestines every hour
Apoptosis
Necrosis vs Apoptosis
Necrosis: Acute injury; Swell and burst; Inflammatory response
Apoptosis:
- Cell shrinks and condenses
- Cytoskeleton collapses, nuclear envelope disassembles
- DNA fragmentation
- Cell surface altered for rapid phagocytosis
- Loss of cell membrane phospholipid morphology
- Condensation of chromatin
- Reduction in nuclear size
- Internucleosomal DNA cleavage (DNA ladder)
- Shrinkage of the cell
- Membrane blebbing
- Breakdown into apoptotic bodies
Apoptotic Cell Morphology
- Family of proteases responsible for apoptosis
- Cysteine in active site and cleave proteins at aspartic acid
- Synthesized as procaspases
- Once activated cleave different proteins
- Inactive Dnase - > activated -> cut up DNA
- Nuclear lamin -> breaks down nuclear lamina
Caspases
Caspase Cascade
- Initiator caspases – Caspase 8, 9, 10, 12
* Effector caspases – 3, 6, 7
Diverse group of signals induce apoptosis
- UV or gamma irradiation
- Chemotherapeutic drugs
- Growth factor withdrawal
- Cytokines TNF-α and TGF-β
2 Pathways (Cancer)
- Death Receptor and Mitochondrial
- Both converge -> caspase 3 activation
- Then branch into many pathways leading to cell death
- Trigger apoptosis by binding to receptors located on cell surface
(Death receptors) - Receptors: Extracellular cysteine-rich domain; Intracellular death domain (DD)
- Once bound to receptors -> oligomerization of DD’s -> recruit adaptor proteins
Nerve Growth Factor/Tumor Necrosis Factor
-External signals initiating apoptosis include tumor necrosis factor-α (TNF-α) and Fas ligand
- They are transmembrane proteins, some of which interact with adapter proteins (such as FADD [Fas-Associated protein with Death
Domain]).
- It digests important structural proteins such as lamin (this is associated with nuclear condensation), various cytoskeletal proteins, and
enzymes involved in DNA repair, causing cell death.
Extrinsic Pathway (Death Receptor Pathway)
- Extracellular cues and internal insults -> DNA damage
- Diverse pathways converge on mitochondria
*Activation of a pro-apoptotic member of Bcl-2 family - can be initiated by exposure
to reactive oxygen species, DNA damage and other stimuli. - This results in pores forming in the outer mitochondrial membrane, through which cytochrome c escapes into the
cytoplasm.
Intrinsic Pathway (Mitochondrial Pathway)
- Group of intracellular proteins
- Group I – anti-apoptotic (includes Bcl-2)
- Groups II and III – pro-apoptotic
- Bax, Bad, Bak, Bim and Bid
- Stimulate release of cytochrome c from mitochondria
Bcl-2 Family
- Associates with Apaf-1 (apoptotic protease activating factor 1) (activator protein)
- Binding triggers apoptosis
Cytochrome C
- Discovered as proteins induced by viruses to prevent cell death before viruses can replicate their DNA
- 2 mechanisms
- Bind to procaspases -> inhibit activation
- Bind to caspases -> inhibit activity
- In mitochondrial pathway, protein is released to block IAPs
IAP (Inhibitors of Apoptosis) family
- Molecules or molecular fragments that contain one or more unpaired electrons in its outer orbit and has an independent existence
- is designated by a superscript dot (R’).
- Associated with many conditions from cancer, inflammatory conditions, atherosclerosis, and aging
FREE RADICALS
- Are mostly products of oxygen metabolism
- Includes superoxide (O2 -), hydroxyl radicals (OH), and peroxy radicals (ROO)
- Are collectively referred to as reactive oxygen species (ROS)
FREE RADICALS
__ is less reactive than superoxide, which in turn is less reactive than hydroxyl radicals (OH)
Hydrogen peroxide
HYDROGEN PEROXIDE IS NOT A FREE RADICAL
- All reactive oxygen metobolites are erroneously believed to be free radicals, though technically speaking, the latter term is reserved for only those substances which contain a single, unpaired electron.
- Thus, superoxide is a free radical but hydrogen peroxide is not.
– chemical compounds and reactions capable of generating the above mentioned toxic oxygen species
Prooxidants
– compounds and reactions disposing of these toxic species
Antioxidants
- Balance can be shifted towards the pro-oxidants when:
- the production of oxygen species is greatly increased
- the levels of the antioxidants are diminished
- Can result in cell damage or even cell death if massive
OXIDATIVE STRESS
DELETERIOUS EFFECTS OF
FREE RADICALS
- Free radicals can react with the polyunsaturated fatty acids found mostly in membranes in a process called LIPID PEROXIDATION
- Can lead to loss of membrane integrity not only to the plasma membrane but also to the mitochondrial membrane
- Destruction of the mitochondria undermines the function of the respiratory chain and the production of ATP
DELETERIOUS EFFECTS OF FREE RADICALS (2)
- Direct interaction of ROS with nucleotides in DNA can cause misreading of the sequence and if left uncorrected, can lead to mutation
- Damage to DNA in ovaries and testes can lead to heritable mutations and in somatic cells can lead to activation of protooncogenes to oncogenes resulting in the initiation of cancer
DELETERIOUS EFFECTS OF FREE RADICALS (3)
- Reactions with amino acids in proteins, either by direct radical action or as a result of reaction with the products of radical-induced lipid peroxidation leads to modification of proteins that are recognized as non-self by the immune system
- The resultant antibodies will also cross-react with normal tissue proteins initiating autoimmune disease
DELETERIOUS EFFECTS OF FREE RADICALS (4)
- Oxidation of the proteins or lipids in plasma LDL by ROS leads to abnormal LDL that is not recognized by the liver LDL receptor and therefore not cleared by the liver
- The modified LDL is taken up by macrophages through scavenger receptors. These macrophages that has taken up the lipids becomes the foam cells that infiltrates the endothelium of the blood vessels leading to the development of atherosclerotic plaques
Many natural processes in the cells that require enzyme-catalyzed oxidation of organic molecules by molecular oxygen generate the __.
reactive oxygen species
PRODUCTION OF FREE RADICALS
- Hydroxylation reactions that occur in cytochrome P-450 for the detoxification of xenobiotics
- Synthesis of steroid hormones
- Degradation of purines to uric acid
- Reoxidation of the prosthetic groups of flavin-containing
enzymes - Transport of electrons in the respiratory chain and the reduction of oxygen which is the final acceptor of the electrons
Pathways by which the less destructive ROS can transform to the more highly reactive ROS in the presence of iron ions
- FENTON REACTION, a non-enzymatic reaction, ferrous ion can transform hydrogen peroxide to hydroxyl radical
- Iron-catalyzed HABER-WEISS REACTION, hydroxyl radical can also be generated when superoxide and hydrogen peroxide reacts.
