Essentials of Medical Biochemistry & Molecular Biology for Dental Students 2024-2025 PDF

Zagazig National University Faculty of Dentistry Medical Biochemistry Department Essentials of Medical Biochemistry & Molecular Biology For Dental Students Volume I 2024-2025 ...

Zagazig National University Faculty of Dentistry Medical Biochemistry Department Essentials of Medical Biochemistry & Molecular Biology For Dental Students Volume I 2024-2025  Preface “Today’s Biochemistry is Tomorrow’s Medicine”. The development of molecular biology has revolutionized our understanding of the biochemistry underlying biology and medicine. The primary aim of this book is to integrate general biochemistry into topics that specifically pertain to dental health and disease.  Contents 1- Carbohydrate Chemistry……………...………………………………………… 1 2- Lipid Chemistry……………………..………………………………………… 15 3- Protein Chemistry………………………..……………………………………. 29 4- Enzymes……………………………..………………………………………… 42 5- Vitamins……………………………….……….……………………………… 53 6- Molecular Biology ……..……………...……………………………………… 66 Carbohydrate Chemistry Carbohydrate Chemistry Definition - Carbohydrates may be defined as polyhydroxy aldehydes or ketones or compounds which produce them on hydrolysis. - They are primarily composed of the elements carbon, hydrogen and oxygen. Biological importance of Carbohydrates 1. Carbohydrates constitute the main energy source for the human body. 2. Some carbohydrate acts as a lubricant of joints and tendons. 3. Storage form of energy (starch and glycogen). 4. Excess carbohydrate is converted to fat. 5. Carbohydrates are used to form structural elements, such as chitin in animals and cellulose in plants. 6. Carbohydrates in the form of fibers can help to lower blood glucose and cholesterol levels and prevent constipation. Classification and Nomenclature of Carbohydrates Carbohydrates are divided into four major groups monosaccharides, disaccharides, oligosaccharides and polysaccharides. 1. Monosaccharides: are molecules having only one sugar unit. 2. Disaccharides: Those sugars which yield two molecules of monosaccharide on hydrolysis. 1 Carbohydrate Chemistry 3. Oligosaccharides: Those sugars which yield 3 to 10 monosaccharide units on hydrolysis. 4. Polysaccharides: Those sugars which yield more than 10 molecules of monosaccharides on hydrolysis. Polysaccharides are further divided into two groups: - Homopolysaccharides: Having only one type of monosaccharide units. - Heteropolysaccharides: Having different types of monosaccharide units. Monosaccharides Monosaccharides serve as the building units of carbohydrates. They are the simplest sugars and cannot be hydrolyzed. They can be further subdivided: 1. According to the presence of aldehyde or ketone group (active sugar group) into: a. Aldoses: They contain aldehyde group (CHO) e.g. glucose and galactose. b. Ketoses: They contain ketone group (CO) e.g. fructose. 2 Carbohydrate Chemistry 2. According to the number of carbon atoms: - Trioses: Contain 3 carbon atoms e.g. glyceraldehyde and dihydroxyacetone. - Tetroses: Contain 4 carbon atoms. - Pentoses: Contain 5 carbon atoms e.g. ribose and deoxyribose. - Hexoses: Contains 6 carbon atoms e.g. glucose, fructose and galactose. 3. According to both the number of carbon and the type of active sugar group: e.g.: Aldopentose contains 5 carbon atoms and has an aldehyde group. e.g.: Ketohexose contains 6 carbon atoms and has a keto group. Examples of Monosaccharides: 3 Carbohydrate Chemistry Importance of monosaccharides Ribose is a component of ribonucleic acid (RNA). Deoxyribose is a component of deoxyribonucleic acid (DNA) Glucose is the sugar of blood and main source of energy in body. Fructose is the sugar of semen. Galactose share in formation of lactose (milk sugar). Biomedical Importance Seminal fluid is rich in fructose and sperms utilize fructose for energy. Important derivatives of monosaccharides 1-Sugar acids  Oxidation of monosaccharides produce sugar acids.  Depending on the oxidizing agent used, the terminal aldehyde (or keto) or the terminal alcohol or both the groups may be oxidized. a) Aldonic acids - The aldehyde group (C1 group) of aldoses is oxidized to form the corresponding aldonic acid. - Glucose is oxidized to form gluconic acid. b) Uronic acids - The alcohol group (C6 group) of monosaccharides is oxidized to form the corresponding uronic acid. - Glucose is oxidized to form glucuronic acid. c) Saccharic acids - These are monosaccharides in which both the aldehyde and alcohol groups are oxidized to form the corresponding saccharic acid. - Glucose is thus oxidized to glucosaccharic acid. 4 Carbohydrate Chemistry Importance of glucuronic acid: - Glucuronic acid is conjugation with toxic substance (bilirubin, steroid hormones, drugs and pollutants) and converts them to a soluble nontoxic substance which is excreted in urine. 2-Sugar Alcohols: - Formed by reduction of sugars. - They are widely used as artificial sweeteners, due to their ability to stimulate sweet receptors on the tongue. - The most important members of this group include the followings: a) Glycerol: It is the alcohol of glyceraldehyde or dihydroxyacetone. b) Ribitol: It is the alcohol of ribose. It enters in the structure of riboflavin (Vitamin B2). c) Sorbitol: It is the alcohol of glucose and fructose. d) Mannitol: It is the alcohol of fructose. 5 Carbohydrate Chemistry Clinical correlations: - Glycerol can be used as a moisturizer and is used to improve smoothness and taste of medicines. - Sorbitol is a laxative. It is used to treat constipation. - Adding mannitol to lidocaine significantly increases the percentage of total pulpal anesthesia. 3- Amino Sugars - They are formed from the corresponding monosaccharide by replacing the OH group at C2 with amino group (NH2) e.g. D-glucosamine and D- galactosamine. Clinical correlations: - Glucosamine has a pain relief effect in patients with dental pain. - Glucosamine is taken for treatment of osteoarthritis, joint pain, jaw pain and back pain. - Certain antibiotics, such as erythromycin. 6 Carbohydrate Chemistry 4- Deoxy sugar - These are the sugars that contain one oxygen less than that present in the parent molecule. CHO CHO H C OH H C H H C OH H C OH H C OH H C OH CH2OH CH2OH Ribose Deoxyribose - Deoxyribose is the most important one. It is present in the structure of DNA. Glycosides These are compounds resulting from condensation between monosaccharides and other compounds. These compounds might be: Another monosaccharide A non-carbohydrate compound (Aglycon). Physiologically important glycosides include: 1. Some antibiotics e.g. streptomycin. 2. Cardiac glycosides (Digitalis). 3. Stevia (Steviol glycosides). 4. Glucovanillin (vanillin-D-glucoside). Clinical correlations:  Cardiac glycosides: Digitalis stimulate muscle contraction. Used as a treatment for heart failure.  Stevia (steviol glycosides): A sweetener extracted from the stevia plant. It is 250-300 times sweeter than sugar. It is safe to be used by diabetics.  Glucovanillin is a natural substance that has vanilla flavor. 7 Carbohydrate Chemistry Disaccharides  When two monosaccharides are combined together by glycosidic linkage, a disaccharide is formed.  The disaccharides are of two types: - Reducing disaccharides with free aldehyde or keto group e.g. maltose, lactose. - Non-reducing disaccharides with no free aldehyde or keto group e.g. sucrose. Disaccharides include: 1. Sucrose (Table sugar): - Sucrose is a plant disaccharide and is present in high concentration in sugar cane and sugar beet. - Sucrose is used for sweetening purpose. - Sucrose consists of glucose and fructose. 2. Lactose (Milk sugar): - It is synthesized in mammary gland and during lactation may appear in the urine. - Lactose consists of galactose and glucose. - Lactose is the suitable sugar for babies feeding because it is nonfermentable, so it does not produce gases that result in gastric colic and pain. 8 Carbohydrate Chemistry 3. Maltose (Malt sugar): - Maltose does not occur in free state but is formed as an important product of the digestion of starch and glycogen. - Maltose consists of two molecules of glucose. 4. Lactulose: Lactulose is a synthetic disaccharide containing galactose and fructose. It is neither digested nor absorbed in the intestine. Clinical correlations: - Lactulose is used clinically in medicine as an osmotic laxative. - Lactulose is useful for the treatment of hepatic encephalopathy. Polysaccharides - They are composed of more than 10 monosaccharide units linked by a glycosidic bond. Polysaccharides can be divided into two groups: 1. Homopolysaccharides: are made up of one type of monosaccharides. They may constitute a linear chain, e.g., cellulose; or moderately branched; e.g., starch; or highly branched, e.g., glycogen. 2. Heteropolysaccharides: are made of different types of monosaccharide units. 1- Homopolysaccharides: These are composed of repeated units of similar monosaccharide units. It includes: 1. Starch: Starch is widely distributed in plants. It is the storage form of carbohydrate material in plant. Starch granules contain two forms, amylose in the inner part and amylopectin in the outer layer. a) Amylose (20%) - It represents the inner part of starch. - Nonbranched polysaccharide formed of -glucose molecules united by 1- 4 glucosidic bonds. 9 Carbohydrate Chemistry b) Amylopectin (80%) - It is a branched polysaccharide, each branch is formed of - glucose molecules. - Within the branch the glucose molecules are united by 1- 4 glucosidic bonds, and at the branching points they are united by 1- 6 glucosidic bonds. 2. Glycogen - Major storage form of carbohydrates in animals. - Present mainly in muscle and liver. - It is a highly branched chain homopolysaccharide. - Formed of α-glucose units linked by α-1,4glycosidic bonds with many branches at α-1,6 glycosidic bonds. 