MMD FINAL EXAM OUTLINE.pdf

Molecular Mechanisms of Diseases Fall 2023 The third exam will mainly focus upon the following topics: Heme biosynthesis, Molecular Endocrine, Signal Transduction and Receptors involved, DNA qReplication, RNA Transcription, and protein translation, Medical Genetics, Techniques in Biochemistry, the B...

Molecular Mechanisms of Diseases Fall 2023 The third exam will mainly focus upon the following topics: Heme biosynthesis, Molecular Endocrine, Signal Transduction and Receptors involved, DNA qReplication, RNA Transcription, and protein translation, Medical Genetics, Techniques in Biochemistry, the Biochemistry of Cancer, and Nutrition. And remember, we are layering knowledge; so be prepared. Review: Lecture Review for third Exam: CHAPTER 9: 1. What are porphyrins and where would you find them? ● Porphyrins nitrogen containing compounds cyclic compounds that bind to metal ions (Fe2+), (Fe3+) ● Porphyrin consists of 4 pyrrole rings (N-containing rings) linked together by methylene bridges ● Heme is the best-known example (iron porphyrin). Heme is the prosthetic ● Heme proteins are rapidly synthesized and degraded to keep up with losses through normal turnover of red blood cells and other hemeproteins 2. HEME biosynthesis, its precursors, and locations of reactions. ● Heme biosynthesis takes place in mitochondrion and cytosol ● Heme synthesis takes place mainly in liver and bone marrow ● Precursors: glycine, succinyl CoA (a TCA intermediate) ● The metabolic pathway is regulated at the first step, which is catalyzed by the enzyme δ-aminolevulinic acid (δ-ALA) synthase located in the mitochondria: 2 isoforms ○ X chromosome linked ● Intermediates in the pathway include: ○ δ-aminolevulinic acid (δ-ALA/5-ALA) ○ Porphyrinogens ● In the cytosol: ○ 2 molecules of δ-ALA/5-ALA condense to form a molecule containing a pyrrole ring: porphobilinogen (PBG) ○ Then 4 PBG molecules combine to form a linear tetrapyrrole compound ○ Cyclizes to yield uroporphyrinogen III and then coproporphyrinogen III ● In the mitochondria: ○ Heme exits mitochondria and associates with globin chains ○ ● There are a series of decarboxylation and oxidation of side chains: yields protoporphyrin IX ○ At the final stage iron (Fe2+) is added by ferrochelatase to protoporphyrin IX to form heme Heme synthesis regulation ○ When heme is abundance in the cell, heme biosynthesis will decrease ○ Two ways of repression: HEME represses the synthesis of ALA synthase by transcriptional regulation and inhibits the activity of ALA synthase ○ When heme levels fall in the cell, heme biosynthesis is accelerated 3. Be familiar with the deficiency of HEME biosynthesis and some clinical manifestations. ● Porphyrias: ○ Rare deficiency of an enzyme in the heme biosynthetic pathway ○ Some are autosomal dominant disorders others are recessive ○ Leads to an accumulation of porphyrin intermediates ○ Excretion of the intermediates in the urine and feces ○ 3 types: hepatic, erythropoietic, and both (classification is based on location of the enzyme deficiency, clinical results vary) ○ Disease manifestation of Porphyrias depend on where the cycle is disrupted ○ Features of abdominal pain, psychiatric manifestations, neurologic manifestations generally occur – enzyme defect upstream of tetrapyrroles ○ Skin rashes and sunlight sensitivity are usually enzyme defect associated with accumulations of tetrapyrrole intermediates ● Lead poisoning ○ Ferrochelatase and ALA dehydratase are particularly sensitive to inhibition by lead ○ Protoporphyrin and ALA accumulate in urine ● Acute intermittent porphyria ○ An acute disease caused by a deficiency in hydroxymethylbilane synthase ○ Porphobilinogen and δ-aminolaevulinic acid accumulate in the urine ○ Urine darkens on exposure to light and air ○ Patients are NOT photosensitive ● Erythropoietic protoporphyria ○ The disease is due to a deficiency in ferrochelatase ○ Protoporphyrin accumulates in erythrocytes, bone marrow, and plasma ○ Patients are photosensitive ● Variegate porphyria ○ An acute disease caused by a deficiency in protoporphyrinogen oxidase ○ Protoporphyrinogen IX and other intermediates prior to the block accumulate in the urine ○ Patients are photosensitive ● Hereditary coproporphyria ○ ○ ● ● An acute disease caused by a deficiency in coproporphyrinogen oxidase Coproporphyrinogen III and other intermediates prior to the block accumulate in the urine ○ Patients are photosensitive Porphyria cutanea tarda ○ A chronic disease caused by a deficiency in uroporphyrinogen decarboxylase ○ Uroporphyrin accumulates in the urine ○ It is the most common porphyria ○ Patients are photosensitive: complement/mast cell; sun exposed hands Congenital erythropoietic porphyria ○ This disease is caused by a deficiency in uroporphyrinogen III synthase ○ Uroporphyrinogen I and coproporphyrinogen I accumulate in the urine ○ Patients are photosensitive 4. Comprehend the structure of hemoglobin and its capacity to carry oxygen. ● Hemoglobin ○ Globular protein: tetramer consisting of 4 polypeptide chains ○ Adult Hb, hemoglobin A1, has 2 alpha chains and 2 beta ○ Each of the 4 polypeptide chains has a heme associated ● Heme ○ Porphyrin ring containing iron (Fe2+, ferrous) in the center ○ Iron is bound to N’s of porphyrin ring and to a histidine residue of the polypeptide chain ○ Iron can form an additional bond with O2 ● Quaternary Structure of HB ○ Deoxy Hb= taut structure ■ Alpha-beta dimers are connected by many salt bridges ○ Oxy Hb= relaxed structure ■ Fewer salt bridges ■ 2,3 BPG released from its association with the Beta chains ● Myoglobin ○ An oxygen storage protein ○ Located in the cytosol of skeletal, cardiac and some smooth muscle cells ○ Binds O2 that has been released by Hb in the tissue capillaries and subsequently diffused into tissues ○ This Stored O2 is available to the mitochondrion ● Oxygen binding curve ○ Sigmoid curve shows cooperative binding of O2 and Hb ○ The binding of O2 to 1 polypeptide chain of Hb increases the binding of another O2 to the 2nd polypeptide chain, etc ○ As O2 binds, the conformation of Hb changes from the deoxy conformation to the oxy conformation ● ● ○ Curve shows how effectively Hb releases its O2 to the tissues Factors causing a shift to the right on hemoglobin curve ○ Decreased O2 affinity, greater release of O2 to tissues ○ Decreased pH, increased pCO2, increased 2,3 BPG Factors causing a shift to the left on hemoglobin curve ○ Increased O2 affinity, less O2 released to tissues ○ Increased pH, decreased pCO2, decreased 2,3 BPG 5. Diseases associated with hemoglobin: Sickle cell anemia and Thalassemia’s. ● Sickle cell anemia ○ Autosomal recessive disease ○ Sickle cell disease (sickle cell anemia): homozygous (both copies of mutated β gene) ○ Sickle cell trait: heterozygous (1 copy of mutated β gene) – a variant of the beta-globin gene called sickle hemoglobin (Hb S) ○ ~10% of African Americans have sickle cell trait ○ Chronic hemolytic disease occurring ○ Hemolytic anemia and recurrent pain ○ Vaso-occlusive lead to pain in the chest, bones, abdomen ○ Mis-shaped, sickled red blood cells become trapped in small capillaries and block circulation ○ Precipitating factors – low