- is a chain reaction involving polyunsaturated fatty acids that produces a continuous supply of more free radicals.
- Generates malondialdehyde which by itself can also covalently reacts with DNA, protein and lipids forming adducts that can produce more cellular damage
Lipid peroxidation
MECHANISMS FOR PROTECTION AGAINST FREE RADICALS
- Metal ions that can initiate free radical formations are bound to proteins for which they provide the prosthetic group or to their transport or storage proteins
- Iron: transferrin, ferritin, hemosiderin
- Copper: ceruloplasmin
- Other metal ions: metallothionein
- Most enzymes that produce and degrade free radicals are confined in peroxisomes
MECHANISMS FOR PROTECTION AGAINST FREE RADICALS 2
• Enzymes
- Superoxide dismutase, Catalase, Glutathione peroxidase
• Vitamins/Minerals
- Vitamin A, Vitamin C, Vitamin E, β-Carotene
- Selenium
• Phytochemicals
- Flavonoids, other polyphenols
- Excessive production of hydrogen per oxide by monoamino oxidase (MAO) has been implicated as a major factor for the neuronal degeneration in patients with Parkinson’s disease.
- The dopaminergic nigrostriatal neurons that are destroyed in this disease have shown high MAO activity.
- Thus, treatment with MAO inhibitors has been found to be an effective mode of therapy in this disorder
PARKINSONISM
- Oxidized low density lipoproteins (LDL), formed by action of free radicals, are readily taken up by the macrophages, producing foam cells.
- These cells accumulate beneath the endothelial layer of the arterial wall, and this triggers onset of atherogenesis
ATHEROSCLEROSIS
- Free radicals induce destruction of the pancreatic BETA cells, and this has been implicated in etiopathogenesis of type-1 diabetes.
DIABETES MELLITUS
- Males are at a greater risk of incurring free radical damage because they tend to accumulate large body stores of iron
- These free radicals are known to reduce sperm viability and motility, hence contributing to male sterility.
- Women are protected till menopaus
Male Infertility
- Compounds that contain several hydroxyl groups in aromatic rings
- Occur in fruits, vegetables, tea, coffee, beer and wine
- Are generally present in plants to protect them against ultraviolet radiation or aggression by pathogens
- Have antioxidant properties
POLYPHENOLS
HOW ANTIOXIDANTS CAN BECOME PROOXIDANTS (Vitamin E)
- When vitamin E reacts with lipid peroxides in lipoproteins, it forms relatively stable tocopheroxyl radical that persist long enough to penetrate deeper in to the lipoproteins and tissues, causing further radical damage rather than interacting with a water-soluble antioxidant at the surface of the lipoproteins or membranes
HOW ANTIOXIDANTS CAN BECOME PROOXIDANTS (Vitamin C)
- Vitamin C reacts with superoxide and hydroxyl to yield monodehydroascorbate and hydrogen peroxide or water
- It can also be a source of superoxide radicals by reaction with oxygen or hydroxyl radicals by reaction with Cu2+ ions
HOW ANTIOXIDANTS CAN BECOME PROOXIDANTS (β-carotene)
- For β-carotene, although it a radical-trapping antioxidant under conditions of low partial pressure of oxygen, as in most tissue, at high partial pressure of oxygen as in the lungs and especially in high concentrations, it is an autocatalytic prooxidant, and hence can initiate radical damage to lipids and proteins
WHAT IS A XENOBIOTIC?
- A xenobiotic (Gk Xenos meaning “stranger”) is a compound that is foreign to the body
- It may include drugs, chemical carcinogens, and various other compounds
- The xenobiotic may be excreted unchanged, or it may me metabolized by the body’s enzymes to other compounds
Metabolism of Xenobiotics
- Occurs in mostly in liver (enzymatic prosesses)
- Convertion into more hydrophilic substances - excretion urine
- May convert procarcinogenics into cytotoxic, muthagenic compounds
- Different persons may have differences in metabolism
(genetic diff., physiol. factors) - Metabolism of one xenobiotic may influence metabolism of another
2 PHASES OF XENOBIOTIC METABOLISM
- Hydroxylation/Biotransformation
2. Conjugation
- Attachment of new functional groups, transformation of existing functional groups
- Oxidation, reduction, hydroxylation, hydrolysis etc
Biotransformation
- Masking of an existing functional group by acetylation, glycolysation, attachment of an amino acid, or other mechanism -> More hydrophilic drug -> Renal excretion
Conjugation
- Catalyzed by cytochrome P450, a monooxygenase
- Hydroxylation may sometimes terminate the action of a drug
- Other phase 1 reactions may include deamination, dehalogenation, desulfuration, epoxidation, peroxygenation, and reduction
- Reactions involving hydrolysis also occur in phase 1
HYDROXYLATION
• The reaction catalyzed by a monooxygenase is as follows:
RH + O2 +NADPH + H+ → R-OH + H2O + NADP
• Approximately 50% of the drugs humans ingest are metabolized by isoforms of cytochrome P450
CYTOCHROME P450
PARTICIPATION OF THE CYP ENZYMES IN METABOLISM OF SOME CLINICALLY IMPORTANT DRUGS (1)
1A1: Caffeine, Testosterone, R-Warfarin
1A2: Acetaminophen, Caffeine, Phenacetin, R-Warfarin
2A6: 17-Estradiol, Testosterone
2B6: Cyclophosphamide, Erythromycin, Testosterone
PARTICIPATION OF THE CYP ENZYMES IN METABOLISM OF SOME CLINICALLY IMPORTANT DRUGS (2)
2C-family: Acetaminophen, Tolbutamide (2C9); Hexobarbital, S- Warfarin (2C9,19); Phenytoin, Testosterone, R- Warfarin, Zidovudine (2C8,9,19);
2E1: Acetaminophen, Caffeine, Chlorzoxazone, Halothane
2D6: Acetaminophen, Codeine, Debrisoquine
3A4: Acetaminophen, Caffeine,
Carbamazepine, Codeine, Cortisol, Erythromycin, Cyclophosphamide, S- and R-Warfarin, Phenytoin, Testosterone, Halothane, Zidovudine
• Compounds produced in phase 1 are converted by specific enzymes to various polar metabolites by conjugation with glucuronic acid, sulfate, acetate, glutathione, or certain amino acids, or by methylation
CONJUGATION
- UDP-glucuronic acid is the glucuronyl donor, and glucuronosyltransferases are the catalysts
- Synthesized in the gluronic acid pathway
- This is the most frequent conjugation reaction
GLUCORONIDATION
• Sulfate donor is adenosine 3-phosphate-5-phosphosulfate (PAPS), called “active sulfate.”