3. Cellulose - Cellulose is the most abundant organic material in nature. - Cellulose is a linear polymer of β glucose united by β1- 4 glucosidic linkages with no branches. - Cellulose cannot be utilized for energy purposes by human beings, because the enzyme which cleavage β-(1,4) linkage is missing in the gastrointestinal tract. - Herbivorous animals contain microorganisms in the gut which produce enzymes that can cleave β -glycosidic bonds. 10 Carbohydrate Chemistry 4. Chitin - It is a homopolysaccharide of the glucose derivative. - Chitin makes up the hard exoskeleton of crustaceans and insects. 5. Inulin - Inulin is made up of fructose units. Biomedical Importance: - Inulin is not utilized by the body. It is used for assessing kidney function through measurement of glomerular filtration rate (GFR). 2- Heteropolysaccharides: Formed of different sugar units. 1. Agar -agar - It is prepared from sea weeds. It contains galactose and glucose. - It is dissolved in water at 100°C, which upon cooling sets into a gel. Biomedical Importance - Agar cannot be digested by bacteria and hence used widely as a supporting agent to culture bacterial colonies. - Agarose is the common support of agarose gels used to separate DNA fragments in electrophoresis. Culture media Electrophoresis 11 Carbohydrate Chemistry 2. Pectin: - Pectin is found in fruits. Biomedical Importance - Pectin decreases the absorption of glucose and cholesterol from the intestine. - Used as gelling agents, so used in treatment of infantile diarrhea. - Pectin nanocomposite is used as filler material for dental applications. 3. Glycosaminoglycans (GAGs)(Mucopolysaccharides): - Mucopolysaccharides are heteroglycans made up of repeating units of sugar derivatives, namely amino sugars and uronic acids. - Most of GAGs are covalently conjugated to a protein, the products are termed proteoglycans. 1. Hyaluronic acid - A sulphate free mucopolysaccharide. - Present in synovial fluid, embryonic tissue, cartilage and skin. Function: - It acts as a lubricant and shock absorber. - Hyaluronic acid has an important role in healing of periodontal lesion. 12 Carbohydrate Chemistry Clinical correlations: - Hyaluronic acid is ingredient of oral gel which is used to relieve pain and reduce gingival inflammation. - Hyaluronic acid is also used as a lip filler in plastic surgery. Hyalurondiase enzyme - This enzyme hydrolyses hyaluronic acid. It is secreted by invasive bacteria. - It helps the spread of bacteria through subcutaneous tissue. It is also present in sperm and helps fertilization. Clinical correlations: - Hyaluronidase compared to plain lidocaine with epinephrine increases in the onset of the inferior alveolar nerve block. 2. Heparin - Heparin is an anticoagulant (prevents blood clotting). - Heparin also increases the release of lipoprotein lipase enzyme from capillary wall which helps in clearance of plasma from lipids after a fatty meal. 3. Keratan sulfate is important for transparency of the cornea. 4. Dermatan sulfate is important for maintenance of structure and shape of the sclera of the eye. 5. Heparan sulfate plays an important role in cell membrane receptors and cell-cell interactions. 13 Carbohydrate Chemistry 6. Chondroitin sulfate is important for binding of collagen of cartilage and compressibility of cartilage. Clinical correlations: Chondroitin sulfate and glucosamine are used as adjuvant treatment in cases of temporomandibular dysfunction. Glycoproteins and mucoproteins - When the carbohydrate chains are attached to a polypeptide chain it is called a proteoglycan. - If the carbohydrate content is less than 10%, it is generally named as a glycoprotein. If the carbohydrate content is more than 10% it is a mucoprotein. - They are seen in almost all tissues and cell membranes. About 5% of the weight of the cell membrane is carbohydrates. - Functions include their role as enzymes, hormones, transport proteins, structural proteins and receptors. 14 Lipid Chemistry Lipid Chemistry Lipids are organic substances in plant and animal tissues, insoluble in water, but soluble in fat solvents such as ether, chloroform. Biomedical Importance: 1. Lipids are important dietary constituent and acts as fuel in the body. 2. Can be stored in the body in unlimited amount in contrast to carbohydrates. 3. Lipids may exert an insulating effect on internal organs like kidney. 4. Subcutaneous fat help in keeping body temperature constant. 5. Lipids supply body with essential fatty acids (EFA) and steroid hormones. 6. Supply body with fat soluble vitamins (A, D, E and K). 7. Phospholipids are important constituents of many membranes. Lipids are classified into three groups: 1. Simple lipids: Esters of fatty acids with alcohols. 2. Conjugated (compound) lipids: Fatty acids + alcohols + other group. 3. Derived lipids: Compounds produced by hydrolysis of simple or conjugated lipids or present associated with lipids in nature. 15 Lipid Chemistry 16 Lipid Chemistry Fatty acids - A fatty acid consists of a hydrophobic (water insoluble) hydrocarbon chain with a terminal carboxyl group. Numbering of carbon atoms: - It starts from the carboxyl carbon which is taken as number 1. The carbons adjacent to this (carboxyl C) are 2, 3, 4 and so on or alternately α, β, γ and so on. - The terminal carbon containing methyl group is known omega (ω) carbon. Starting from the methyl end, the carbon atoms in a fatty acid are numbered as omega 1, 2, 3 etc. Classification of fatty acids 1. Depending on total number of carbon atoms a. Even chain: - They have carbon atoms 2,4,6 and similar series. - Most of the fatty acids are of even carbons (usually 14C – 20C). - Palmitic acid (16C) and stearic acid (18C) are the most common. b. Odd chain: - They have carbon atoms 3, 5, 7, etc. - Among the odd chain fatty acids, propionic acid (3C) and valeric acid (5C). - Odd numbered fatty acids are seen in microbial cell walls and are present in milk. 17 Lipid Chemistry 2. Depending on length of hydrocarbon chain a. Short chain with 2 to 6 carbon atoms. b. Medium chain with 8 to 14 carbon atoms. c. Long chain with 16 to 22 carbon atoms. d. Very long chain fatty acids (more than 24 carbon). 3. Depending on the nature of the hydrocarbon chain a. Saturated fatty acids: no double bonds e.g. Palmitic acid (C16). b. Unsaturated fatty acids: contain one or more double bonds. - Unsaturated fatty acids are ω3, ω6 and ω9 fatty acids according to the position of the first double bond in relation to the omega (ω) carbon (CH3). - According to the number of double bonds unsaturated fatty acids are classified into two types:  Mono unsaturated having single double bond e.g.ω9: Oleic acid. 18 Lipid Chemistry  Polyunsaturated fatty acid (PUFA) with 2 or more double bonds e.g. - ω 3: Linolenic acid. - ω 6: Linoleic acid and Arachidonic acids. 4. Depending on nutritional status of fatty acids a. Essential Fatty Acids (EFA): These are polyunsaturated fatty acids that cannot be synthesized in the body and must be taken in diet. Examples: Linolenic, linoleic and arachidonic acids. Clinical importance of polyunsaturated fatty acids 1. Normal growth. 2. Enter in the formation of phospholipids and cholesterol ester. 3. Arachidonic acid is a precursor of a group of substances called eicosanoids. 4. High content of polyunsaturated fatty acids tends to lower serum level of cholesterol. Clinical Correlations: - Deficiency of essential fatty acids produces fatty liver. - The deficiency of essential fatty acids results in toad skin. b. Non-essential Fatty Acids: They are formed inside the human body. 19 Lipid Chemistry Simple Lipids Simple lipids are esters of fatty acids with various alcohols. According to the types of alcohol they are classified into:  Neutral fat (triglycerides): They are glycerol + three fatty acids.  Waxes: Fatty acids + higher monohydroxy aliphatic alcohols. Triacylglycerols (TAG) - Neutral fats are also called triacylglycerols (TAG) or triglycerides (TG). A triacylglycerol molecule is an ester of three fatty acids and glycerol. Function of triglycerides: 1. Main storage form of fat. 2. Can be stored in large amount. 3. It provides the body with energy. 4. It acts as insulator for heat. 5. It supports internal organs. 20 Lipid Chemistry Compound Lipids Conjugated lipids contain in addition to fatty acids and alcohol, other groups. According to the other group conjugated lipids are classified into: I- Phospholipids: Containing phosphate. II- Glycolipids: Containing carbohydrates (Non-phosphorylated lipids). III- Proteolipids. 21 Lipid Chemistry I-Phospholipids Phospholipids are classified according to the type of alcohol into: A. Sphingophospholipids: - This type of phospholipid contains sphingosine. - The fatty acid is linked to this alcohol by amide bond (not ester bond) to form ceramide, which is connected to phosphocholine to form sphingomyelin. - Sphingomyelins are important constituents of myelin and are found in good quantity in brain and nervous tissues. 22 Lipid Chemistry B. Glycerophospholipids: Containing alcohol glycerol 1. Phosphatidic acid (diacylglycerol phosphate): - Consists of: Glycerol + Saturated FA + Unsaturated FA + Phosphate. - Basically, phosphatidic acid is an intermediate in the synthesis of triacylglycerols and phospholipids. 2. Lecithin (phosphatidyl choline): Phosphatidic acid + Choline. Functions: a. Lung surfactant (Dipalmityl-lecithin): - A substance formed by lung cells to decrease the surface tension of the fluids lining the lung alveoli and helps easy expansion of the lung during inspiration. Clinical correlations: - Lack or decrease of surfactant (dipalmityl-lecithin) leads to difficulty in breathing and called respiratory distress syndrome in premature babies. b. Lecithin represents the storage form of body’s choline. 3. Cephaline(phosphatidyl ethanolamine):Phosphatidic acid + Ethanolamine. Functions: - Cephalin is necessary for blood coagulation. 