oxygen (exercise, high altitudes), high pCO2, low pH, concentration of HbS ● Thalassemia’s ○ An imbalance in the synthesis of one of the two globin chains ○ As a consequence of abnormal or non-functioning globin genes, the insufficient synthesis of either hemoglobin chain leads to an anemia ○ β thalassemia: decreased synthesis of β globin chains ○ Homozygous β-thalassemia: Cooley’s anemia 6. HEME degradation and Hyperbilirubinemia and jaundice. ● Heme degradation ○ After 4 months, RBC’s are turned over in the blood stream ○ The first step in this process of turnover and heme degradation is the formation of bilirubin within the macrophages of the reticuloendothelial system ○ Bilirubin is delivered to the liver bound to plasma albumin ○ Heme→ biliverdin→ bilirubin ○ The iron of the heme is recycled, rather than excreted from the body ○ In the hepatocytes, bilirubin is conjugated. This refers to the conversion of bilirubin to bilirubin di-glucuronide ○ ● ● Conjugated bilirubin is transported through the bile ducts of the liver and into the bile – bile emulsifies fats ○ Some of the conjugated bilirubin is converted to urobilinogen in the intestine by bacteria ○ Urobilinogen can then be converted to stercobilin which gives feces their brown color ○ Some Urobilinogen is absorbed into the portal circulation ○ Some will travel back to the liver and be re-secreted in the bile ○ Some Urobilinogen makes its way to the kidneys where it is converted to urobilin and excreted in the urine Hyperbilirubinemia ○ Normal serum bilirubin levels = <0.8 mg/dl, most of which is unconjugated ○ Hyperbilirubinemia, with levels >2.5 mg/dl, leads to jaundice (icterus) Jaundice ○ Is a manifestation of skin and eye yellowing (sclearae) deposition of bilirubin from increased bilirubin levels in the blood ○ In cases where massive hemolysis takes place, the liver is unable to conjugate the heme fast enough, leading to high unconjugated bilirubin in the bloodstream ○ Damage to liver cells has occurred, unconjugated bilirubin levels in the blood will rise ○ Inefficiency in conjugation activity ○ Some of the bilirubin entering the liver via the portal circulation from the gut will not be re-secreted into the bile. It will find its way to the kidney, darkening the urine while the stool is pale in color ○ In bile ducts-obstruction, bilirubin will not pass to the intestine, yielding a pale stool and darkening the urine due to delivery of conjugated bilirubin from the bloodstream ○ Causes of jaundice ■ Prehepatic: ● Hemolysis ● Autoimmune diseases ● Abnormal hemoglobin ■ IntraHepatic: ● Infection ● Drug ● Neonatal (bilirubin uridine diphosphate glucuronosyl transferase) ■ PostHepatic ● Intrahepatic bile duct obstruction ● Extrahepatic bile duct obstruction ● Gallstones 7. ● ● ● ● Nucleotide synthesis and their components in synthesis: Amino acids, Phosphoribosylpyrophosphate (PRPP, activated ribose), Tetrahydrofolate (FH4) and diseases associated with deficiencies therein. Purine and pyrimidine nucleotides synthesized de novo from amino acids and carbohydrate precursors (remember the pentose phosphate pathway) Purine synthesis: ○ 1) Amination of PRPP via displacement of its PPi with amine group. PRPP is also a precursor for pyrimidine biosynthesis ○ 2) Multiple phosphorylations via ATP which activate CO groups for displacement with amine group. Sources of atoms include Gly, Gln, Asp, THF-formyls, CO2 ○ 3) Resulting IMP is aminated to yield AMP and GMP (ribose sugars). ○ 4) As with the pyrimidines, the AMP and GMP are easily converted to ATP and GTP via phosphotransfer by specific and general kinases Pyrimidine synthesis: ○ (1) Synthesis of carbamoyl phosphate from bicarbonate and Gln-derived ammonia ○ (2) Formation of orotate ring from carbamoyl phosphate & aspartate ○ (3) Coupling to activated ribose PRPP to form Orotidylate (Orotidine 5'-monophosphate) (OMP) ○ (4) Decarboxylation of OMP to form uridine (UMP): Important product!! ○ (5) Phosphotransfer to UMP to form UTP ○ (6) Amination of UTP to form CTP Deficiencies: ○ HGPRT deficiency: Lesch-Nyhan Syndrome ■ sex -linked congenital defect ■ Mostly Males ■ X linked recessive inherited disorder associated with virtually complete deficiency of hypoxanthine-guanine phosphoribosyl-transferase and therefore, the inability to salvage hypoxanthine or guanine ■ The enzyme deficiency results in increased levels of PRPP and decreased levels of IMP and GMP, causing increased de novo purine synthesis ■ This results in the excessive production of uric acid, plus characteristic neurologic features, including self-mutilation and involuntary movements ■ Neurological abnormalities - spasticity, mental retardation, aggressive and destructive behavior including self harming ■ The excess of PRPP activates amidophosphoribosyl transferase (this is reaction #2 of purine synthesis) ○ Gout ■ Characterized by hyperuricemia with recurrent attacks of acute arthritic joint inflammation caused by deposition of monosodium urate crystals ■ Urate crystals are deposited in cartilage, joints and in kidneys ■ Gout can be genetic and may be due to overproduction of uric acid, excessive purine nucleotide synthesis, or defective renal excretion of uric acid ■ Gout can also be secondary to other diseases like cancer ■ In gout, the hyperuricemia results primarily from the underexcretion of uric acid. Overproduction of uric acid is less common, and known causes involve certain inborn errors of metabolism or increased availability of purines ○ Adenosine deaminase (ADA) - look at question #10 8. Understand how cells acquire nucleotides: de novo, ingestion, salvage pathway. ● de novo: ○ de novo synthesis of inosine monophosphate (IMP): a purine derivative containing the base hypoxanthine ○ Performed nucleotides obtained from the diet ● Salvage pathways ○ Recycle of endogenous nucleic acids ● Ingestion 9. Know the reason some intracellular pathogens that are blocked in the de novo pathways can survive as intracellular pathogens. ?? ● 10. ● ● ● ● ● ● ● Be familiar with Adenosine Deaminase Deficiency resulting in immunodeficiency. Adenosine deaminase is one of the enzymes of purine nucleotide degradation. Autosomal recessive6 A deficiency of this enzyme causes severe combined immunodeficiency (SCID). There is a lack of T cells and B cells, making it impossible to fight infection. Mechanism ○ Deoxyadenosine is phosphorylated to yield extremely high levels of dATP ○ This concentration of dATP inhibits ribonucleotide reductase ○ Result: Synthesis of the other dNTPs is greatly reduced. ○ DNA synthesis is inhibited ○ Cell proliferation decline ○ SCIDs The deficiency causes a type of severe combined immunodeficiency (SCID), involving T cell and B cell depletion (lymphocytopenia) Untreated ADA-deficient children usually die before two years of age from overwhelming infection CHAPTER 10 11. What is the significance of homeostasis by way of the endocrine system? ● ● Endocrine system influences other organ systems in maintaining homeostasis through cellular metabolism, growth and development Endocrine system homeostasis – secretes hormones into the blood, slower response, more prolonged response 12. Remember receptor/hormone specificities. ● ● ● Hormones