SULFATION
• Glutathione (γ-glutamyl-cysteinylglycine) is a tripeptide consisting of glutamic acid, cysteine, and glycine
• Electrophilic xenobiotics (such as certain carcinogens) are conjugated to the nucleophilic
GSH:
R+GSH→R—S—G
• The enzymes catalyzing these reactions are called glutathione S-transferases and are present in high amounts in liver cytosol
GLUTATHIONE
X+Acetyl-CoA→Acetyl-X + CoA
• Addition of acetyl group catalyzed by acetyltransferases present in the cytosol of various tissues, particularly the liver
• The drug isoniazid, used in the treatment of tuberculosis, is subject to acetylation
• Depending on your genetic makeup: Polymorphic types of acetyltransferases exist, resulting in individuals who are classified as slow or fast acetylators
ACETYLATION
• Filipinos have high resistance to isoniazid treatment because we are mostly fast acetylators (we metabolize isoniazid fast kaya
hindi sya masyadong effective)
• Slow acetylators are more subject to certain toxic effects of isoniazid because the drug
persists longer in these individuals
*Kaya din daw side effects are adjusteddepending sa race.
ACETYLATION
• Methyl donor is S-adenosylmethionine (SAM)
- SAM is “Active methionine”
• Catalyzed by methyltransferases
METHYLATION
- Liver has several soluble UDP-Gluc-transferases
- Present in both endoplasmic reticulum and cytosol
- 2-acetylaminofluorene (a carcinogen), aniline, benzoic acid, meprobamate (a tranquilizer), phenol, and many steroids are excreted as glucuronides
- The glucuronide may be attached to oxygen, nitrogen or sulfur groups of the substrates
- Probably the most frequent conjugation reaction
GLUCURONIDATION
PURPOSE OF XENOBIOTIC METABOLISM
- To increase the water solubility of the xenobiotic and facilitate excretion from the body
- If xenobiotic is not metabolized, it would remain in the nonpolar state and would be stored indefinitely in the adipose tissues
A METABOLIZED XENOBIOTIC MAY HAVE THE FOLLOWING FATES:
- It may be converted from an inactive to a biologically active compound (prodrug or procarcinogen)
- Additional phase 1 reactions may convert the active compounds to less active or inactive forms prior to conjugation.
- In other cases, it is the conjugation reactions that convert the active products of phase 1 reactions to less active or inactive species, which are subsequently excreted in the urine or bile
- In a very a few cases, conjugation may increase the biologic activity of a xenobiotic
PROPERTIES OF HUMAN CYTOCHROME P450 (1)
- All are hemoproteins
- Exhibit broad substrate specificity
- Found mostly in the liver but is also present in other tissues
- A mitochondrial or endoplasmic reticulum enzyme
- Activity may be induced or depressed by certain drugs, causing drug interactions
PROPERTIES OF HUMAN CYTOCHROME P 450 (2)
- Exhibits genetic polymorphism, so different individuals metabolize drugs differently
- Activity is affected also by tissue or organ disease (e.g. Cirrhosis)
- NADPH, not NADH, is involved in the reaction mechanism
- The preferred lipid in its structure is phosphatidylcholine
CYTOCHROME NOMENCLATURE
- CYP – cytochrome
- Arabic number – family (40% or more sequence identity)
- Capital letter – subfamily (>55% sequence identity)
- Individual P450s are given arabic numerals
- Ex. CYP1A1 means it is a member of family 1 subfamily A and is the first individual member of the subfamily
RESPONSE TO XENOBIOTICS
- May be affected by age, sex, and genetic factors (Pharmacogenomics)
- Reaction to drugs: Pharmacogenetics
- May result in cell injury, allergies, or carcinogenesis
- Covalent bonding of xenobiotic metabolites to DNA, RNA and protein macromolecules can lead to cell injury (cytotoxicity)
- If severe enough, can lead to cell death
- DNA damage -> DNA repair mechanisms -> transfer of ADP-ribose units to DNA binding protein catalyzed by ADP-ribose polymerase -> depletion of NAD (source of ADP-ribose) -> impaired ATP formation -> cell death
Cell injury
- The reactive xenobiotic metabolite may bind to a protein as a hapten, altering its antigenicity
- Haptens are small molecules that elicit an immune response only when attached to a large carrier such as a protein
- The resulting antibodies react with both modified and unmodified proteins, imitating autoimmune disease
Allergies
Why care about biotransformation?
- Development of a toxicological method
- Interpretation of toxicological findings
- Understanding of drug effects
- Correct and effective therapy, reduction of adverse drug effects
- Primarily occurs in the liver
- Efffect may be negligible but is sometimes fatal
- Primarily occurs in the liver and in the wall of the small intestine
- Stimulants of Cyt P450: ex. Barbiturates, Carbamazepine, Phenytoin,, Rifampin, St. John’s Wort – does not take place quickly (7-10 days)
- Some drugs also inhibit Cyt P450 – effect is FASTER
DRUG METABOLISM
- Major biochemical transducer
- Potential chemical energy kinetic energy
- Largest single tissue
- Types:
- Skeletal
- Cardiac
- Smooth
Muscle
Ultrastructure of a Myofibril
- A muscle is made up individual cells called MUSCLE FIBERS
- Longitudinally within the muscle fibers, there will be bundles of myofibrils
- A myofibril can be subdivided into sarcomeres, demarked by a Z LINE
- Sarcomeres are composed of thin and thick filaments creating bands
- Contraction causes no change in the length of the A band, a shortening of the I band, and a shortening of the H zone (band)
Striated muscle is composed of multinucleated muscle fiber
cells surrounded by an electrically excitable plasma membrane, the __.
sarcolemma
An individual muscle fiber cell, which may extend the entire length of the muscle, contains a bundle of many myofibrils arranged in parallel, embedded in intracellular fluid termed __. Within this fluid is contained glycogen, the high-energy compounds ATP and phosphocreatine, and the enzymes of glycolysis.
sarcoplasm
- Functional unit of skeletal muscle
- Under Electron Microscopy
*Alternating dark and light bands ( anisotropic and isotropc)
A and I bands
H band : central region of the A band; appears less dense
Z line : bisects the I band; very dense and narrow - Region between 2 Z- lines
- Repeated along the axis of a fibril (depending on the state of contraction)
- Striated appearance (voluntary and cardiac)
- High degree of organization
- Muscle fibers aligned with their sarcomeres in parallel register
Sarcomere
- Confined to A- band
- Protein: MYOSIN
Thick Filaments
- Lies in the I- band
- Extends to A band but not into its H zone
- Protein: ACTIN, TROPOMYOSIN, TROPONIN
Thin Filaments
Sliding Filament Cross- Bridge Model
Henry Huxley and Andrew Huxley et al.