23 Lipid Chemistry 4. Phophatidyl inositol: Phosphatidic acid + Inositol. Function - Generation of second messengers. 5. Cardiolipins (diphosphatidyl glycerol): Two molecules of phosphatidic acids attached by glycerol. - Cardiolipins is abundant in the cardiac muscle and is used in diagnosis of syphilis. Functions of Phosphoglycerates 1. Membrane lipids: Phosphoglycerates are easily the most abundant membrane lipids. 2. Biologic detergents: Phosphoglycerates are excellent biologic detergents. 3. Lipoprotein structure: Lipids are transported as lipoproteins and phosphoglycerates are essential structural components of lipoproteins 24 Lipid Chemistry II. Glycolipids - A glycolipid consists of sphingosine and fatty acid (ceramide) attached to it one or more carbohydrate radicals 1- Cerebrosides: Sphingosine + fatty acid + Glucose or galactose. 2- Sulfolipids: They are cerebrosides in which sulfate group is attached. 3- Gangliosides: Sphingosine + fatty acid + complex carbohydrate. - Glycolipids are important components of myelin sheath and red blood cell membrane N.B. Sphingolipids are lipids containing sphingosine (Sphingomyelin, cerebrosides, sulfolipids and gangliosides). 25 Lipid Chemistry Functions of glycolipids 1. The glycolipids are essential components of biological membranes. 2. Sphingolipids are highly antigenic, and have been identified in several tumor antigens, blood group antigens. 3. Gangliosides act as receptors for toxic agents and some pathogens such as Vibrio cholerae and influenza virus Derived Lipids Derived lipids are substances produced by hydrolysis of simple or conjugated lipids or substances associated with lipids in nature. Derived lipids include: 1- Fatty acids 2- Alcohols: Glycerol, Sphingosine 3- Steroids 4- Fat soluble vitamins( A, D, E and K ) 5- Carotenoids. Steroids Steroids are complex molecules consisting of four fused carbon rings. Cholesterol is the most important sterols. Cholesterol  Cholesterol is the most important animal steroid from which other steroid compounds are formed.  Sources of cholesterol: 1- Exogenous sources: Dietary sources are egg yolk, liver, and brain. 26 Lipid Chemistry 2- Endogenous sources: every cell in the body can synthesize its own cholesterol but blood cholesterol is formed by the liver. Occurrence of cholesterol inside the body: It is widely present in body tissues, but it is present in high concentration in liver, brain, ovary, and testis. Blood cholesterol: - Blood cholesterol level: 150 - 220 mg/dl. - 25% of cholesterol is carried on HDL. - 75% of cholesterol is carried on LDL. Functions of cholesterol: 1. Cell membranes: Cholesterol is a component of membranes and has a modulating effect on the fluid state of the membrane. 2. Nerve conduction: Cholesterol has an insulating effect on nerve fibers. 3. Bile acids and bile salts are derived from cholesterol. Importance of bile salts: - Emulsification of lipids. - Activation of pancreatic lipase enzyme. - Absorption of lipids and fat soluble vitamins (A, D, E, K). 4. Steroid hormones: Glucocorticoids, androgens and estrogens are from cholesterol. 5. Vitamin D3 is from 7-dehydro-cholesterol. 27 Lipid Chemistry Eicosanoids - Eicosanoids include prostaglandins, prostacyclins, thromboxane and leukotrienes. - Arachidonic acid is the precursor of the prostaglandins, prostacyclins and thromboxane. 1. Prostaglandins (PGs) Inhibits hair follicle growth and vasodilator. Exerts effects in labor (uterine contraction). Induces fever. 2. Thromboxane Thromboxane is produced by platelets. Thromboxane plays roles in clot formation and thrombosis. They are potent vasoconstrictors and facilitate platelet aggregation. The anti-clotting effects of aspirin is due to inhibition thromboxane synthesis. 3. Prostacyclin Prostacyclin counters the effects of thromboxane, inhibiting platelet activation and acting as vasodilators. 4. Leukotrienes Leukotrienes are associated with the production of histamines which act as mediators of inflammation. Over production of leukotrienes may play a role in asthma and allergic reactions. Some treatments for asthma aim at inhibiting production or action of leukotrienes. 28 Protein Chemistry Amino Acids - Proteins are made up of a combination of 20 different subunits called amino acids linked by peptide bonds. All proteins are polymers of amino acids. - Most of the amino acids (except proline) are alpha amino acids, which means that the amino group is attached to the same carbon atom to which the carboxyl group is attached. Occurrence of amino acids: All the standard amino acids occur in almost all proteins. Cereals are rich in acidic amino acids Aspartic and Glutamic while collagen is rich in basic amino acids and proline and hydroxyproline. Classification of Amino Acids A. Classification Based on Nutritional Requirement: 1. Essential amino acids: These are amino acids that cannot be formed in the body and must be taken into the diet for normal growth. A deficiency of one or more essential amino acids in the diet gives rise to a decrease in protein synthesis resulting in a failure in the growth of the child and a fall in plasma proteins and hemoglobin levels. They include 8 amino acids: 1. Valine 2. Leucine 3. Isoleucine 4. Lysine 5. Phenylalanine 6. Tryptophan 7. Methionine 8. Threonine. 2. Semi-essential amino acids: Can be synthesized in enough amounts in adults but must be taken with diet in growing infants and children. They are: Histidine and arginine. 29 Protein Chemistry 3. Nonessential amino acids: Can be synthesized in the body: Include the remaining amino acids. These amino acids are derived from carbon skeletons of lipids and carbohydrates during their metabolism or from the transformation of essential amino acids. 4. Conditionally essential amino acids: These amino acids are normally non- essential, but become essential during times of physiological stress, and chronic illness. B. Classification Based on Metabolic Fate: 1. Purely Ketogenic: Leucine is purely ketogenic because it is converted to ketone bodies. 2. Glucogenic and ketogenic: Phenylalanine, tyrosine, tryptophan, and isoleucine are partially ketogenic and partially glucogenic. These amino acids will enter the ketogenic pathway and the other part into the glucogenic pathway. 3. Purely Glucogenic: This gives glucose to the body. All the remaining 14 amino acids are purely glucogenic. Non-standard amino acids : 1. Non-standard amino acids found in proteins: After the synthesis of proteins, some of the amino acids are modified, e.g. Hydroxyproline and hydroxylysine are important components of collagen. 2. Non-standard amino acids not found in proteins: (Non-protein amino acids). These are produced during the metabolism of amino acids and seen free in cells, e.g. Ornithine which is intermediate of the urea cycle. 3. Non-alpha amino acids:  Gamma amino butyric acid (GABA) is derived from glutamic acid, and acts as a neurotransmitter.  Beta-alanine, where the amino group is in the beta position, is a constituent of pantothenic acid (vitamin) and co-enzyme A. 30 Protein Chemistry Peptides - Peptides are short sequences of amino acids, linked by a peptide bond. The peptide bond is formed by a condensation (dehydration) reaction between an α-carboxyl group of one amino acid and an α-amino group of another amino acid (CO-NH). - Peptides consist of a few (3 to 50) amino acids. Biologically important Peptides 1. Hormones: ACTH of the anterior pituitary, vasopressin, and oxytocin of the posterior pituitary. 2. Gastrin and Secretin: Gastrointestinal peptides that act as hormones that stimulate the secretion of bile and other enzymes of digestive juices. 3. Endorphins: (5 amino acids) They are natural painkillers that are produced in the body. They interact with receptors in the brain to inhibit the transmission of pain signals. 4. Aspartame: dipeptide is used as a sweetening agent. 5. Glutathione: It is a tripeptide formed of glutamic acid- cysteine- glycine. It is abbreviated as G-SH. 31 Protein Chemistry Importance of glutathione: 1. Detoxification of some drugs and carcinogens. 2. Protect the wall of RBCs against toxic H2O2. 3. It plays a role in the transport of amino acids across the intestinal membrane (absorption of amino acids). 4. Activator for some enzymes. Cyclic peptides 1. They differ from normal peptides. 2. In these peptides N-terminus and C-terminus are linked by peptide bond resulting in cyclization of peptide. 3. An antibiotic gramicidin-S is a cyclic peptide. It consists of ten amino acids. Toxic peptides 1. Some peptides act as toxins. 2. α-amanitin is a bicyclic octapeptide present in a particular variety of mushrooms. It is extremely toxic to humans. It is responsible for mushroom poisoning cases around the world. 3. When the mushrooms are consumed it causes pain in the gastrointestinal tract, vomiting, diarrhea, and nausea. 4. Death occurs within a week due of impairment of liver and kidney functions. 32 Protein Chemistry Proteins - Protein are macromolecules formed from amino acids (greater than 50 a.a.) united by peptide bonds. - Some Proteins are formed of two or more polypeptide chains. - Proteins contain Carbon, Hydrogen, Oxygen, and Nitrogen as the major components while Sulphur and Phosphorus are minor constituents. Nitrogen is characteristic of proteins. On average, the nitrogen content of ordinary proteins is 16% by weight. Functions of proteins: 1. Structural element:  Cell membrane: contain protein in the form of glycoprotein,  Skin and bone: contain protein in the form of collagen. 2. All enzymes are proteins. 3. Defense: antibodies (immunoglobulins) are proteins. 4. Blood clotting: Coagulation factors are proteins. 5. Some hormones are protein in nature (e.g. insulin). 6. Transport: Hemoglobin is an oxygen carrier. 