that bind to cell membrane receptors ○ These are lipophobic/hydrophilic Hormones that bind to intracellular receptors and activate genes ○ These are lipophilic/hydrophobic Hormones (the ligands) bind to receptors specific for them only ● These receptors may be present in the cell membrane or within the cell itself 13. Be familiar with the classification of hormones: lipophilic and lipophobic. ● Classes of hormones 1. Peptides and proteins - Glycoproteins 2. Amino acid derivatives - Catecholamines, Thyroid hormones, Melatonin 3. Steroids - Sex steroids, Corticosteroids ● Hormones may be categorized as: ○ Lipophilic / Hydrophobic (nonpolar) – fat-soluble ■ Steroid hormones and thyroid hormones ■ Travel on transport proteins in blood ■ Tend to act over brief time period ■ Circulate in the blood bound to transport proteins ■ Dissociate from carrier at target cells ■ Pass through the cell membrane ■ Bind to an intracellular receptor, either in the cytoplasm or the nucleus ■ Hormone-receptor complex binds to hormone response elements in DNA ■ Regulate gene expression ○ Hydrophilic / Lipophobic (polar) – water-soluble ■ All other hormones - peptide, protein, glycoprotein, and catecholamine hormones ■ Freely soluble in blood ■ Bind to extracellular receptors ■ Tend to have much longer active period ■ Too large or polar to cross cell membrane ■ Bind to receptors on plasma membrane ■ Initiate signal transduction pathways ■ Activation of protein kinases (activate or deactivate intracellular proteins by phosphorylation) ■ Production of second messengers Hormones and Homeostasis ● ● ● ● Production of most hormones controlled by two things: ○ Negative feedback ■ Sensitive to either the condition it regulates or the blood level of the hormone that it is producing ○ Action of other hormones ■ Insulin and glucagon The blood concentration of a substance prompts an endocrine gland to secrete its hormones ○ Ex: The parathyroid gland secretes a hormone when blood Ca+2 level falls below normal ○ Osteoclasts respond to hormone by slowly releasing Ca+2 from bone ○ It takes time for the response, but it is long-lasting Example of negative feedback ○ As the blood glucose level rises, the pancreas secretes insulin ○ Insulin causes the liver to store glucose, and glucose is removed from the blood ○ The stimulus for insulin production is, thereby, inhibited ○ The pancreas stops secreting insulin Hormone regulation by release of an antagonistic hormone ○ ○ The effect of insulin is offset by the secretion of glucagon Insulin lowers the blood sugar level, while glucagon raises it RAA Pathway Atrial Natriuretic Hormone (ANH) ● ● ● ● ● ● Hormone secreted by the atria when the cardiac cells are stretched due to increased blood volume Another hormone regulating sodium Secreted by right atrium of heart in response to stretching - indicates increased blood volume Inhibits renin secretion by juxtaglomerular apparatus Inhibits aldosterone release Promotes diuresis and sodium excretion - natriuresis The Action of Hormones ● ● ● Hormones act on target cells ○ Only targets with receptor can respond Hormones fall into two chemical classes ○ Peptide hormones – peptides, proteins, glycoproteins or modified amino acids ○ Steroid hormones – same complex of four rings, but varying side chains Hormones function as chemical signals. ○ Typically affect the metabolism of target cells with appropriate receptors ■ For peptide hormones, receptors on cell surface ■ For steroid hormones, receptors inside cell (cytoplasm or nucleus) Different Classes of Hormones ● Intracellular receptors have a domain for the hormone binding (ligand binding domain). ○ They also have a DNA binding domain which is covered by a heat shock protein (DNA binding domain) ○ The receptor hormone complex will translocate to the nucleus where the exposed DNA binding domain can bind to DNA ○ Long process (45 mins) – not as fast as the cell membrane associated secondary messenger systems in general ● Hormones using cAMP dependent pathways ○ Anti-Diuretic Hormone (ADH) – is secreted by the hypothalamus. It promotes water retention by the kidneys ○ Growth Hormone Releasing Hormone (GHRH) – is secreted by the hypothalamus. It stimulates the synthesis and release of Growth Hormone (GH) ○ Corticotropin Releasing Hormone (CRH) – is secreted by the hypothalamus. It stimulates the synthesis and release of Adrenocorticotropic Hormone (ACTH) ○ Adrenocorticotropic Hormone (ACTH) – produced by anterior pituitary gland. It stimulates the synthesis and release of Cortisol ○ Thyroid Stimulating Hormone (TSH) – Secreted anterior pituitary gland. It stimulates the synthesis and release of a thyroid hormone ○ Luteinizing Hormone (LH) – Released by the anterior pituitary. It stimulates follicle maturation and ovulation. In men, it stimulates testosterone synthesis and spermatogenesis ○ Follicle Stimulating Hormone (FSH) – Released by the anterior pituitary. It stimulates follicular development in women and spermatogenesis in males ○ Parathyroid Hormone (PTH) – Secreted by the Parathyroid gland. It increases blood calcium levels ○ Calcitonin – Secreted by the Parafollicular Cells of the thyroid. It lowers blood calcium levels ○ Glucagon –Secreted by the alpha cells of the Pancreas. It stimulates glycogenolysis. ○ Epinephrine –Released by the adrenal medulla. It stimulates a variety of tissue and organ responses including glycogenolysis, increases in heart rate, relaxation of bronchial smooth muscle ○ Gαi inhibits the production of cAMP from ATP. ○ Insulin works through Gαi (inhibitory) second messenger proteins. ● Hormones using PIP2/Ca2+ dependent pathways ○ Anti diuretic Hormone (ADH) -Released by the Posterior Pituitary. It causes vasoconstriction, platelet aggregation, uterine contraction, glucocorticoid release ○ Thyrotropin Releasing Hormone (TRH) – Produced in the hypothalamus. It stimulates the synthesis and release of TSH ○ Thyroid Stimulating Hormone (TSH) – Released from the anterior pituitary. It stimulates the synthesis and release of Thyroid Hormone ○ Angiotensin II – Final processing occurs in the lungs. It stimulates Aldosterone synthesis and release ○ Gonadotropin Releasing Hormone (GnRH) – Produced by the hypothalamus. It stimulates the synthesis and release of FSH and LH What is AIS? ● ● A genetic condition: affected people have male chromosomes and male gonads with complete or partial feminization of the external genitals Inheritance: X-linked recessive disease with a mutation in the Androgen Receptor (AR) gene resulting in: ○ Functioning Y sex chromosome ● ● ○ Abnormality on X sex chromosome Types ○ CAIS (completely insensitive due to AR gene) ■ External female genitalia ■ Lacking female internal organs ■ Grade 7: CAIS ● Female genitalia with little to no pubic/underarm hair ○ PAIS (partially sensitive-varying degrees) ■ External genitalia appearance on a spectrum (male to female) ○ MAIS (mildly sensitive, rare) ■ Impaired sperm development and/or impaired masculinization Also called Testicular Feminization 14. And understand how the different classified hormones interacts cellularly. Some hormones are lipophobic and acts externally to the cell. While lipophilic hormones