Resting, extending, contracting
Arrays of interdigitating filaments must slide past one another during contraction.
(Sliding Filament Cross- Bridge Model)
- link thick and thin filaments at certain stages in the contraction cycle
- generate and sustain the tension
Crossbridges
(Sliding Filament Cross- Bridge Model)
Tension developed during muscle contraction is proportionate to
- filament overlap
- # cross bridges
2 Major proteins of Muscle
- Actin
- G- Actin -> 25% of muscle CHON by weight
- At physiological pH + Mg2+
* G- Actin polymerizes con covalently -> F- Actin (insoluble double helical filament) - MYOSIN
- 55% of muscle CHON
- Forms thick filaments
- Has fibrous tail (2 intertwined helices)
- Aggregates of insoluble a- helical fibers from tail of myosin
- No ATPase activity
- Does not bind to F- actin
Light meromyosin (LMM)
- Soluble protein w/c has both a fibrous and globular portion
- Exhibits ATPase activity
- Binds to Actin
- Papain digests HMM -> 2 subfragments
Heavy meromyosin (HMM
- Enhances the rate by which myosin ATPase releases its products (ADP and Pi)
- Does not affect hydrolysis step
- Ability to promote release of products greatly accelerates the overall rate of catalysis.
F- Actin
- Cyclic attachment and detachment of the S1 head of myosin to F- actin filaments (Making and breaking of Cross bridges)
- Conformational changes
- Dependent upon wc/c nucleotide is present (ATP or ADP)
- Results to POWER STROKE
Muscle Contraction
- Head of myosin hydrolyzes ATP -> ADP + Pi
- ADP-Pi-myosin complex energized (high energy conformation)
Relaxation Phase
- Involves Ca, troponin, tropomyosin and actin
- Actin becomes accessible, S1 head of myosin binds with it and forms a complex
Contraction stimulated
- initiated by release of Pi
- Release of ADP -> conformational change in myosin -> pulling actin towards the center of sarcomere
- Myosin (low energy state)
Power Stroke
- Another molecule of ATP binds to S1 head
Actin- Myosin- ATP complex
Myosin-ATP has a low affinity for actin, and actin is thus released. This last step is a key component of relaxation and is dependent upon the __.
binding of ATP to the actinmyosin complex
- Drive the cycle
- Actual power stroke -> conformational change in the S1 head -> upon release of ADP
Hydrolysis of ATP
Drop in the intracellular level of ATP (DEATH)
- ATP not available to bind w/ S1 head
- Actin does not dissociate relaxation does not occur
- RIGOR MORTIS
Tropomyosin vs Troponin
TROPOMYOSIN
- Fibrous molecule
- 2 chains (alpha and beta) attach to F- actin
- Present in all muscular and muscle like structures
TROPONIN complex
- Unique to striated muscles
- 3 polypeptides
- TpT, TpI, TpC
Troponin T vs Troponin I vs Troponin C
Troponin T: Binds to tropomyosin as well as to other 2 troponin components
Troponin I: Inhibits the F- actin- myosin interaction
Troponin C: Calcium binding polypeptide ; Structurally and functionally analogous to CALMODULIN
2 mechanism of regulation of muscle contraction
- ACTIN- based: Skeletal and cardiac
2. MYOSIN- based: smooth
- ATP – only limiting factor in the cycle of muscle contraction
- Skeletal muscle system INHIBITED at rest
- Troponin system : inhibitor of striated muscle
Actin based regulation
- Regulates intracellular levels of Ca in the skeletal muscle
- Resting state : Ca is pumped into the SR through an active transport, Ca2+ ATPase -> relaxation
Sarcoplasmic Reticulum
- Sarcoplasmic Ca falls below 10^-7 , resequestration into the SR by Ca ATPase
- Troponin inhibits further interaction between actin and myosin
- Myosin head detaches from F- actin in the presence of ATP
RELAXATION
Effects of decreased ATP in sarcoplasm
- Ca pump in the SR ceases to maintain the low concentration of Ca in the sarcoplasm
- Interaction of myosin heads w/ F- actin promoted
- ATP dependent detachment of myosin heads from F actin cannot occur and rigidity sets in.
- Exposure to certain anesthetics (halothane) and depolarizing skeletal muscle relaxants (succinylcholine)
- Rigidity of skeletal muscles + high fever
- High cytosolic concentration of Ca
- Vfib -> Death
- Tx:
- Stop anesthetic
- Dantrolene: Inhibit release of Ca form SR into cytosol; Preventing increase of cytosolic Ca
Malignant Hyperthermia
- Mutation in gene encoding the protein dystrophin
- Milder form Becker muscular dystrophy
- Also in some cases Dilated cardiomyopathy
- Dystrophin (Links the actin cytoskeleton to the ECM; Needed for assembly of synaptic junction)
Duchenne Muscular Dystrophy
- Resembles that of skeletal muscle
- Striated
- Uses Actin-myosin-tropomyosin-troponin system
- Exhibits intrinsic rhythmicity
- Indiv myocytes communicate through its syncytial nature
- T- tubular system more developed
- SR less extensive hence intracellular supply of Ca for contraction is less.