7. Storage: as ferritin which is the storage form of iron. 8. Under certain conditions proteins can be catabolized to supply energy. Protein Structure (conformation) Proteins have different levels of structural organization (primary, secondary, tertiary, and Quaternary structure). The spatial arrangement of atoms in a protein is called conformation. Any change in the conformation may lead to disorder in function and disease. 33 Protein Chemistry Definitions of Levels of Organization 1. Primary structure: - Definition: The linear sequence of amino acids. Each protein has unique sequence, determined by the sequence of the gene. - Bond responsible: The primary structure is maintained by peptide bonds. 2. Secondary structure: - Definition: it is the spatial relationship of adjacent amino acid residues. - Bond responsible: Hydrogen bonds between the hydrogen of -NH group of one amino acid and the oxygen of C=O group of the fourth one. - Forms: The folding of the polypeptide chain into α-helices and β- pleated sheets. N.B. Marfan syndrome and Alzheimer’s disease: results from a disturbance in the secondary structure of the protein. β- pleated sheets α-helices 3. Tertiary structure (three-dimensional structure): - Definition: The final arrangement of a single polypeptide chain resulting from spatial relationship of more distant amino acids. - Bonds responsible: hydrogen bonds, disulfide bonds, and hydrophobic interactions. - Forms: Fibrous (e.g keratin, collagen and elastin) and globular (e.g. myoglobin). 34 Protein Chemistry 4. Quaternary structure: - Definition: Some of the proteins are composed of two or more polypeptide chains. Each polypeptide chain is called subunits. Each subunit has its own primary, secondary and tertiary structure. The spatial arrangement of these subunits is known as the quaternary structure. - The protein loses its function when the subunits are dissociated. - Bonds responsible: hydrogen bonds, disulfide bonds, and hydrophobic interactions. - Examples of proteins having a quaternary structure:  Insulin: 2 subunit.  Creatine kinase ''dimer'': 2 subunits  Lactate dehydrogenase ''tetramer: 4 subunits  Hemoglobin: 4 subunits ''tetramer'' (two α and two β subunits).  Immunoglobulin: 2 heavy chains and 2 light chains. 35 Protein Chemistry Classification of Proteins 1. Classification according to nutritional value: - Nutritionally rich proteins high biological value proteins: Contain all essential amino acids and easily digested e.g. meat, milk, fish, liver. Soy protein: is the only vegetable protein that provides all essential amino acids. It is considered to heart healthy, since it lower cholesterol. it is also taste neutral and can flavor to prepared savory or sweet products. - Incomplete proteins: They lack one essential amino acid. They cannot promote body growth in children; but may be able to sustain the body weight in adults. Proteins of cereals lack in lysine. - Poor proteins: They lack in many essential amino acids. Zein from corn lacks tryptophan and lysine. 2. Classification based on the shape: - Globular proteins: They are spherical or oval in shape. They are easily soluble. Most enzymes, antibodies, and hormones are globular proteins. - Fibrous proteins (Scleroproteins): The molecules are elongated, or needle-shaped. They are insoluble and resist digestion. e.g. Collagen, Elastin, Keratins. - Intermediate proteins: Their structure is intermediate between globular proteins and fibrous proteins. They are soluble in water. e.g. fibrinogen 3. Classification based on the function of protein: - Contractile proteins e.g. myosin, actin. - Transport proteins e.g. hemoglobin, albumin, transferrin. - Regulatory proteins or hormones e.g. insulin and growth hormone. - Genetic proteins e.g. histones. - Protective proteins (protect from any diseases) e.g. immunoglobulins. - Catalytic proteins e.g. enzymes. - Structural proteins e.g. collagen, elastin. 36 Protein Chemistry 4. Classification according to chemical composition and properties: Proteins may be divided into three major groups: simple, conjugated, and derived. A. Simple Proteins: they contain only amino acids. - Albumins & Globulins: High biological value proteins present. - Scleroproteins: They form supporting tissues e.g. Collagen, Elastin, Keratins. B. Conjugated Proteins: They are formed of a protein part (apoprotein) and a non-protein part (prosthetic group). According to the nature of the prosthetic group, they are further classified into: - Phosphoproteins: These are proteins conjugated to a phosphate group e.g. Casein of milk (caseinogen). - Lipoproteins: These are proteins conjugated to lipids e.g. plasma lipoproteins. - Glycoproteins: These are proteins conjugated to carbohydrates e.g. Blood group antigens and immunoglobulins. - Nucleoproteins: These are proteins attached to nucleic acids e.g. Histones. - Chromoproteins: These are proteins with colored prosthetic groups e.g. Hemoglobin (Heme, red). - Metalloproteins: These are proteins conjugated to metals (Fe, Cu & Zn). Metalloproteins Containing Iron (Fe): The iron may be in the form of heme or non heme iron (NHI). a) Hemoproteins: They are proteins containing iron in the form of heme. Hemoproteins include hemoglobin, myoglobin, and cytochromes. b) Non-heme iron containing proteins: They include ferritin, transferrin and hemosedrin. 37 Protein Chemistry Structure-Function Relationship 1. Keratin: It is a fibrous protein. Present in hair and nails. The properties of the keratin present in different tissues are due to the differences in the number and position of disulfide bonds. 2. Hemoglobin: The transporter of oxygen. The binding of oxygen to one heme facilitates oxygen binding by other subunits. Even a single amino acid substitution alters the structure and thereby the function. For example, in sickle cell anemia (HbS). 3. Collagen: Function and location: - It is the most abundant protein in mammals. - The main fibrous component of bone, tendon, cartilage, and teeth. Structure and solubility:  Collagen is a simple protein; consist of 3 polypeptide chains are wound around each other forming triple helix molecules.  The 3 polypeptide chains are held together by hydrogen bond.  Collagen is insoluble in all solvents. Low biological value protein. Not digested.  When collagen is heated (denaturation) gelatin is formed. Gelatin is soluble in water and digestible. 38 Protein Chemistry The amino acid composition and sequence:  Composition: Collagens contain 33% Glycine, 10% proline, 10% hydroxyproline and 10% hydroxy lysine.  Sequence: every third amino acid in chain is Glycine. the repeating sequence is Gly-X-Y sequence, where X are mainly proline and Y is often hydroxyproline or hydroxylysine. Post-translational modifications in collagen:  Vitamin C is important for the hydroxylation of proline to hydroxyproline and lysine to hydroxylysine.  In vitamin C deficiency, failure of hydroxylation of proline/lysine leads to reduced hydrogen bonding and consequent weakness of collagen. Collagen has a very firm structure due to:  Each helical turn contains only 3 amino acids. Other protein contains 3.6 amino acids in each turn.  Glycine form 33% of total molecules. This makes the polypeptide chain compact.  The high content of hydroxyproline and hydroxylysine increases the number of hydrogen bonds. Importance of collagen fibers in the gingiva  The ends of the collagen fibers of the periodontal ligament are embedded in calcified cementum and bone to anchor the tooth into the bony socket.  Provide structural support to the gingival tissue.  Maintain the alignment of the teeth.  Absorb masticatory forces. 39 Protein Chemistry Plasma Proteins  The normal value of plasma proteins is 6 to 8 g/100 ml.  Plasma proteins include albumin, globulin, and fibrinogen. Functions of Plasma Proteins 1. Nutritive: They are simple proteins that are a good source of protein. 2. Osmotic pressure: albumin contributes to the osmotic pressure of the blood. 3. As buffers: Proteins can combine with acids or bases. So, help in maintaining pH of the body. 4. Viscosity of blood: The viscosity of blood provides resistance to flow of blood in the blood vessels to maintain blood pressure in the normal range. Globulins and fibrinogen, which are large in size account for the viscosity of blood. 5. Reserve proteins: Proteins serve as a source of proteins for the tissues when the need arises. 6. Role in blood coagulation and fibrinolysis: e.g. prothrombin and fibrinogen. 7. As a carrier of certain metabolites: albumin serves as nonspecific transporter for insoluble substances such as bilirubin, free fatty acids, steroid hormones and lipids. 8. As immunoglobulins: The property of antibodies formation resides in γ- globulin fraction of the proteins. Acute Phase Proteins The level of certain proteins in blood may increase 50 to 1000 folds in various inflammatory and neoplastic conditions. Such proteins are acute phase proteins. 1. C-Reactive Protein (CRP). 2. Ceruloplasmin. 3. Haptoglobin. 4. α1-antitrypsin. 5. Fibrinogen. 40 Protein Chemistry Denaturation of proteins (Loss of protein conformations) Definition: unfolding and loss of secondary, tertiary, and quaternary structures of proteins. Does not affect primary structure. Denaturing factors: - Physical: Mild heating, X-ray, ultraviolet rays, high pressure, vigorous shaking, repeated freezing and thawing. - Chemical: strong acids and alkalis and salicylate. - Digestive enzymes. Effect of protein denaturation: - Decreased solubility. - Increased viscosity. - Loss of biological activity as in case of enzymes, hormones, antigens, or antibodies. - Increased digestibility due to unfolding of the peptide chains. 41 Enzymes Enzymes  Enzymes are specific protein catalysts that accelerate the rate of chemical reaction. They are produced by the living organism and are usually present in a very small amount in various cells.  Lack of enzymes will lead to blocks in metabolic pathways causing inborn errors of metabolism.  The substance upon which an enzyme acts, is called the substrate. The enzyme will convert the substrate into the product or products General properties of enzymes are: 1. All enzymes are proteins with exception of ribozymes. 