interact with intercellular receptors. ● Two Main Types: ○ Lipophobic/hydrophilic ■ Hormones that bind to cell membrane receptors ■ These ligands (the hormones) must bind to a cell membrane receptor to get its message across to the cell interior ■ The cell membrane receptor will be a Tyrosine Kinase monomer ■ Signal transduction ■ CAN’T cross the cell membrane directly ■ A method to get your signal across the cell membrane ○ Lipophilic/hydrophobic ■ Hormones that bind to intracellular receptors and activate genes ■ For some peptide hormones (like insulin) the receptor itself is a kinase ● Can directly phosphorylate intracellular proteins, altering cellular activity ■ For other peptide hormones (like growth hormone) the receptor itself is not a kinase ● Rather, it activates intracellular kinases ■ Able to cross the cell membrane and the nuclear membrane ■ Binds to a target receptor in the cytosol or the nucleus 15. We spoke about differences between G-couple protein receptors and Tyrosine kinase receptors. ● ● Hormones that bind to cell membrane receptors ○ Tyrosine kinase activators ○ Hormones that act through G-proteins G Protein-Coupled Receptors ○ Second-messenger systems ■ Receptors are linked to a second-messenger-generating enzyme via membrane proteins called G proteins ● G protein–coupled receptors (GPCRs) ○ ○ ● ● 16. ● ● ● 17. When the G protein activates the enzyme, the second-messenger molecules increase Cellular response depends on the type of G protein activated ■ Some activate while others inhibit their second-messenger-generating system ■ A single hormone can have distinct actions in 2 different cells ○ Single largest category of receptor type in animal cells is GPCRs ○ Receptors act by coupling with a G protein ○ G protein provides link between receptor that receives signal and effector protein that produces cellular response ○ All G proteins are active when bound to GTP and inactive when bound to GDP ○ Effector proteins are usually enzymes G Protein and cyclic nucleotide signaling ○ Another signal transduction system ○ For lipophobic molecules ○ These ligands must bind to a cell membrane receptor to get its message across to the cell interior ○ The cell membrane receptor is a G Protein Receptor ○ The G Protein receptor, when activated, will interact with the G Protein ■ The G Protein will lose its alpha sub unit ■ The alpha sub unit will migrate and bind to Adenylyl Cyclase ○ Examples: receptors for glucagon, angiotensin, vasopressin (antidiuretic hormone-ADH) Tyrosine Kinase ○ Signal transduction system ○ For lipophobic/hydrophilic molecules ○ Ligands must bind to a cell membrane receptor to get its message across to the cell interior ○ The cell membrane receptor will be a tyrosine kinase monomer ○ Receptors with intrinsic tyrosine kinase activity are capable of autophosphorylating themselves as well as phosphorylation of other substrates Understand the cAMP pathway and the significance of Phosphodiesterase. Adenylyl Cyclase will produce a secondary messenger known as cAMP cAMP is broken down by Phosphodiesterase cAMP activated Protein Kinase A Review secondary messengers. ● cAMP ● Second-messenger systems ○ Receptors are linked to a second-messenger-generating enzyme via membrane proteins called G proteins ■ G protein–coupled receptors (GPCRs) ○ When the G protein activates the enzyme, the second-messenger molecules increase ● Different receptors can produce the same second messengers ○ Hormones glucagon and epinephrine can both stimulate liver cells to mobilize glucose ■ Different signals, same effect ■ Both act by same signal transduction pathway ● 2 effectors ○ ○ Adenylyl cyclase ■ Produces cAMP ■ cAMP is generated and then broken down by Phosphodiesterase ■ cAMP binds to and activates the enzyme protein kinase A (PKA) ■ PKA adds phosphates to specific proteins Phospholipase C ■ PIP2 is acted on by effector protein phospholipase C ■ Produces IP3 plus DAG ■ Both act as second messengers 18. Remember, Remember, Remember the role of phosphodiesterase. Be able to apply your knowledge to circumstances. ● cAMP is simultaneously being degraded by Phosphodiesterase ● cellular cAMP levels will be low ● Break downs cAMP to regulate ** 19. Understand the Phospholipase C pathway and the importance of calcium as a secondary messenger. ● Phospholipase C pathway ○ Splits PIP2 into DAG and IP3 ○ IP3 binds to ligand gated Calcium Channels on Smooth ER which initiates release of calcium ■ Calcium binds to Protein Kinase C, fully activating it ○ DAG activated Protein Kinase C ● Calcium ○ Ca2+ serves as second messenger ○ Intracellular levels normally low ○ Extracellular levels quite high ○ IP3 binds to receptors on the ER, signals the release of Ca2+ ○ Ca2 initiates some cellular responses by binding to calmodulin 20. Be sure of the difference of the types of diabetes: Type I, Type II, and diabetes insipidus. Know the mechanisms of cause. ● Diabetics cannot take up glucose from blood ● Diabetes Type I ○ Type I (insulin-dependent diabetes) ○ Individuals lack insulin-secreting b cells ○ Treated by daily injections of insulin ● Diabetes Type II ○ Type II (noninsulin-dependent diabetes) ○ Most patients have this form ○ Very low number of insulin receptors ○ Treated by diet and exercise ● Diabetes Insipidus ○ Diabetes insipidus is a disorder of a large volume of urine (diabetes) that is hypotonic, dilute ○ This is opposed to the hypertonic and sweet urine of diabetes mellitus (honey) ○ ○ Diabetes insipidus is caused by absence of the hormone vasopressin or inadequate response to vasopressin Patients with diabetes insipidus who have inadequate thirst can rapidly become dehydrated and develop severe hypernatremia with devastating effects on the central nervous system (CNS) CHAPTER 11 21. During lecture we spoke about the Griffith's experiment and other experiments contributing support that DNA is the inheritable molecular information. ● Griffith (1927) ○ Provided foundation for Avery, Macleod, and McCarty’s research ○ Showed avirulent strains of Streptococcus pneumoniae could be transformed to virulence ○ Speculated transforming principle could be part of polysaccharide capsule or compound required for capsule synthesis ● ● Avery, Macleod, and McCarty (1940s) ○ First direct experimental proof that DNA is a biomolecule responsible for heredity ○ Published results of their experiment DNase (deoxyribonuclease) utilized to destroy transforming activity ○ Demonstrated transforming principle was DNA, not protein ○ Control: IIIS contains active factor Hershey and Chase ○ Used Escherichia coli and bacteriophage T2 ○ Demonstrated that DNA, not protein, is the genetic material ○ ○ Used radioisotopes 32P and 35S Demonstrated DNA enters bacterial cell during infection and directs viral reproduction 22. Review DNA replication and RNA transcription. ● DNA Replication ○ Replication of DNA will occur during the S (Synthesis) phase of cell replication ○ The DNA bases on each strand act as a template to synthesize a complementary strands ○ The process is semiconservative ○ The new double-stranded DNA contains one old strand from the template and one new synthesized complementary strand ○ The process is bidirectional. The