- Relies on extracellular Ca for contraction
- cAMP more prominent role in cardiac muscle
Cardiac Muscle
(Extracellular CA 2+ : important role in contraction)
- Portal of entry is the L type or slow CA 2+ channel
- Voltage gated
- Equivalent to DHP receptors in skeletal muscle
- Regualated by cAMP dependent protein kinases and cGMP dependent kinases
- Ca channel blockers (Verapamil) inhibits these channels
- Fast or T CA 2+channels are also present
CA 2+ channels
(Extracellular CA 2+ : important role in contraction)
- Principal route of exit of Ca from myocytes
- Resting myocytes (helps maintain low level of free intracellular Ca: 1Ca for 3Na)
- Any increase IC Na -> Ca to rise -> more forceful contraction (positive inotropic effect)
CA 2+ - Na + Exchanger
- inhibits sarcolemnal Na K ATPase
- Diminishing exit of Na
- Promotes inflow of Ca via Ca Na exchange
Digitalis
(Extracellular CA 2+ : important role in contraction)
- Also contributes to Ca exit
CA 2+ ATPase
- any structural or functional abnormality of the ventricular myocardium due to an inherited cause
Inherited Cardiomyopathies
Inherited Cardiomyopathies: Classes
- Disorders of cardiac energy metabolism
- mutations in genes encoding enzymes or proteins involved in FA oxidation (a major source of energy for the myocardium) and oxidative phosphorylation - Mutations in genes encoding proteins involved in or affecting myocardial contraction
- Hypertrophy, often massive, of 1 or both
- Autosomal dominant
- Missense mutation in the β- myosin heavy chain gene
Familial Hypertrophic Cardiomyopathy
Regulation of Smooth Muscles
- Sarcomeres are not aligned (not striated appearance)
- α-actinin and tropomyosin molecules
- No troponin system
- MYOSIN based
- Contraction is also regulated by Ca
Phosphorylation of Myosin Light Chains Initiates Contraction of Smooth Muscle
- no detectable ATPase activity when smooth muscle myosin is bound to F-actin
- Smooth muscle myosin contains light chains that prevent the binding of the myosin head to F-actin
- must be phosphorylated - hydrolyzes ATP about 10-fold more slowly than in skeletal muscle
- calcium dependent
- requires binding of calmodulin-4Ca2+ to its kinase subunit
myosin light chain kinase
- increase the contraction of smooth muscle
- phosphorylates myosin light chain phosphatase
- directly phosphorylates the light chain of myosin
Rho kinase
- binds to tropomyosin and actin at low concentrations of Ca2+
- prevents interaction of actin with myosin
- Muscle relaxation
- may also participate in organizing the structure of the contractile apparatus in smooth muscle
Caldesmon
- acetylcholine
- Endothelium derived relaxing factor (EDRF)
- formed by the action of the enzyme NO synthase
- arginine
- five redox cofactors:
NADPH, FAD, FMN, heme, and tetrahydrobiopterin - very short half-life (approximately 3-4 seconds) in tissues
Nitric Oxide
Replenishing ATP in Muscle
- glycolysis, using blood glucose or muscle glycogen
- oxidative phosphorylation
- creatine phosphate
- 2 molecules of ADP in a reaction catalyzed by adenylyl kinase
*amount of ATP in skeletal muscle is only sufficient to provide energy for contraction for a few seconds
- Release of glucose via glycogen phophorylase
- Activated: CA, epinephrine, AMP
- McArdle disease : glycogen phosphorylase b is inactive
Glycogen
- Synthesis of ATP during aerobic state
Oxidative phosphorylation
- Major energy reserve
- Prevents rapid depletion of ATP by providing readily available high energy phosphate
- Creatine kinase
Creatine Phosphate
SKELETAL MUSCLE CONTAINS
SLOW (RED) and FAST (WHITE)
TWITCH FIBERS
- Type I (slow twitch)
- Type IIA (fast twitch-oxidative)
- Type IIB (fast twitch-glycolytic)
- contain myoglobin and mitochondria
- Aerobic metabolism
- Maintain relatively sustained contractions
Type I (slow twitch)
- lacking myoglobin
- Few mitochondria
- derive their energy from anaerobic glycolysis
- exhibit relatively short durations of contraction
Type II (fast twitch-glycolytic)
- An extensive intracellular network of filamentous structures involved in cell:
- self propulsion,
- Morphogenesis
- cleavage
- endocytosis
- exocytosis,
- intracellular transport
- changing cell shape
Cytoskeleton
all eukaryotic cells contain three types of filamentous structures:
- actin filaments (also known as microfilaments)
- microtubules,
- intermediate filaments
- integral component of the cellular cytoskeleton
- Cytoplasmic tubes 25 nm in diameter
- present in all eukaryotic cells
- necessary for the formation and function of the mitotic spindle
- endocytic and exocytic vesicles
- structural components of cilia and flagella
- component of axons and dendrites
Microtubules
- An absence of dynein in cilia and flagella results in immotile
cilia and flagella, leading to male sterility, situs inversus
and chronic respiratory infection - Mutations in genes
affecting the synthesis of dynein have been detected in individuals with this syndrome
Kartagener syndrome
Aging vs Senscence
Ageing/ Aging - gradual decline in the capacity of physiologic systems
Senescence – decline of an individual’s fitness components, internal deterioration with increasing age
Gerontology vs Geriatrics
Gerentology – study of biological, psychological and social phenomena associated with aging and old age
Geriatrics – branch of medicine involved with the treatment and diagnosis of diseases associated to the aged
– study of the biology of aging and longevity
Biogerentology
- Remarkable growth and physical development
- Development of motor and intellectual skills
- Dependence for food, water, shelter, protection
Infancy, childhood
- Tremendous pace in growth
- Developmental changes – physical, hormonal
Adolescence
- Longest stage
- Decline in physical growth and development except during pregnancy
Adulthood
Lifespan vs. Life expectancy
Life span
- Normal or average duration of life of an individual
- Maximum number of years that a person can potentially expect to live
Life expectancy
- Number of years a person is expected to live
- Life EXpectancy where “e” represents the expected number of years remaining and “x” represents the person’s present age
Life expectancy
Philippines’ data:
Females - 73.24 years
Males: 68.93 years
Maximum life potential
- Oldest age which a human has lived
- Jeanne Louise Calment (122 years, 164 days)
Factors affecting Aging
- Hydrolytic reactions
- UV radiation
- AGE/ Protein glycation
- Reactive oxygen species
Factors affecting aging: UV radiation
- UV radiations is absorbed by DNA or RNA bases, amino acids, heme groups etc.