2. Enzymes accelerate the rate of reaction. 3. They are heat labile, soluble in water. 4. Enzymes possess active sites at which interaction with substrate takes place. Enzyme structure According to the chemical nature of enzymes, it can be classified into two main categories: 1. Simple enzymes: are formed of protein only e.g. lipase, amylase. 2. Conjugated enzymes (holoenzymes) consisting of : a) Protein part of the enzyme known as apoenzyme. b) Non-protein parts are known as coenzymes or prosthetic groups.  If such a compound is firmly attached to enzyme proteins, then it is called a prosthetic group.  If its attachment to protein is not very firm, then it is called a coenzyme. Holoenzyme = Apoenzyme + Coenzyme 42 Enzymes Apoenzyme Coenzyme Structure Protein Non-protein Effect of heat Heat labile Heat stable Molecular weight High Low Dialysis Non- dialyzable Dialyzable Coenzymes - A coenzyme is a low molecular weight, non-protein, organic substance, which is essential for the biological activity of some enzyme. - Coenzymes are commonly derived from vitamins. Therefore, vitamins are essential in our diet. They act as carriers for certain groups, which must be added to or removed from the substrate. 43 Enzymes Classification of Enzymes 1. Oxidoreductases: They catalyze the transfer of hydrogen in oxidation and reduction reactions e.g. catalase. 2. Transferases: They catalyze the transfer of groups other than hydrogen from one molecule to another molecule e.g. transaminases. 3. Hydrolases: They catalyze the removal of groups from the substrate by the addition of water molecules e.g. peptidases and phosphatases. 4. Lyases: They catalyze the removal of groups from the substrate without the addition of water e.g. decarboxylases. 5. Isomerases: They catalyze the conversion of a compound into an isomer, e.g. racemases, epimerases, isomerases and mutases. 6. Ligases: They catalyze the condensation of two molecules coupled with the breaking of pyrophosphate bound in ATP e.g. glutamine synthetase. Enzyme Specificity There is various degree of enzyme specificity: 1. Absolute specificity: One enzyme acts only on one substrate e.g. Glucokinase. 2. Group specificity: Enzyme active site can recognize many substrates, all belonging to the same group of compounds e.g. Trypsin catalyzes the hydrolysis of the peptide bonds in several proteins. 3. Reaction specificity: The enzyme catalyzes only one type of reaction e.g. Oxidoreductases. 4. Stereospecificity: Some enzymes show specificity towards D- and L- form of the same substrate e.g. D-amino acid oxidase. 44 Enzymes Mechanism of Action of Enzymes - Catalytic (Active) sites: This is the substrate binding site. - Michaelis and Menten’s hypothesis for enzyme action  According to their hypothesis, the enzyme molecule (E) first combines with a substrate molecule (S) to form an enzyme substrate (ES) complex which further dissociates to form product (P) and enzyme (E) back.  Enzyme once dissociated from the complex is free to combine with another molecule of the substrate and form a product in a similar way. Models of enzyme-substrate complex formation These interactions have been described basically as two types. 1. Template or Lock-and-Key Model - The substrate fits into the active site of an enzyme. This model states that the active site by itself provides a rigid, pre-shaped template fitting with the size and shape of the substrate molecule. 2. Induced-Fit or Koshland Model - The important feature of this model is the flexibility of the region of active site. 45 Enzymes - The substrate during its binding induces conformational changes in the active site to attain the final catalytic shape and form. This model is more consistent with a wider range of enzymes. Factors influencing the rate of enzymatic reactions 1. Concentration of the substrate: The velocity of enzyme action is directly proportional to the concentration of the substrate. 2. Concentration of the enzyme: The velocity of the reaction is directly proportional to the enzyme concentration but also up to certain point. 3. Effect of temperature: For each enzyme there is an optimum temperature at which the action of enzyme is maximum and above or below which the activity gradually decrease. 4. Effect of pH: Each enzyme has an optimum medium pH above and below which the activity gradually decrease. 5. Activators: Enzyme activators are inorganic ions. Chloride ions activate amylase and magnesium ions activate kinases. 46 Enzymes Enzyme Activation Mechanisms for enzyme activation include the following: 1. Auto-activation - Conversion of an inactive proenzyme or zymogen to an active enzyme. - Activation requires proteolysis (removal of a part of the polypeptide chain which masks the active site or substrate site). This results in the unmasking of the active center. These changes are not reversible. e.g. pepsinogen is activated by pepsin, and trypsinogen is activated by trypsin. - All gastrointestinal enzymes are synthesized in the form of proenzymes, and only after secretion into the alimentary canal, they are activated. This prevents the autolysis of cellular structural proteins. 2. Metal ion activation The metal interacts with the enzyme and helps its binding with the substrate e.g. chloride ion activates salivary amylase. 3. Allosteric activation Certain enzymes contain specific site (allosteric site) other than the catalytic site. The binding of an allosteric activator with the allosteric site produces conformational changes in the protein structure of the enzyme which results in increased velocity of the reaction. 47 Enzymes 4. Covalent Modification - The process of altering the activity of enzymes by adding or removing groups is called a covalent modification. - Many enzymes are activated by phosphorylation and inactivated by dephosphorylation and vice versa. Enzyme Inhibition An enzyme inhibitor is defined as a substance that binds with the enzyme and decrease the catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature. There are two broad categories of enzyme inhibition - Reversible inhibition. - Irreversible inhibition. 1. Reversible inhibition: The inhibitor binds non-covalently with the enzyme and the enzyme inhibition can be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into I. Competitive inhibition. II. Non-competitive inhibition. I. Competitive inhibition: - A competitive inhibitor is structurally similar to the substrate. The inhibitor competes with the substrate to bind reversibly at the active site of the enzyme. - As long as the competitive inhibitor holds the active site, the enzyme is not available for the substrate to bind. The reaction velocity is decreased. - The inhibition can be reversed by increasing the concentration of substrate. 48 Enzymes - Examples:  Allopurinol: A drug used for the treatment of gout.  Dicoumarol: Used as an anticoagulant because they are structurally similar to vitamin K (required for activation of blood clotting factors).  Statin: Inhibit the key enzyme of cholesterol synthesis. II. Non-competitive inhibition: - Non-competitive inhibitors are not structurally related to the substrate, and they are not competing for binding to the active site. They can bind either the free enzyme or the enzyme-substrate complex. - They are not overcome by increasing substrate concentration. 1. Inhibition by removal of catalytic ions e.g. Addition of EDTA to blood prevents blood clotting by removal of calcium ions. 2. Inhibition by phosphorylation and dephosphorylation (covalent modification) 3. Allosteric inhibition: The inhibitor binds to the enzyme at a site other than the active site and on a different region in the enzyme molecule called allosteric site. 49 Enzymes 2. Irreversible inhibition: The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. This type of inhibition cannot be reversed by adding more substrate and includes: 1. Nerve gas irreversibly binds with acetylcholine esterase (essential for nerve conduction), resulting in paralysis of vital body functions. 2. Cyanide inhibits cytochrome oxidase (binds to iron atom) of the electron transport chain. 3. Fluoride inhibits enolase (by removing manganese), and thus inhibits glycolysis. Isoenzymes These are a group of enzymes which are characterized by the following: - They are present in different tissues. - They catalyze the same reaction. - They have different polypeptide chains which are produced by different genes. - They have different affinities to the substrate. Examples of isoenzymes 1. Lactate dehydrogenase (LDH) - It is a tetrameric enzyme (4 subunits). The subunit may be either H or M polypeptide chains. The tetrameric molecule is the only active form of the enzyme. The different subunits are combined to form five isoenzymes - Elevated levels of LDH1 and LDH2 indicate myocardial infarction. - Elevated levels of LDH5 indicate possible liver damage. 50 Enzymes 2. Creatine Kinase - It is a dimer formed of two subunits termed M or B. It has three isozymes. - Estimation of CK isozymes in plasma is of clinical importance. CK-BB is present in brain tissues and its plasma level increases in case of brain infarction. CK-MB is present mainly in cardiac muscles and its plasma level increases in myocardial infarction. CK-MM is present mainly in skeletal muscles and its plasma level increases in muscle diseases. Salivary Enzymes Saliva supplies enzymes for digestion. The main enzymes are: 1. Amylase - The major salivary enzyme is alpha-amylase. The amylase acts on carbohydrates. It cleaves the alpha-1,4-glycosidic bonds of starch. - Chloride ion activates salivary amylase. - Amylase also shows weak antibacterial properties as well as buffering properties. 2. Lingual lipase: Acts on triglycerides and is important in the digestion of milk fat in infants. 