replication begins at an origin site and simultaneously moves out in both directions ○ Prokaryotes have one origin site which is AT rich called OriC ○ Eukaryotes have multiple sites of origin on each chromosome ○ DNA polymerase adds new nucleotides. DNA ligase joins the DNA segments together ○ This allows for replication of the much larger eukaryotic genome to occur much more quickly ○ Initiation: ■ Primase (a type of RNA polymerase) builds an RNA primer (5-10 ribonucleotides long) ■ DNA polymerase attaches onto the 3’ end of the RNA primer ○ Initiation: The Replication Fork ■ In Prokaryotes, DnaA binds upstream of OriC and unwinds it in preparation for helicase ○ Helicase and Topoisomerase ■ Helicases unwind the helix ■ The Single stranded binding proteins keep the strands from associating together again ■ Topoisomerase prevent extreme supercoiling of the helix that would result from the unwinding ○ Elongation: DNA polymerase reads (3’ to 5’) EACH strand and elongates EACH new complimentary strand (5’ to 3’) ○ Okazaki Fragment ■ As more and more of the helix is unwound, synthesis on the lagging strand begins from another primer by DNA Polymerase δ ■ These short fragments formed in this process are called Okazaki fragments ■ The RNA Primer will be removed by the nuclease RNase H ■ The gaps will be filled in by DNA Polymerase δ ■ The resulting Okazaki fragment will be joined together by DNA Ligase ○ ● RNA Transcription ○ Transcription Initiation ■ ○ ○ ○ ○ RNA polymerase binds to a region on DNA known as the promoter, which signals the start of a gene ■ Promoters are specific to genes ■ RNA polymerase does not need a primer ■ Transcription factors assemble at the promoter forming a transcription initiation complex – activator proteins help stabilize the complex ■ Gene expression can be regulated (turned on/off or up/down) by controlling the amount of each transcription factor Promoters and transcription factors of RNA polymerase II ■ The typical promoter for RNA polymerase II has a short initiator sequence, consisting mostly of pyrimidines and usually a TATA box about 25 bases upstream from the start point. ■ RNA Polymerase II does not recognize the promoter sequence by itself 1. TFIID finds and binds to the TATA box 2. TFIIF brings the RNA Polymerase II to the promoter site 3. TFIIH opens the DNA double strand 4. After an activation step requiring ATP-dependent phosphorylation of the RNA polymerase molecule, the polymerase can initiate transcription at the start point. Transcription Elongation ■ RNA polymerase unwinds the DNA and breaks the H-bonds between the bases of the two strands, separating them from one another ■ Base pairing occurs between incoming RNA nucleotides and the DNA nucleotides of the gene (template) ■ Recall RNA uses uracil instead of thymine Transcription Termination ■ A region on DNA known as the terminator signals the stop of a gene ■ RNA polymerase disengages the mRNA and the DNA Posttranscriptional modification of mRNA ■ In EUKARYOTES mRNA contains a cap structure on the 5’ end of the transcript. ■ This cap is a methylated Guanine Triphosphate attached to the hydroxyl of the 5’ terminal end of the mRNA ■ This cap protects the mRNA from degradation ■ The 3’ end of mRNA has a Poly adenylated tail (AAAAA……) – protects the mRNA from degradation ■ Newly synthesized mRNA will contains unwanted base sequences and is referred to as heterogeneous nuclear RNA (hnRNA) ■ The unwanted sequences are called introns ■ They are removed from the transcript by a spliceosome protein ■ This leaves behind coding sequences called exons ■ The remaining exons are linked together ■ This process is called splicing ■ Splicing occurs before the message leaves the nucleus 23. Be familiar with the antiparallel nature of DNA and know how a phosphodiester bond in is formed in the 5’ to 3’ direction. ● The #5 carbon of one sugar will form a phosphodiester bond with the #3 carbon of the sugar above it – ribose’s attach to other ribose’s and deoxyribose’s attach to other deoxyribose’s – this forms a sugar backbone for a string of nucleic acids with a 5’ end and a 3’ end ● There is a phosphoester linkage at the #3 carbon of the molecule above and a phosphoester linkage at the #5 carbon below – results in a chain of nucleic acids with a 5’ end and a 3’ end ● The two complimentary chains (associated via the hydrogen bonding of the base pairs) are antiparallel. One chain runs from 3’ to 5’, the complimentary chain runs from 5’ to 3’ 24. Histones: what is the purpose of Histones? ● Histone modifications ○ Complex topic ○ Histones can be modified ○ Acetylation, methylation, phosphorylation are all possible modifications ○ In general acetylation is correlated with active sites of transcription ○ Some transcription coactivators have been shown to be histone acetylases ○ Transcription is increased by removing higher order chromatin structure that would prevent transcription 25. Compare and contrast the reverse transcriptase to RNA polymerase and to DNA polymerases. What direction do all these enzymes read their templates? ● ● DNA and RNA ● DNA ○ ○ ○ ○ ● RNA ○ ○ ○ DNA and RNA polymerase ○ Only reads in the 3’ to 5’ direction ○ Can only add to a new strand already present and do so in the 5’ to 3’ direction ○ 3’ end has a free deoxyribose ○ 5’ end has a free phosphate ○ DNA Polymerase READS DNA in the 3’ to 5’ direction to build a new strand ○ The new strand ELONGATES in the 5’ to 3’ direction Reverse transcriptase – reads RNA and makes DNA (in the 5’ to 3’ direction) Deoxyribose sugar is used for DNA OH is ABSENT on #2 carbon DNA is generally double stranded Thymine will base pair with Adenine Ribose sugar is used in RNA OH is PRESENT on #2 carbon Uracil will base pair with Adenine ● ● ○ RNA is generally single stranded Adenine base pairs with Thymine via 2 hydrogen bonds Cytosine base pairs with Guanine via 3 hydrogen bonds 26. We spoke about reverse transcriptase in the context of telomeres and in retroviruses, specifically, HIV-1. Understanding how HIV-1 enters and replicates in a cell and be familiar with various routes of therapy for HIV-1 infection. ● ● ● ● ● Telomerase is an example of a reverse transcriptase – it reads RNA and makes DNA (in the 5’ to 3’ direction) Reverse transcriptase are seen in virology The retrovirus HIV carries it’s genome as ssRNA and uses reverse transcriptase to produce viral DNA (in the 5’ to 3’) direction the viral DNA is then incorporated into the host genome as the host’s DNA is transcribed, viral RNA is produced eventually yielding new viral proteins Retrovirus inhibition ○ This is an enormous area of research. ○ Retroviral therapy has many therapeutic targets. ○ Approach: drugs that block the assembly of virus Use of nucleoside analogs ○ A modified nucleoside that has its OH group removed from the #3 carbon of the deoxyribose ring results in 2’,3’ dideoxyinosine (didanosine) which suppresses the replication of the human HIV reverse transcriptase inhibitor class ○ The modified nucleoside Thymidine has had its OH group removed from the #3 Carbon of the deoxyribose ring and replaced with N3 results in the drug AZT (Zidovudine) ○ Chain growth of DNA transcribed by reverse transcriptase