- May cause rupture of covalent bonds in proteins, DNA, RNA and generation of free radicals and mutation
- Prolonged exposure leads accumulation of DNA lesions and cancer
Factors affecting aging: Protein glycation
- Advanced Glycoxidation and Lipoxidation Endproducts (AGE/ALEs)
- Crosslink between proteins or biological macromolecules
- Implicated in the pathogenesis of complications of diabetes and atherosclerosis, Alzheimer’s disease, Parkinson’s disease and Creutzfeld–Jakob (prion) disease
- Most common form of progressive cognitive deterioration in the elderly
- Characterized microscopically by the appearance of neurofibrillary tangles and senile plaques
- AGEs and ALEs are increased in tangles and plaques in the brain of AD patients
- Amyloid protein is toxic to neurons in cell culture and catalyzes oxidative stress and inflammatory responses
Alzheimer’s disease
- Shows the limit to replicative capacity of cells
- Demonstrated by fibroblasts cultured in vitro
- Cells continue to divide until maximum density is reached (Contact inhibition)
- After 50 divisions, fibroblasts stops dividing regardless of density (Hayflick’s limit)
- Cells from elderly divide less – loss of replicative capacity
Hayflick’s Limit or Phenomenon
- Sequence of repeating nucleotides at the end of chromosomes that serves as handles as chromosomes move during telophase of meiosis
- Irreversibly shorten every time the cell divides
- Telomeres are too short = no cell division
Telomeres
- In transformed or cancerous cells, the enzyme telomerase lengthens telomeres even after telophase
- Cells become immortal, dividing far beyond Hayflick’s limit
- Presence of telomeres in cancer cells allow them to maintain telomere length while they proliferate
Cancer
- Result of cumulative oxidative damage to biomolecules: DNA, RNA, protein, lipids, and glycoconjugates
- Free radicals and Reactive Oxygen Species (ROS) are ↓ in longer living organisms = better antioxidant properties, better repair and cellular turnover
- Increased oxidative damage to proteins with age
- FR are generated through Fenton and Haber Weiss reaction
Free radical theory of aging
Free Radicals
- Free radicals – stable molecule that loses an electron
- ROS – highly reactive free radicals
e. g. Superoxide, hydroxyl radical, peroxyl radical - Form as a result of stress, inflammation, body’s natural defenses
- Formed in the mitochondria, phagosomes, peroxisomes
- Protein carbonyl groups (glutamic and aminoadipic acid semialdehyde) formed by oxidative deamination of arginine and lysine are formed in proteins exposed to ROS
- Steady-state level of protein carbonyls in intracellular proteins ↑ with age and at a rate inversely proportional to the lifespan of species
- Protein carbonyls are also much higher in fibroblasts from patients with progeria (accelerated aging), e.g. Werner’s or Hutchinson–Gilford syndromes
Free Radicals
- Autosomal recessive diseases caused by mutation of WRN gene
- Gradually show premature aging, graying and loss of hair, thinning of skin, development of early cataracts, impaired glucose tolerance, diabetes, atherosclerosis and osteoporosis, and cancer
Werner’s syndrome
- Mutation in the BLM gene responsible for copying and repairing DNA in cells
- Characterized by growth retardation, reduced fertility and chromosomal instability
- Display sunlight sensitivity, immunodeficiency, and a broad spectrum of cancers that appear early in life
- Death occurs typically in the mid-20s
Bloom’s syndrome
- Rare, fatal, autosomal dominant disease
- Single base mutation in LMNA , which results in the production of a mutant lamin A protein called progerin
- Progerin is ↑ in concentration in skin and the vascular wall
- Children develop progressive atherosclerosis and die of heart attacks or strokes at a median age of 14.5 yr, most often between ages 5 and 20 yrs.
Hutchinson-Gilford progeria syndrome
- Small organisms tend to have high metabolic rates and thus tend to die at an earlier age than larger organisms
- Free radicals and other metabolic by-products accumulate and play a role in senescence
- Prevention of aging: reduction in activity and caloric intake restriction
Rate of Living Theory
- The neuroendocrine or immune system is vulnerable (presumably to entropic processes) during senescence
- Failure of the neuroendocrine system - loss of reproductive function and metabolic regulation
- Failure of the immune system - increased susceptibility to infection and a decreased ability to reject tumor cells
- Failure of the weak system accelerates dysfunction of the whole organism
Weak Link Theory
- Aging is an accumulation of damage and injuries to the body
- Mechanical functions of the body wear away and by-products accumulate and impair the function
- Most agents that cause damage to biomolecules are water, oxygen, sunlight or radiation
e. g. Hydrolytic damage to proteins and nucleotide causes mutation
Wear and Tear Theory
- Errors in DNA transcription or RNA translation eventually lead to genetic errors that promote senescence
- Errors build up over time up to a catastrophe that may lead to the death of the cell or to the whole organism
Error Catastrophe Theory
- Aging is under direct genetic control
- Cells divide to its maximum level then can no longer divide further
- Individual variation develops because of maladaptation, exposure, and lifestyle
- Maladapted individuals tend to die out and the well-adapted ones persist, altering longevity in the best interest of the species
Master Clock Theory
- Cell membrane becomes less lipid as we age - impeding its efficiency to conduct normal function to transfer chemicals, heat etc.
- Toxic accumulation of lipofuscin deposits are present in the brain, heart and lungs and also in the skin
Membrane theory of aging
- Gradual and spontaneous change resulting in maturation through childhood, puberty, young adulthood and decline through middle and late age
- Deterioration in the function of an organism resulting to resulting changes in cellular structure and function, biochemistry and metabolism
- Leads to increased susceptibility in diseases and probability of death
Aging
Prevention of Aging
- Apoptosis in the mitochondria
- Repair of ROS through antioxidants
- Stem cells
- ROS, viral dsRNA, DNA damage, heat shock triggers the opening in the mitochondria
- Apoptosome initiate activation of capsase that breaks down proteins and lead to death of the affected cell
- Eliminated by phagocytosis
- Presence of intrinsic mechanisms in the cell leads to elimination of transformed or cancer cells
Apoptosis
- Stabilize free radicals by sharing an electron to the unstable compounds
- Glutathione peroxidase, superoxide dismutase, catalase (enzymatic antioxidants)
- Vitamin E and C, thiol antioxidants (glutathione, thioredoxin and lipoic acid), melatonin, carotenoids, natural flavonoids (non-enzymatic antioxidants)
Antioxidants
- Master antioxidant
- Composted of cysteine, glycine, and glutamate
- Found in virtually every cell of the human body
- Highest concentration of glutathione is in the liver, making it critical in the body’s detoxification process
- Central role in biotransformation and elimination of xenobiotics and protects cells against oxidative stress
- Fortify immune system and prevents photoaging
- Skin whitening by preventing tyrosinase activation of melanogenesis
Glutathione
- Stem/progenitor cell endowed with self-renewal and regenerative capacities
- Multipotent differentiation function and paracrine factors, which can whiten, suppress wrinkles and promote anti-oxidation and wound healing of skin
Stem cells
According to the __, ROS are the primary culprits, causing alterations in the sequence of DNA (mutations) and structure of proteins
free radical theory of aging
Molecular and Cellular Therapies
- Recombinant DNA products
- Gene Transfer
- Regenerative Medicine
- Mutations in the factor VIII (FVIII) gene produce __
hemophilia A
- This X-linked disorder demonstrates the many challenges in developing therapeutic products by recombinant DNA (rDNA) means
- The gene is large, with 26 exons and a genomic structure extending over 186 Kb.
- The protein contains 2332 amino acids and is synthesized as a single chain
- The mid-portion (B subunit) of the molecule is excised since it is not required for hemostatic function
The heterodimers formed are held together by calcium.
Hemophilia
- The development of __ is a serious complication, occurring in approximately 30% of patients with severe hemophilia A and about 5% of patients with severe hemophilia B
- are antibodies against coagulation FVIII or FIX, and they occur as a consequence of:
- Genetic predisposition, since the risk is higher if there is a positive family history.
- immunoregulatory molecules such as interleukins
- Environmental factors including exposure to new antigens (neoantigens) in blood products to which immunological tolerance has not been developed.
Hemophilia Inhibitors
Hemophilia Inhibitors
- Treatment of patients with inhibitors may involve an attempt at inducing tolerance by exposing them to regular and long term administration of factor concentrates
- If this does not work, activated, plasma-derived, prothrombin complexes (mixture of factors II, VII, IX, X) can be used
- These overcome the block on FVIII activation resulting from the development of antibodies
However, these products are expensive and they have the same infection risks as plasma-derived FVIII
Hemophilia Inhibitors 2
- They can also increase the risk of thrombosis.