3. Carbonic anhydrase: Responsible for the buffering action of saliva. 4. Lysozyme: Has antimicrobial action. The bactericidal effect is by breaking down the muramic acid present in bacterial cell walls. Clinical correlations: The levels of alanine amino transferase, aspartate amino transferase, gamma glutamyl transferase, and alkaline phosphatase are raised in the saliva of patients with gingivitis and periodontitis. 51 Enzymes Uses of Enzymes 1. Diagnostic uses: Enzyme estimation in serum and body fluids is used in the diagnosis and prognosis of diseases. e.g. CK-MB increases in myocardial infarction. 2. Therapeutic uses: e.g. Streptokinase and urokinase are used in the treatment of acute myocardial infarction and deep vein thrombosis. 3. laboratory uses: A. Enzymes use in laboratory as reagent for investigation - Glucose oxidase enzyme is used for the estimation of glucose in blood. - Uricase enzyme is used for the estimation of serum uric acid. B. Uses in genetic engineering - Restriction endonuclease: DNA fingerprinting - Taq DNA polymerase: polymerase chain reaction (PCR). 52 Vitamins Vitamins Vitamins are small organic molecules which are not synthesized in the body or are synthesized in inadequate amounts and hence must be present in diet. Most vitamins are precursors of coenzymes, they are also precursors of hormones or act as antioxidants. General Characteristics 1. Vitamins are of widespread occurrence in nature, both in plant and animal. 2. Human body can synthesize some vitamins, e.g., Vitamin A is synthesized from its precursor carotene. Vitamin D from ultraviolet irradiation of 7- dehydrocholesterol. Some members of the vitamin B complex and vitamin K are synthesized by microorganisms present in the intestinal tract. 3. They differ from other organic foodstuffs in that they do not enter into the tissue structure and do not undergo degradation for the purpose of providing energy. They are classified into: a. Fat soluble vitamins: Vitamin A. Vitamin D. Vitamin E. Vitamin K. b. Water soluble vitamins: Vitamin C Members of vitamin B complex. 53 Vitamins Fat Soluble Vitamin Vitamin A (Retinol or Anti -Night blindness) Source: Provitamin (beta-carotene) sources: present in yellow, orange and dark green leafy vegetables and fruits such as carrot, tomatoes. Readymade sources: Fish liver oils such as shark cod, halibut fish, liver oils. Functions 1. The most important function of vitamin A is in the visual cycle. 2. Maintenance of proper health of epithelium tissues. 3. Antioxidant. 4. In mouth: - It helps to maintain healthy mucous membranes. - It also promotes saliva production. - Vitamin A helps the growth of teeth and bones in babies and children. Manifestations of Vit A Deficiency 1. Night blindness: Visual acuity is diminished in dim light. 2. Xerophthalmia: The conjunctiva becomes dry, thick and wrinkled. 3. Bitot spots: These are seen as grayish-white triangular plaques firmly adherent to the conjunctiva. 4. Keratomalacia: Softening of the cornea. 5. Xeroderma: Dry & rough keratinized skin. Keratomalacia Dry skin Xerophthalmia 54 Vitamins Oral signs and symptoms of Vitamin A deficiency: 1. Increase in keratin formation. 2. Salivary gland ducts can be blocked. 3. Delay in Eruption of teeth. 4. Enamel is severely affected. 5. Pulpal tissue is invaded by epithelial tissue. Therapeutic uses of vitamin A It has been used in oral leukoplakia, a precancerous condition. It improve the condition by reverting the cells to normal epithelium. Vitamin D3 (Cholecalciferol or Antirickets) Sources: Fish liver oils (i.e. cod liver oil, shark liver oil, halibut liver oil), egg yolk and milk. Endogenous source: - Vitamin D3 produced in skin by UV irradiation of 7-dehydrocholesterol (an intermediate in cholesterol synthesis) present in subcutaneous fats to produce cholecalciferol. - The cholecalciferol is activated in liver and kidney to calcitriol. - Calcitriol is the active form of vitamin. 55 Vitamins Function: 1- The main function of calcitriol is maintenance of serum calcium level through its effect on intestine, kidney and bones. a. It increases calcium absorption by intestinal cells. b. It increases renal tubular reabsorption of calcium. 2- It is essential for normal bone calcification. 3- The activate form of vitamin D acts as a hormone and regulating cell growth. 4- Regulates insulin secretion, parathyroid and thyroid hormones. Deficiency: Deficiency of vitamin D gives rise to osteomalacia in adults and rickets in children. Clinical Features of Rickets a. Rickets is seen in children. There is insufficient mineralization of bone. Bones become soft. The bone growth is markedly affected. b. The classical features of rickets are bone deformities. Weight bearing bones are bent. c. The clinical manifestations include bow legs, knock- knee, rickety rosary, bossing of frontal bones, and pigeon chest. 56 Vitamins Clinical features of osteomalacia The bones are softened due to insufficient mineralization and increased osteoporosis. Patients are more prone to get fractures. Oral signs and symptoms of Vitamin D deficiency: 1. Delayed eruption of primary and permanent teeth. 2. Developmental anomalies of dentin and enamel. 3. Pulp is also affected; the pulp horns are elongated. 4. Malocclusion of teeth is seen in the oral cavity. Vitamin E (Tocopherols or Rat Antisterility Vitamin) Sources: Wheat germ oil, corn oil, peanut oil, soyabean oil and sunflower oil. Function 1. Antioxidant: Vitamin E is the most potent and most biological antioxidant. 2. Protection of erythrocyte membrane from oxidants. Vitamin E protects RBC from hemolysis. 3. It reduces the risk of atherosclerosis by reducing oxidation of LDL. 4. In mouth: Prevent periodontal disease and works against it in two ways: a. Through decreasing inflammation in the mouth. b. Being an antioxidant. It has also been used to relieve soreness of the gums during infant teething. Deficiency: 1. Anemia in newborn infants and in pregnant and lactating females, due to decrease life span of RBC’s. 2. Muscle weakness. 3. Neurological dysfunction. 57 Vitamins Vitamin K (Anti- Hemorrhage Vitamin) Sources: - Vitamin K1 is present chiefly in green leafy vegetables, such as spinach. - Vitamin K2 is a product of metabolism of normal intestinal bacteria. - Vitamin K3 is water soluble synthetic vitamin. Function: 1. Vitamin K is necessary for coagulation: - Factors dependent on vitamin K are Factor II ; Factor VII; Factor IX; Factor X. 2. Vitamin K dependent gamma carboxylation is also necessary for the functional activity of osteocalcin. Osteocalcin (calcium binding protein in bone) is synthesized by osteoblasts and seen only in bone. Deficiency of Vitamin K: Causes for Deficiency: Vitamin K is formed normally by intestinal bacteria so its deficiency is a rare condition may result from: 1. Prolonged administration of oral antibiotics that kill the intestinal bacteria. 2. Failure to absorb vitamin K in cases of steatorrhea (failure to absorb fat and fat soluble vitamins). 3. Liver diseases: failure to utilize vitamin K. 4. New born infants have tendency to bleed due to vitamin K deficiency as their intestinal flora is not well developed and vitamin K cannot pass the placental barrier. Clinical Manifestations of deficiency 1. Easy bruising and bleeding. 2. Hemorrhagic disease of the newborn. Clinical correlations: Dicumarol and warfarin act as anticoagulants because they are structurally similar to vitamin K. They produce competitive inhibition of clotting enzymes. 58 Vitamins Spontaneous gingival hemorrhages Bleeding nose Oral Signs and Symptoms of Vitamin K deficiency: - Spontaneous gingival hemorrhages. Water soluble vitamins Water-soluble vitamins include vitamin B complex and vitamin C. I – Vitamin C (L- Ascorbic Acid or Antiscurvy) Sources: Citrus fruits such as lemon, orange, pineapple, green pepper and tomatoes. Function: 1. Antioxidant a. Regenerates vitamin E. b. ↓ Oxidation of LDL. 2. Keeps iron in Fe2+ reduced state. 3. Collagen synthesis - Essential for hydroxylation of proline and lysine to prolyl and lysyl hydroxylases. Deficiency: 1. Scurvy - Swollen gums, loose of teeth, bruising, perifollicular hemorrhage, poor wound healing, glossitis, ↑ bleeding time. 2. Anemia due to combined iron and folate deficiency. 59 Vitamins Bruising Perifollicular hemorrhage Lose of teeth II- Vitamin B complex These vitamins are chemically not related to one another. They are grouped together because all of them function in the cells as coenzymes. Vitamin B1 (Thiamine or Anti- Beri Beri) Sources: Yeast, outer coating of seeds, cereals, legumes, wheat and eggs. Function: Thiamine it is converted into its active form, thiamine pyrophosphate (TPP) which act as a cofactor for several enzymes such as a. Pyruvate dehydrogenase (glycolysis). b. Transketolase (HMP shunt). Deficiency: Beriberi a. Dry beriberi - Peripheral neuropathy due to demyelination. - Symmetrical muscle wasting. b. Wet beriberi - Dilated cardiomyopathy. - Oedema. 60 Vitamins Vitamin B2 (Riboflavin) Sources: Rich sources are liver, dried yeast, egg and whole milk. Function: - Riboflavin is converted to its active coenzyme forms flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). - FMN and FAD serve as coenzymes for the enzymes called flavin-dependent enzymes. FMN-dependent enzymes: FMN is a cofactor for L-amino acid oxidase. FAD-dependent enzymes: FAD is a cofactor for xanthine oxidase. Deficiency Manifestations: Symptoms are confined to skin and mucous membranes. 1. Seborrheic dermatitis. 2. Conjunctival injection, vascularization of cornea, burning of eyes, photophobia and cataract. 