can be inhibited by incorporation of these modified nucleoside analogs ○ The non-functional nucleoside analogs compete with the naturally occurring nucleotides for incorporation into the viral DNA strand ○ The incorporation of non-functional nucleoside analogs stops chain elongation due to its inability to bind with other adjacent nucleotides CHAPTER 12 ● ● ● 27. Understand the significance of a karyotype in diagnostics. A karyotype is a photographic inventory of an individual’s chromosomes A karyotype is an ordered display of magnified images of an individual’s chromosomes arranged in pairs Karyotypes ○ Are often produced from dividing cells arrested at metaphase of mitosis ○ Allowing for the observation of ■ Homologous chromosome pairs, ■ Chromosome number ■ Chromosome structure ■ Therefore, karyotypes can render information on chromosomal abnormalities 28. Variations in Chromosome Number: Aneuploidy in trisomy and monosomy. ● ● ● Aneuploidy ○ Variations in chromosome number ○ Individual may gain or lose one or more chromosomes (not entire set) ■ Monosomy: Loss of single chromosome in diploid genome ■ Trisomy: Gain of single chromosome ○ Mechanism: ■ Nondisjunction ● Gives rise to chromosomal variation ● Paired homologs fail to disjoin during segregation ● Nondisjunction during meiosis I or II Monosomy ○ Loss of one chromosome ○ Produces 2n - 1 complement ○ Although one copy remains, can be lethal, organism is not viable ○ Monosomy unmasks recessive lethal ○ Haploinsufficiency: When one copy is not sufficient for organism to survive Trisomy ○ 2n + 1 chromosomes ○ Addition of chromosome produces more viable organisms ○ Trisomies for autosomes are often lethal 29. Be able to describe the non-disjunction process defining aneuploidy. ● Nondisjunction ○ Gives rise to chromosomal variation ○ Paired homologs fail to disjoin during segregation ○ Nondisjunction during meiosis I or II Human Aneuploidy ● ● ● ● Patau syndrome (trisomy 13) Edwards syndrome (trisomy 18) ○ Both trisomies survive to term ○ Manifest severe malformations and early lethality ○ Shows abnormal karyotype 30. Understand the significance of trisomy twenty-one and mechanism of occurrence and methods of diagnostics. Down syndrome: Trisomy of chromosome 21 ○ Extra chromosome (three number 21s) ○ Has 12 to 14 characteristics ○ Affected individuals express 6 to 8 on average ○ Produces a characteristic set of symptoms, including ■ Characteristic facial features ■ Short stature ■ Heart defects ■ Susceptibility to respiratory infections, leukemia, and Alzheimer’s disease, and ■ Varying degrees of developmental disabilities ○ The incidence increases with the age of the mother. DSCR: Down syndrome critical region ○ Critical region of chromosome 21 ○ Genes are dosage sensitive ○ Responsible for many phenotypic-associated syndromes 31. Know the significance of monosomies in the population and the reason there are so few in the population. ● ● Trisomies ○ Often found in spontaneously aborted fetuses ○ Autosomal monosomies are seldom found ■ Suggests monosomic gametes may be functionally impaired Monosomy (Do not see monosomy which may suggest monosomic gametes are functionally impaired) 32. Non-disjunction of sex chromosomes: Turner syndrome and Klinefelter syndrome and some phenotypic characteristics associated with each syndrome. ● Klinefelter’s syndrome ○ An ovum with an extra X chromosome is fertilized by a sperm with a Y chromosome- XXY genotype ○ Most common (1 out of 700 to 1000) ○ Tall stature, slightly feminized physique, mildly impaired IQ (15 points less than average), tendency to lose chest hairs, female-type pubic hair pattern, frontal baldness absent, poor beard growth, breast development (in 30% of cases), small testes ● Turner syndrome ○ 1 out of every 10,000 female births (more likely to spontaneously abort)- XO ○ Short stature, low hairline, shield-shaped thorax, widely spaced nipples, shortened metacarpal IV, small fingernails, brown spots (nevi), characteristic facial features, fold of skin, constriction of aorta, poor breast development, elbow deformity, rudimentary ovaries gonadal streak (underdeveloped gonadal structures), no menstruation 33. Be able to differentiate between dominant and recessive disorders both autosomal and X chromosome linked. When considering genetically inherited diseases, consider if the disease X-dominate, X-recessive, autosomal dominate or autosomal recessive? ● X-linked Inheritance ○ There is no male-to male transmission of the phenotype ○ Unaffected males do not transmit the phenotype ○ All daughters of an affected male are heterozygous carriers ○ Some mothers of affected males will not themselves be heterozygotes (they will be homozygous normal)- but will have a germinal mutation ○ Heterozygous women transmit the mutant gene to 50% of their sons, who are affected, and to 50% of their daughters, who are heterozygotes ○ If an affected male mates with a heterozygous female, 50% of the male offspring will be affected, giving the false impression of male-to male transmission. ○ Among the female offspring of such mating, 50% will be affected as the average hemizygous male (XY) ■ In small pedigrees this pattern may simulate autosomal dominate inheritance ■ Why? Because the mother is not a known carrier ○ A female may inherited hemophilia A when both X chromosomes are affected or one is affected and the other is missing or non-functioning ● ● ● ● ● ● ● X-linked recessive ○ Hemophilia A ○ X chromosome-linked recessive coagulation/bleeding disorder and is due to mutations or deletions in the Factor VIII gene (F8) ○ Ratio in population: 1:5,000 males ○ The severity (mild to severe) of disease is defined by the percentage of FVIII levels to normal levels ■ Severe:<1% ■ Moderate:2-5% ■ Mild: 5-30% ○ Heterozygous females may exhibit a mild form of the disease due to X-chromosome inactivation Autosomal Dominant Inheritance ○ Vertical pattern is observed with multiple generations affected ○ Heterozygotes for the mutant allele show an abnormal phenotype ○ Males and females are affected with equal frequency and severity ○ Only one parent must be affected for an offspring to be at risk for developing the phenotype ○ When an affected person mates with an unaffected one, each offspring has a 50% chance of inheriting the affected phenotype Autosomal Recessive Inheritance ○ Horizontal pattern is observed – a single generation being affected ○ Males and females are affected with equal frequency and severity ○ Inheritance is from both parents (each of whom is a heterozygote carrier) and each of whom is usually clinically unaffected by his or her carrier status 34. Be familiar with the genetic disease and how to read pedigree charts. How to read pedigree charts ○ Generation I is on the top, generation II, and III, and so on ○ Shaded square = affected male, shaded circle = affected female ○ Unshaded square = unaffected male, unshaded square = unaffected female ○ Half shaded and half not = carrier Dominant pedigree – juvenile glaucoma ○ Disease causes degeneration of optic nerve leading to blindness ○ Dominant trait appears in every generation Dominant Traits and their Phenotypes ○ Middigital hair – presence of hair on