- A solution to inhibitors was found with the development of an rhFVIIa (FVIIa is activated factor VII)
- This product is now approved in many countries. It can lead to thrombosis but this is more of an issue if used for off-label indications, i.e. other uses apart from treating inhibitors in hemophilia
DNA Vaccines
- Nucleic acid (DNA) vaccines predominantly utilize genes in the form of plasmid DNA
- Such genes express proteins to produce a sustained antigenic stimulus, and so generate an ongoing immune response
- There are various routes for administration including parenteral, topical or a gun that delivers tiny amounts of DNA-coated gold beads
- The first DNA vaccine was used in 1990
- This approach to vaccination has provoked interest because of the relatively simple way in which vaccines can be prepared to deliver a range of protein antigens for immunization
- In animal studies, these vaccines stimulate both humoral and cell-mediated immune mechanisms, comparable to what occurs with live attenuated vaccines
- Thus, they could be an alternative, but safer, approach to live viral vaccines and should be better than inactivated (dead) vaccines in the breadth of the immune response they
DNA Vaccines
Subunit Vaccines
- Hepatitis B Vaccine (HbsAgn)
- Human Papilloma Vaccine (L1 protein)
- Gene therapy can be defined as “the transfer of genetic material (DNA or RNA) into the cells of an organism”.
- It aims either to produce a therapeutic effect or to mark a cell with a gene so that it can be followed or identified as part of a research protocol
- An example would be marking cells in a transplantation scenario to determine if cancer relapse occurs in host (patient) or donor cells
Somatic Cell Gene Transfer
- Therefore, gene transfer is probably a better description than gene therapy, since a therapeutic intent is not necessary.
- Gene therapy in humans refers to somatic cell gene therapy, meaning the target is a somatic cell, and transmission to future generations cannot occur
- Germline gene therapy, an example of which would be a transgenic animal, is prohibited
Somatic Cell Gene Transfer
Criteria have been proposed to identify the types of genetic disorders for which gene therapy might be appropriate
- A life-threatening condition for which there is no effective treatment
- A condition in which the cause of the defect is a single gene and the gene has been cloned
- A condition in which regulation of the gene need not be precise
- A condition in which technical problems associated with delivery and expression of the gene have been resolved
Ways to Transfer DNA/RNA
- Physical
2. Viral
- The cell and nuclear membranes can be made more permeable to DNA following co-precipitation of DNA with calcium phosphate, or an electric shock (called electroporation). Using micropipettes, it is possible to inject DNA into the cell’s nucleus.
Physical
More novel approaches to facilitate movement of DNA into a cell include:
- Injection of DNA directly into muscle cells;
- Insertion of DNA via cationic liposomes in a process known as lipofection; i.e. it uses synthetic spherical vesicles which have lipid bilayers and so are able to cross the cell membrane, and
- Coating of DNA with proteins and using a gene gun – DNA-coated microprojectiles
- The preferred method of gene transfer involves the use of viruses particularly the retroviruses.
Viral (biological)
Pros and Cons of Using Viral Transfer (Advantages)
Advantages
- A single virus infects one cell
- The virus is usually non-immunogenic
- Integration into the host genome means there is the potential for long-term expression of the inserted gene
Pros and Cons of Using Viral Transfer (Challenges)
Challenges
- The target cell must be dividing before the retrovirus can integrate into the cell’s genome
- Transduction efficiency is usually inadequate
- DNA insert size is limited which can be a problem if a large gene is involved
- Retroviral vectors are produced from living cells so there is worry that contaminants from these cells will be present
Pros and Cons of Using Viral Transfer (Risks)
Risks
- Integration is random, and so there is always the worry that a normal gene is inactivated or an oncogene is activated
- There is the potential for retroviruses to revert to replication-competent organisms and so induce cancer
- is defined in a 2011 UK report as a “therapeutic intervention that replaces or regenerates human cells, tissues or organs to restore or establish normal function”
- utilizes small molecule drugs, biological products, medical devices and cell-based therapies
- Non-regenerative applications of the same technology include drug discovery and toxicity testing
Regenerative Medicine
- are non-specialized cells that can self-renew and transform into other cells
Stem cells
Stem Cells
Unipotent – forms one differentiated cell type
Multipotent – forms all cell types that constitute an organ, e.g. a hematopoietic stem cel
Pluripotent – forms most if not all of the adult cell types in the body.
Totipotent – forms all cell types including adult, embryo and placenta.
Important Stem Cell Property
- Self renewal – the capacity to make more stem cells
2. Differentiation – the ability to give rise to different progeny when exposed to the appropriate transcription factors
- describes the application of computational tools and analysis to capture, store and interpret biological data
- It intersects a number of disciplines, including biology, medicine, computer science, information technology and mathematics
- There are many related terms used interchangeably with bioinformatics, including informatics, computational biology, medical informatics, eHealth and health information technology
Bioinformatics
The most important was the discovery of the structure of DNA itself by Watson and Crick (1953) and another female __ who is an X-ray crystallographer who uses X-ray crystallography refraction to discover the structure of the DNA which is a double helix
Rosalind Franklin
Recombinant DNA discoveries
Kornberg - DNA polymerase
Arber - DNA restriction
Gellert - DNA ligase
Recombinant DNA discoveries 2
Sanger and Batrell and Maxam and Gilbert - DNA sequencing method
Mullis - PCR
Hood and Hunkapillar - automated DNA sequence
- purified from bacteria
- cut the DNA double helix at specific sites defined by the local nucleotide sequence
- cleaving a long, double-stranded DNA molecule into fragments of strictly defined size
RESTRICTION ENDONUCLEASES
Nomenclature of Restriction enzyme:
- The first letter of the name is from the genus of the bacterium.
- The next two letters are from the name of the species.
- An additional letter indicates the type or strain, and a final number is appended to indicate the order in which the enzyme was discovered in that particular organism
2 types of restriction enzyme:
the blunt-end (straignt) and the sticky end
(Major Classes of Restriction enzyme)
- Less common
- Cut both strands at nonspecific location (>1000bp) away from recognition site
- Three-subunit complex individual recognition
- not useful in recombinant DNA research
Type I
(Major Classes of Restriction enzyme)
- more common
- cut strand at specific, usually palindromic, recognition site (4-8 bp)
- endonuclease and methylase
- very useful in recombinant DNA research
Type II
(Major Classes of Restriction enzyme)
- rare
- cleavage of one strand only (24-26 bp downstream of the 3’ recognition site)
- not useful in recombinant DNA research
Type III
- simpler than for proteins because each nucleotide in a nucleicacid molecule already carries a single negative charge (on the phosphate group)
- no need to add the negatively charged detergent SDS that is required to make protein molecules move uniformly toward the positive electrode (hindi mo na kelangan ichange ang pH nya kasi meron na syang charge)
- Larger DNA fragments will migrate more slowly because their progress is impeded to a greater extent by the gel matrix.