3. Oral Signs and Symptoms of Vitamin B2 deficiency: a) Cheilosis: It is the inflammation or cracking of the corners of the mouth. b) Glossitis: It is the inflammation of the tongue. c) Stomatitis: It is inflammation of the mouth and lips. Cheilosis Glossitis Stomatitis 61 Vitamins Vitamin B3 (Nicotinic acid, Niacin) (Pellagra Preventive Factor) Sources - Yeast, meat, liver, kidney, eggs, fish and legumes. - About half of the requirement is met by the conversion of tryptophan to niacin; 60 mg of tryptophan gives rise to 1 mg of niacin. This conversion requires vitamin B6. Function: 1. Oxidation and reduction: Nicotinamide adenine dinucleotides (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are active coenzymes of dehydrogenases for various oxidation reduction reactions. Deficiency: Pellagra Causes for niacin deficiency - Farmers who use maize flour for preparation of bread are more liable to suffer from pellagra than those using wheat flour. - Maize is deficient in both tryptophan and nicotinic acid. - Vitamin B6 deficiency may be a factor in the development of pellagra, as it is required for conversion of tryptophan to nicotinic acid. Symptoms of pellagra comprise three D’s: Diarrhea, dermatitis and dementia. The fourth D, i.e. death, follows in the untreated cases. Therapeutic Uses - Nicotinic acid is used in the treatment of hyperlipidaemia. - Hypoglycaemic effects with large doses have also been observed. 62 Vitamins Vitamin B6 (Pyridoxine) Sources: Yeast, liver, egg yolk and rice polishing. Function: 1. Converted to pyridoxal phosphate (PLP) which acts as coenzyme for many reactions, especially for amino acid metabolism. Transamination: PLP is a coenzyme for transaminases, where it acts as an amino group carrier. Decarboxylation: All decarboxylation reactions of amino acid metabolism require PLP. 2. Required for heme synthesis. 3. Required for the synthesis of niacin from tryptophan. 4. Necessary for the absorption of amino acids from the intestinal mucosa. Deficiency: - Hematological manifestation: Hypochromic microcytic anemia may occur due to impaired heme synthesis. - Dermatological manifestation: B6 deficiency leads to niacin deficiency which is manifested as pellagra - Neurological manifestation: - Convulsions. - Peripheral neuritis. Therapeutic uses: Vitamin B6 has been used in treatment of nausea and vomiting of pregnancy (morning sickness) and radiation sickness. 63 Vitamins Vitamin B12 (Cobalamin) (Anti-pernicious Anemia Factor) Sources: Liver is the richest source, kidney, meat, fish and egg yolk. Absorption of Vitamin B12 : Vitamin B12 combines with the intrinsic factor (IF). Intrinsic factor is secreted by the gastric parietal cells. This IF-B12 complex is attached with specific receptors on mucosal cells. The whole IF-B12 complex is absorbed. Function: 1. Important for nucleic acid synthesis. 2. Helps in neuronal metabolism and brain function. 3. Helps in the production of red blood cells. Deficiency Manifestations: 1. Hematological abnormalities: Megaloblastic anemia, leucopenia and thrombocytopenia. 2. Gastro-intestinal manifestation: glossitis and atrophy of gastric mucosa. 3. Neurological manifestations which include peripheral neuritis. Oral Signs and Symptoms of Vitamin B12 deficiency: Hunter’s glossitis Folic Acid Sources: Yeast, liver, kidney and green vegetables. Function: The folic acid is reduced to tetrahydrofolic acid (THFA). The THFA is the carrier of one-carbon groups. One-carbon groups play a pivotal role in donating carbon atoms for synthesis of different types of compounds: Synthesis of purine nucleotides. 64 Vitamins Synthesis of glycine and serine amino acids. Methyl group in methyl THFA is used for synthesis of active methionine, which takes part in transmethylation reactions. Manifestations of deficiency 1. Macrocytic, megaloblastic anemia. 2. Homocysteinemia. 3. ↑ Risk of deep venous thrombosis and atherosclerosis. 4. Deficiency in pregnancy causes fetal neural tube defects. Oral Signs and Symptoms of Folic Acid deficiency: Glossitis. 65  Molecular Biology Nucleic Acid Chemistry and Metabolism  Nucleic acids (DNA and RNA) are polynucleotides (polymers of nucleotides).  Their building block is Nucleotides, found in all cells, which participate in the storage, transmission, and translation of genetic information. Differences between DNA & RNA DNA RNA Types One Three Site Nuclei and mitochondria Cytoplasm, Nuclei, mitochondria Structure Double-helix Single strand Sugar Deoxyribose Ribose Base A,G,C,T A,G,C,U Function  DNA is the permanent storage place  RNA synthesized by DNA for for genetic information. transportation of genetic information  DNA controls the synthesis of RNA. to the protein building apparatus in  The sequence of the nitrogenous the cell. base in DNA determines the protein development in new cells.  The function of double helix formation of DNA is to ensure that no disorder occurs. This is because the second identical strand of DNA is a backup in case of lost or destroyed genetics. 66 Molecular Biology Nucleotide metabolism Nucleotide roles  Formation of DNA and RNA.  Energy : ATP.  Physiological mediators e.g. cAMP, cGMP: second massenger Sources of nucleotides  Diet (exogenous).  Biochemical synthesis (endogenous) - Direct synthesis. - Salvage pathway. Composition of Nucleotides  A nucleotide has three structural components: - Nitrogenous base (a purine or a pyrimidine), - Pentose sugar (either ribose or deoxyribose) and - Phosphate groups.  A nucleoside (No Phosphate groups) is formed from - Nitrogenous base (a purine or a pyrimidine), - Pentose sugar (either ribose or deoxyribose). 67 Molecular Biology Nitrogenous Bases The nitrogenous base is linked to the ribose or deoxyribose sugar via a glycosidic bond. The nitrogen bases in nucleotides consist of two general types :  Purines: adenine (A) and guanine (G).  pyrimidines: cytosine (C), thymine (T) and Uracil (U) N.B. THYMINE is the base present in DNA while THIAMINE is the vitamin B1. Purine metabolism Synthesis of Purine Nucleotides: The purine nucleotides are synthesized by most of the tissues. However, the major site of purine synthesis is in the liver. This pathway operates in the cytoplasm. Purine ingredients: - Ribose phosphate (HMP shunt). - Amino acids. - Carbons (Folic acids, Co2). 68 Molecular Biology There are two synthetic pathways: 1. De novo synthesis: Major pathway, the purine ring is synthesized from different small components. 2. Salvage pathways: - This pathway ensures the recycling of purines formed by the degradation of nucleotides. - The salvage reactions reduce the requirement for the energetically expensive de novo biosynthesis and prevent wastage of raw materials. - This is of special importance in tissues like RBCs and the brain where the de novo pathway is not operating. Degradation of purine nucleotides (Formation of uric acid) - Uric acid is the final product of human purine degradation. - Normal concentration of uric acid in serum is 3–7 mg/dl in males and 2-6 mg/dl. The uric acid concentration in urine is 0.7gm/day, this amount decreases in cases of a protein-free diet. Disorders of Purine Metabolism A. Hyperuricemia refers to increased serum uric acid concentration. It may or may not be associated with increased excretion of uric acid in the urine, which condition is called uricosuria. B. Gout is a clinical syndrome that is characterized by hyperuricemia and recurrent acute arthritis. Renal disease (nephropathy) is also a common complication. Not all patients with hyperuricemia develop gout. Risk factors of gout: - Hyperuricaemia has been identified as the most important risk factor for the development of gout. - Older age. - Male sex - Diet rich in meat. - Genetics. 69 Molecular Biology - Dehydration: uric acid cannot be diluted and efficiently released through the kidneys. - Soft drinks: Fructose which is added as a sweetener to sodas can increase uric acid levels. - Seafood: Mackerel, sardines, tuna, and Shellfish are high in purine. - Obesity is often linked to gout because excessive fat leads to insulin resistance which makes your body less efficient at removing uric acid. Types of gout: Gout may be either metabolic or renal 1. Metabolic gout: can occur from - Overproduction of uric acid. - High cell turnover e.g. cancer and hemolytic anemia. 2. Renal gout: can occur from - An inherited defect in the kidney that leads to failure of tubular excretion of uric acid and an increase in blood uric acid. - Lead poisoning or chronic renal decrease in uric acid excretion. Symptoms of gout :Clinical manifestations of abnormal purine catabolism arise from the insolubility of the degradation by-product, uric acid. 1. Gouty arthritis: Crystal deposition in joints triggers the inflammatory response (hot red and swollen joints with severe pain). 2. The crystals can be deposited in subcutaneous tissues also, most commonly in the external ear, over the knees and elbows, and along the tendons. 3. Nephropathy: uric acid may precipitate in the kidneys and cause renal damage. Being of limited solubility, it tends to crystallize as urinary stones. Treatment of Gout: 1. Diet: Reduce dietary purine intake e.g. meat, liver, kidney. The chief source of proteins for these patients should be milk, cheese, yogurt, and eggs. 2. Drugs: a) Allopurinol: Reduce uric acid production. 70 Molecular Biology b) Uricosuric drugs: Increase renal excretion of urate e.g. salicylates and cortisone. c) Colchicine: an anti-inflammatory agent to arrest arthritis. Metabolism of pyrimidine nucleotides Pyrimidine ingredients: - Ribose phosphate (HMP shunt). - Amino acids. - Carbons (Folic acids, Co2). Synthesis of Pyrimidine Nucleotides: The pyrimidine ring (unlike the purine) formed first then ribose sugar was added. Catabolism of pyrimidine bases: - The pyrimidine ring is degraded to highly soluble products. - The end products of pyrimidine catabolism are β-alanine and β- aminoisobutyrate, with the production of NH3 and CO2. Disorders of pyrimidine metabolism: Because the products of pyrimidine catabolism are soluble, few disorders result from excess levels of their synthesis or catabolism. 