middle segment of fingers ○ Brachydactyly – short fingers ○ Huntington disease – degeneration of nervous system, starting in middle age ○ Phenylthiocarbamide (PTC) sensitivity – ability to taste PTC as bitter ○ Camptodactyly – inability to straighten the little finger ○ Hypercholesterolemia (most common human Mendelian disorder) – elevated levels of blood cholesterol and risk of heart attack ○ Polydactyly – extra fingers and toes Recessive pedigree – albinism (TYR gene) predominant form ○ Condition in which the pigment melanin is not produced ○ Pedigree for this form of albinism due to a nonfunctional allele of the enzyme tyrosinase ● ● ○ Males and females affected equally ○ Most affected individuals have unaffected parents Recessive Trains and their Phenotypes ○ Albinism – lack of melanin pigmentation ○ Alkaptonuria – inability to metabolize homogentisic acid ○ Red-green color bindless – inability to distinguish red or green wavelengths of light ○ Cystic fibrosis – abnormal gland secretion, leading to liver degeneration and lung failure ○ Duchenne muscular dystrophy – wasting away muscles during childhood ○ Hemophilia – inability of blood to clot properly, some clots form but the process is delayed ○ Sickle cell anemia – defective hemoglobin that causes red blood cells to curve and stick together Xeroderma Pigmentosum – autosomal recessive inheritance ○ Malfunctioning NER (nucleotide excision repair) system ○ XP-A gene for example ○ UV radiation and accumulation of error over time ○ Deficiency in at least one of the DNA repair enzymes ○ Accumulation of DNA damage ○ Cancers ○ Early death 35. Understand the difference between Tumor suppressor genes and proto-oncogenes. Be able to describe the significance of the various genes mentioned that interferes with tumorigenesis. Comprehend the natural roles of these genes. ● Cancer ○ Unrestrained, uncontrolled growth of cells ○ Failure of cell cycle control ○ Two kinds of genes can disturb the cell cycle when they are mutated ■ 1. Tumor-suppressor genes ■ 2. Proto-oncogenes ● Tumor-suppressor genes ○ p53 gene and many others ○ Both copies of a tumor-suppressor gene must lose function for the cancerous phenotype to develop ○ First tumor-suppressor identified was the retinoblastoma susceptibility gene (Rb) ○ Predisposes individuals for a rare form of cancer that affects the retina of the eye ○ p53 plays a key role in checkpoint ○ p53 protein monitors integrity of DNA ■ If DNA damaged, cell division halted and repair enzymes stimulated ■ If DNA damage is irreparable, p53 directs cell to kill itself ○ Purpose: Prevent the development of many mutated cells ○ p53 is absent or damaged in many cancerous cells ● Rb gene ○ Inheriting a single mutant copy of Rb means the individual has only one “good” copy left ■ During the hundreds of thousands of divisions that occur to produce the retina, any error that damages the remaining good copy leads to a cancerous cell ■ Single cancerous cell in the retina then leads to the formation of a retinoblastoma tumor ○ Rb protein integrates signals from growth factors ■ Role to bind important regulatory proteins and prevent stimulation of cyclin and cyclin-dependent kinase ● Breast cancer susceptibility protein ○ Tumor suppressor gene: BRCA1 and BRCA2 ○ Expressed in breast tissue ○ Repair of damaged DNA and apoptosis ○ Mutation: Improper repair of DNA ■ Increased risk of breast cancer ● Proto-oncogenes ○ Involved in signal transduction and mitogenic signals, triggering mitosis ○ Normal cellular genes that become oncogenes when mutated ■ Oncogenes can cause cancer ○ Some encode receptors for growth factors ■ If receptor is mutated in “on,” cell no longer depends on growth factors ○ Some encode signal transduction proteins ○ Only one copy of a proto-oncogene needs to undergo mutation for uncontrolled division to take place Cancer and Viruses ● ● ● ● ● ● ● Oncogenic viruses Activated oncogenes transform normal cells into cancerous cells Transformed cells have increased growth, loss of contact inhibition, tumor-specific transplant antigens, and T antigens (nucleus) The genetic material of oncogenic viruses becomes integrated into the host cell’s DNA Cancer may develop years after initial infection Oncogenic DNA viruses ○ Adenoviridae ○ Herpesviridae ○ Poxviridae ○ Papovaviridae ○ Hepadnaviridae Oncogenic RNA viruses ○ Retroviridae ○ Viral RNA is transcribed to DNA, which can integrate into host DNA ○ Human T cell lymphotropic viruses ■ HTLV-1 ■ HTLV-2 36. And know the molecular tools used in genetic diseases diagnostics. ● Family history ○ All first-degree relative (parents, siblings and offspring) ■ Second-degree (grandparents, aunts, uncles, nieces and nephews and grandchildren) ■ Third-degree (first cousins) ○ ● ● ● ● ● ● ● ● This information can be further analyzed utilizing a pedigree diagram to identify mode of inheritance for a disease process Study of chromosomes utilizing light microscopy ○ Obtained from peripheral blood, amniotic fluid, trophoblastic cells (chorionic villus) bone marrow and cultured fibroblasts (usually obtained from a skin biopsy) DNA fingerprinting for identification (Restriction Enzymes) to generate and RFLP Analytical techniques useful during DNA and RNA investigation ○ Absorption of UV light ○ Denaturation and renaturation of nucleic acids ○ Molecular hybridization ○ FISH: Fluorescent in situ hybridization ○ Electrophoresis of nucleic acids Nucleic acids ○ Absorb UV light strongly at 254–260 nm due to interaction between UV light and ring systems of the bases ○ Use of UV critical to isolation of nucleic acids following separation Molecular hybridization ○ Denaturation and renaturation of nucleic acids are the basis for molecular hybridization ○ Example: Single strands of nucleic acids combine duplex structures, yet are not from same source ■ If DNA is isolated from two distinct sources, double- stranded hybrids will form Fluorescent in situ hybridization (FISH) ○ Uses fluorescent probes to monitor hybridization ○ Mitotic cells fixed to slides and subjected to hybridization ○ ssDNA is added and hybridization is monitored ○ Probes are nucleic acids that will hybridize ONLY with specific chromosomal areas ○ Fluorescence in situ hybridization (FISH) has made it easier to visualize and map chromosomal (gene) abnormalities and the presence of infectious agents ○ Using fluorescent dye-labeled RNA or DNA ○ Quick test ○ Clinically: used in diagnostics and in genetic counseling ■ Circulating tumor cells ■ Infectious particles Nucleic acid electrophoresis ○ Separates DNA and RNA fragments by size ○ Smaller fragments migrate through gel at faster rate than large fragments ○ Agarose gel ■ Porous matrix restricts migration of larger molecules more than it restricts smaller ones Agarose Gel Electrophoresis ○ Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA ○ ● Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field ○ How fast will the DNA migrate? ■ Strength of the electrical field, buffer, density of agarose gel ■ Size of the DNA – *Small DNA move faster than large DNA, gel electrophoresis separates DNA according to size Polymerase Chain Reaction (PCR) ○ Make numerous copies of DNA fragments ○ PCR is a laboratory