- DNA fragments become spread out across the gel according to size, forming a ladder of discrete bands
GEL ELECTROPHORESIS
- makes it possible to separate extremely long DNA molecules, even those found in whole chromosomes.
- travel end-first through the gel in snakelike configurations at a rate that is independent of their length
- the direction of the electric field changes periodically, which forces the molecules to reorient before continuing to move snakelike through the gel
- Similar to the ordinary gel electrophoresis however you use pulse electrical fiel
PULSED-FIELD GEL ELECTROPHORESIS
PURIFIED DNA MOLECULES CAN BE SPECIFICALLY LABELED WITH RADIOISOTOPES OR CHEMICAL MARKERS IN VITRO
- If you’re trying to do the DNA hybridization or you want to determine the gene of interest, you actually use probes. Probes are nucleotides that contain radioisotope or protein or fluorescent molecule.
How can the DNA sequence of interest be picked out of a mixture of thousands or even millions of irrelevant DNA fragments?
The answer lies in the use of a probe—a short piece of ssDNA, labeled with a radioisotope, such as 32 P, or with a nonradioactive molecule, such as biotin. The sequence of a probe is complementary to a sequence in the DNA of interest, called the target DNA.
- are used to identify which band on a gel or which clone in a library contains the target DNA, a process called screening.
Probes
GENES CAN BE CLONED USING BACTERIA
- Any DNA fragment can be cloned
- In molecular biology, the term DNA cloning is used in two senses
1. act of making many identical copies (typically billions) of a DNA molecule—the amplification of a particular DNA sequence
- the isolation of a particular stretch of DNA (often a particular gene) from the rest of the cell’s genome
- may carry genes that convey antibiotic resistance to the host bacterium, and may facilitate the
transfer of genetic information from one bacterium to another.
Plasmids
Do you know how we can insert double stranded recombinant DNA?
- Lipofection
- Heat Shocking
- Electroporation
- is a linear, double-stranded DNA molecule approximately 50 kb in length
- The modified strain used in most cloning experiments contains two cleavage sites for the enzyme EcoR1, which fragments the genome into three large segments
lambda genome
- naturally occuring multicopy plasmids
-
Plasmid
Basis: Bacteriopage lambda
Size limits of insert: 5-20 kb
Major Application: Genomic DNA cloning, cDNA cloning and expression libraries
Phase
Basis: Plasmid containing a bacteriophage lambda
Size limits of insert: 35-45 kb
Major Application: Genomic library construction
Cosmid
Basis: Escherichia coli F factor plasmid
Size limits of insert: 75-300 kb
Major Application: analysis of large genomes
BAC (bacterial artificial chromosome)
Basis: Aaccharomyces cerevisiae centromere, telomere and autonomously replicating sequence
Size limits of insert: 100-1000kb (1 Mb)
Major Application: analysis of large genomes, YAC transgenic mice
YAC (yeast artificial chromosome)
Basis: Mammalian centromere, telomere, and origin of replication
Size limits of insert: 100 kb to >1Mb
Major Application: Under development for use in animal biotechnology and human gene therapy
Mammalian artificial chromosome (MAC)
The collection of cloned plasmid molecules is known as a __.
DNA library
Two kinds of libraries are commonly used:
- Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome
- complementary DNA (cDNA) libraries contain those DNA sequences that only appear as processed mRNA molecules, and these differ from one cell type to another.
A pair of primers directs the synthesis of a desired segment of DNA in a test tube. Each cycle of PCR includes three steps:
- Strand Separation
- DNA Hybridization
- DNA Synthesis
- The double-stranded DNA is heated briefly to separate the two strands at 95 C
STRAND SEPARATION/DENATURATION
- The DNA is exposed to a large excess of a pair of specific primers—designed to bracket the region of DNA to be amplified—and the sample is cooled to allow the primers to hybridize to complementary sequences in the two DNA strands at 60 C
DNA Hybridization
- This mixture is incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA can be synthesized, starting from the two primers at 72 C
DNA SYNTHESIS
By using a __ (for example, Taq polymerase from the bacterium, Thermus aquaticus that normally lives at high temperatures), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle. Typically 20–30 cycles are run during this process, amplifying the DNA by a million-fold (2^20) to a billion-fold (2^30).
heat-stable DNA polymerase
So you have a single-stranded DNA fragment then you add a labeled DNA primer. Also add the deoxyribonucleotide triphosphate (dNTPs) and add DNA polymerase. You have to divide it into 4 separate tubes and add a small amount of one chain-terminating dideoxyribonucleotide (ddNTP) to each tube. Then you have a sequence. One is for A (adenine), T (Thymine), C (Cytosine) and G (guanine). This is the __
manual sanger method.
- You wll use a capillary tube. The thing is it already has fluorescein agent or may nag fluorescein na sa kanya. You will load the gel then everytime it cuts yung ddNTP nya contains certain colorimetric agent. Your automated only needs a single tube or gel. Ngayon mahahati na sya to the specific na nucleotide nya. Kaya lang, paano mo malalaman na G, C, A or T sya? Your DNA sequence will already have a probe. They will emit a certain color. Kung kulay red sya, T yon. Pag blue, C. Pag green, A. Tapos pag yellow, G tapos mataas sya.
- Mas mabilis sya.You don’t need 4 tubes, you only need one.
automated sanger method.
- is a second generation DNA Sequencing Technique
- the nucleotides carry a chemical group that blocks elongation by DNA polymerase but which can be removed chemically
- Sequencing is then carried out as follows: the four fluorescently labeled nucleotides along with DNA polymerase are added to billions of DNA clusters immobilized on a slid
Illumina
- DNA Sequencing Uses the Changes in pH
- hydrogen ion (H+) is released (along with pyrophosphate) each time a nucleotide is incorporated into a growing DNA chain and the ion torrent method is based on this simple fact
- DNA sequence on a given bead can be read from the pattern of pH changes observed as nucleotides are washed over them.
Ion Torrent
- bypass the DNA amplification steps altogether and determine the sequence of single molecules of DNA
- you only need a single chromosome from one cell and you don’t need to amplify.
- It will bypass the PCR portion of amplifying it.
It uses electrical signals.
“Third-generation” technologies
DNA Cloning Allows Any Protein to be Produced in Large Amounts
- What we do in cloning is we overexpressed mRNA or the overexpressed protein. The overexpressed mRNA, you can use that in the complementary DNA library. The overexpressed protein, you can use this for protein analysis.