version (“in vitro” – occurs in test tube) vs DNA replication occurs in a living cell (“in vivo”) ○ Real-time PCR: newly made DNA is tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle ○ Reverse-transcription (RT-PCR): reverse transcriptase makes DNA from viral RNA or mRNA ○ Diseases that can be diagnosed with PCR: Huntington’s disease, cystic fibrosis, human immunodeficiency virus ○ Steps of PCR ■ 1. Denature DNA: at 95°C, the DNA is denatured (i.e. the two strands are separated) ■ 2: Primers Anneal: at 40°C- 65°C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA ■ 3: DNA polymerase Extends the DNA chain: at 72°C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers CHAPTER 13 37. What is malnutrition? And what are the two distinct kinds of malnutrition? ● ● ● ● ● ● Protein Energy Malnutrition (PEM) is a deficiency syndrome caused by inadequate macronutrient and micronutrient intake. The causes include social, economic, biologic, and environmental factors. Protein, energy and nutrient intake are all inadequate ○ Characteristics can range from kwashiorkor-like to marasmus-like. Morbidity is due to fluid and electrolyte imbalances, opportunistic infections, decline in cardiovascular function, and anemia. PEM occurs in all parts of the world ○ Africa, south and central America, East and Southeast Asia, Middle east In industrialized countries PEM occurs ○ Populations living in poverty ○ Older adults ○ Hospitalized patients with anorexia nervosa, AIDS, cancer Two forms: Kwashiorkor and Marasmus ○ Kwashiorkor ■ A type of malnutrition seen in developing countries and is caused by a deficiency of protein in a diet that is adequate in calories ■ Kwashiorkor results in lack of cellular development due to the failure to synthesize normal amounts of proteins ■ There is hypoalbuminemia (reduced oncotic pressure) which leads to edema: there is also diarrhea, atrophy of the pancreas and intestinal mucosa ■ Rarely observed in affluent countries ○ Marasmus ■ Caused by a severe deficiency of both protein and total calories: chronic PEM. Protein, energy and nutrient intake are all inadequate ■ This is generalized starvation, with symptoms including muscle wasting (emaciation), weakness, and anemia ■ Seen in children 6 to 18 months who are fed diluted or poorly mixed formulas ■ Anorexia nervosa 38. Consider the two groupings of vitamins (water soluble and fat soluble). ● ● Water soluble ○ Water-soluble vitamins and/or their derivatives function as coenzymes for enzymes involved in metabolic pathways ○ Water-soluble vitamins – B complex and C Fat soluble ○ Some fat-soluble vitamins function as hormones, some as anti-oxidants ○ Fat-soluble vitamins – A, D, E, and K ○ These vitamins are associated with lipids of the diet ○ Absorption of these vitamins requires the normal lipid absorption mechanisms, including emulsification by bile, normal mucosal cells, and transport in chylomicrons ○ These vitamins are stored in the body in the liver and adipose tissue 39. What are some of the physiological significances of vitamins? Water Soluble Vitamins ● ● ● ● ● Thiamine (B1) ○ The coenzyme form is thiamine pyrophosphate (TPP), which is involved in reactions of energy production ○ TPP is a coenzyme for pyruvate dehydrogenase (pyruvate → acetyl CoA) and for a TCA cycle enzyme Riboflavin (B2) ○ Riboflavin is a constituent of the coenzymes FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) ○ These coenzymes function as electron carriers for oxidoreductases ○ Deficiency thereof is not associated with a major human disease Niacin (Nicotinic Acid) (B3) ○ Niacin is a constituent of the coenzymes NAD+ (nicotinamide adenine dinucleotide) and NADP+ (nicotinamide adenine dinucleotide phosphate) ○ These coenzymes function as electron carriers for oxidoreductases ○ Some niacin can be synthesized endogenously from tryptophan--an essential amino acid Pyridoxine (Vitamin B6) ○ The coenzyme form is pyridoxal phosphate (PLP) ○ This coenzyme is required by enzymes involved in amino acid metabolism, such as transaminases ○ In the 1960’s, when infants were fed a formula lacking this vitamin, they developed seizures and hyperirritability ■ Why? Pyridoxal phosphate is a cofactor involved with the enzyme glutamic acid decarboxylase (GAD), which converts glutamic acid to GABA ○ Isoniazid, used in the treatment of TB, can produce a B6 deficiency. ■ How? ■ Its metabolites directly attach to and inactivate pyridoxine species ■ Second, it directly inhibits the enzyme pyridoxine phosphokinase: ■ This enzyme activates pyridoxine to pyridoxal 5' phosphate--the cofactor in many "pyridoxine-dependent" reactions Folic Acid (B9) ○ The coenzyme form is tetrahydrofolate (FH4) and derivatives ○ ● ● FH4 functions as a carrier of 1-carbon groups for enzymes catalyzing 1-C transfer reactions ○ Tetrahydrofolate derivatives are required for nucleotide synthesis and for synthesis of methionine from homocysteine Vitamin B12 (Cobalamin) ○ The coenzyme forms are methylcobalamin and deoxyadenosylcobalamin ○ A reaction that requires B12 is the conversion of homocysteine to methionine ○ Intestinal absorption of B12 requires a glycoprotein called intrinsic factor (IF), which is synthesized in the parietal cells of the stomach ○ Large amounts of B12 are stored in the liver Vitamin C (Ascorbate) ○ Ascorbate is a coenzyme in the hydroxylation of proline and lysine residues in collagen, reactions that are required in the production of mature collagen ○ Vitamin C is also an anti-oxidant (reducing agent) ○ Vitamin C deficiency causes scurvy, which is characterized by bleeding gums, loose teeth, fragile blood vessels, impaired wound healing Fat Soluble Vitamins ● Vitamin A ○ Vitamin A has 3 active forms – retinol, retinal and retinoic acid ○ Function: ■ Important in growth, reproduction, immunity and cell differentiation ■ Help maintain healthy bones, skin and mucous membrane ■ Visual cycle as a constituent of the visual pigment--rhodopsin ○ β-carotene from plants is provitamin A – it is converted in the GI tract to retinal ○ β-carotene functions as an antioxidant ○ Animal sources provide vitamin A in the form of retinol and retinyl esters ○ They are absorbed and incorporated into chylomicrons ○ Stored in the liver as retinyl esters to the tissue ○ Delivered to tissue by retinol-binding protein (RBP) ○ Once delivered to the cell the cell will convert retinol to retinal or retinoic acid ○ In vision, retinol is carried to the retina and converted to retinal. It then combines with a protein opsin to form a pigment known as rhodopsin. It is an integral part of the visual cycle allowing us to see ○ Retinoic acid affects gene expression. It acts like a steroid hormone, binding to retinoid nuclear receptors, and enhancing transcription of certain genes involved in differentiation of stem cell into various cell types ○ Both retinol and retinal support sperm production and fertility ○ All forms of Vitamin A are essential for proper bone growth ● Vitamin D ○ Vitamin D3, cholecalciferol, is from animals (skin UV radiation) ○ Vitamin D2, ergocalciferol, is from yeast ○ The active form of the vitamin is 1,25-dihydroxycholecalciferol, or calcitriol ○ Function: ■ Calcitriol functi