71 Molecular Biology DNA structure - Contains genetic codes. - Found in the nucleus of eukaryotic cells. - Found in the cytoplasm of prokaryotic cells. - Deoxyribonucleic acid (DNA) is composed of four deoxyribonucleotides. It has the following primary, secondary, and tertiary structures. Primary structure:  The four deoxyribonucleotide units are linked by 3' 5'–phosphodiester bonds. The alternating sugar-phosphate units form the backbone of each DNA strand. The nitrogenous bases, which are linked to the pentose units, by a glycosidic bond, are projecting to the inside of the two strands of DNA at a right angle.  The polymer has Polarity: one end has a free phosphate group at 5' end and a free hydroxyl group at the other 3' end. DNA secondary structure (double helix):  DNA exists as two polynucleotide chains wound about a common axis in the form of a double helix.  These two polynucleotide chains are antiparallel, which means that one strand runs in the 5' to 3' direction, while the other is in the 3' to 5' direction.  DNA double helix is stabilized by hydrogen bonding between the purine and pyrimidine bases.  Nitrogenous bases pair with complementary bases. So, the adenine of one strand will pair with the thymine of the opposite strand, while guanine will pair with cytosine. 72 Molecular Biology DNA 3ry structure (Chromatin):  Histones: Peptides which are formed from basic amino acids. Positively charged histones bound to the negatively charged phosphate backbone of DNA.  Nucleosome: Units of histones (key protein) plus DNA. Histones condense to Chromatin. Chromatins condense to chromosomes.  A different forms of 3ry structure DNA - Double-stranded linear (e.g. eukaryotic nuclear chromosome). - Double-stranded circular (e.g. mitochondrial, plasmid, virus). - Single-strand circular DNA (e.g. small viruses). 73 Molecular Biology  Gene: a part of a chromosome, which occupies a specific position (locus) on it.  Exons (expressed regions): The segments of the gene coding for proteins.  Introns (intervening areas): They are silent areas (not translated) in the DNA. Denaturation of DNA helix: separation of the double-stranded DNA by disruption of H-H bonds between the two strands. 1. Alkali treatment led to the ionization of bases and disruption of H bonds. 2. Heating: When the temperature of the medium containing a DNA molecule is raised, the hydrogen bonds linking the complementary base pairs tend to break. Melting Temperature: indicates the temperature at which half of the double- stranded structure is lost. Regions of the double helix that have predominantly A-T base pairs will be less stable than those rich in G-C base pair. 74 Molecular Biology Types of RNA - RNA is unbranched polymeric molecules composed of nucleoside monophosphates joined together by phosphodiester bonds. - There are three major types of RNA all produced in the nucleus from DNA and participate in the process of protein synthesis. - They differ in size, function, and stability. A. Ribosomal RNA: It comprises 50-80% of the total cellular RNA. They are involved in protein biosynthesis and are very stable. B. Transfer RNA: It comprises 10-20% of the total RNA of the cell. There are at least 20 tRNA, one for each A.A. They act as adaptors for carrying A.A. during protein synthesis. C. Messenger RNA: It carries the message (genetic information from the DNA for the synthesis of proteins). After transcription, m-RNA passes into cytoplasm and then on to ribosomes where it serves as a template for the sequence of amino acids during the biosynthesis of proteins. 75 Molecular Biology DNA Replication  DNA replication: The process of copying the DNA during cell division.  DNA replication is semi-conservative i.e. in the daughter cell; one strand is derived from the mother cell; while the other strand is newly synthesized. Inhibitors of DNA Replication - Anti-bacterial agents which inhibit bacterial enzymes but not affect human cells e.g., Ciprofloxacin. - Anticancer agents: arrest new DNA synthesis, and arrest cell division e.g. Adriamycin. 76 Molecular Biology Transcription Transcription is the process of RNA synthesis by RNA polymerase, in which one of the strands of the DNA serves as a template. Inhibitors of RNA Synthesis - Anticancer drugs e.g. Actinomycin D. - Rifampicin is widely used in the treatment of tuberculosis and leprosy. N.B. The replication process occurs only at the time of cell division. But transcription is taking place all the time. Reverse Transcription - Reverse transcription is the process of DNA synthesis from RNA. - The genetic materials of some animal and plant viruses are made up of RNA. Thus genetic information is transferred from RNA to DNA. 77 Molecular Biology Protein biosynthesis (Translation) - Translation of the genetic information carried by the mRNA to synthesize a protein. The translation is a cytoplasmic process. - Inhibitors of Protein Synthesis - Bacteriostatic (Reversible inhibitors) e.g. Erythromycin. - Bactericidal (Irreversible inhibitors) e.g. Streptomycin. DNA Damage and Repair  DNA repair is possible because the DNA molecule consists of two complementary strands, so damage in one strand can be removed and accurately replaced (by using the undamaged complementary strand as a template).  Even within a single organism, repair rates can vary among cells, with the most efficient repair going on in terms (sperm and egg) cell.  Moreover, certain genes are repaired more quickly than others, including those that regulate cell proliferation. Causes of DNA damage - Replication errors. - Toxic Chemicals, radiation, or ultraviolet light. - Spontaneously bases are also altered or lost from DNA. DNA repair and cancer 1. Hereditary nonpolyposis colorectal cancer (HNPCC): Mutation of the proteins involved in the DNA repair system. 2. Xeroderma pigmentosum (XP): the cells cannot repair Pyrimidine dimers caused by UV light, resulting in early and numerous skin cancers. 78 Molecular Biology DNA repair and aging  DNA damage, which gradually accumulates, leads to malfunctioning genes, proteins, cells, and, as the years goes by, deteriorating tissues and organs.  DNA repair processes decline with age while damage accumulates, it could help explain why cancer is so much more common among older people.  Photoaging: both normal aging and photoaging initiated by sunlight (as Ultraviolet light can damage DNA) the skin becomes drier and loses elasticity due to damage of collagen and elastin (the two proteins that give skin its elasticity). Mutations  The mutation may be defined as an abrupt spontaneous origin of a new character.  Mutation is the replacement of a nitrogen base with another in one or both the strands or the addition or deletion of a base pair in a DNA molecule. Mutagens: Any agent which will increase DNA damage or cell proliferation can cause an increased rate of mutations. These can be chemicals, radiation, or viruses. Antimutagens: These are substances that will interfere with tumor promotion. - Vitamin A and carotenoids are shown to reverse precancerous conditions. - Vitamin E acts as an antioxidant, preventing the damage made by free radicals. - Beans and leafy vegetables are shown to interrupt tumor promotion. - Curcumin, the yellow substance in Turmeric is known to prevent mutations. - The beneficial effect of the fiber content of the diet. A low protein, low fat, diet decreases the risk of cancer in animal studies. 79 Molecular Biology Classification of mutations A. Substitution: Replacement of one base with a different one. Can result in a change in one amino acid of a protein.  HbS or sickle-cell hemoglobin leading to sickle-cell anemia.  HbM or methemoglobinemia which considerably decreases the oxygen carrying capacity of hemoglobin. B. Deletion:  Gene deletions, e.g. alpha thalassemia (entire gene).  Deletion of a codon, e.g. cystic fibrosis (one amino acid is missing). C. Insertion:  In Huntington's chorea, CAG trinucleotides are repeated 30 to 300 times. The severity of the disease is increased as the numbers of repeats are more. 80 Molecular Biology Manifestations of Mutations A. Beneficial Mutations: Although rare, beneficial spontaneous mutations are the basis of evolution. Such beneficial mutants are artificially selected in agriculture. B. Silent Mutations: Alteration at an insignificant region of a protein may not have any functional effect. C. Partially acceptable mutation: The protein function may be altered or deficient. Clinical manifestations also are present, but compatible with life e.g. HbS. D. Unacceptable Mutation: The protein becomes nonfunctional and the condition is incompatible with normal life e.g. HbM. E. Carcinogenic Effect: The mutation may not be lethal, but may alter the regulatory mechanisms. Such a mutation in a somatic cell may result in uncontrolled cell division leading to cancer. 81 Molecular Biology Biochemistry of Cancer Carcinogenesis: Change of normal cell to malignant [cancer] cell Carcinogens: Any substance causing an increased rate of mutation can also increase the probability of cancer. Thus, all carcinogens are mutagens. Etiology of Cancer: All cancers are multifactorial in origin. 1. Physical agents: X-Ray, UV, γ-Rays. 2. Chemical carcinogens act cumulatively. Tobacco, food additives, coloring agents. 3. Biological Viruses: HBV. 4. Inherited Mutation. Proto-Oncogenes: they are normal cellular genes. Codes for proteins that is important for normal growth and differentiation. They can be activated (over- expressed) to Oncogenes [Cancer-producing genes]. Anti-oncogenes or Oncosuppressor Genes:  These are the genes, which normally protect the individual from getting cancer. When the gene is deleted or mutated, cancer results. E.g.  The p53: It blocks cell division until the damage is repaired. Most tumors have a complete absence of p53, whereas others show mutant nonfunctional p53. 82 Molecular Biology Recombinant DNA technology  The fusion of the DNA’s from two species into one DNA molecule is known as Recombinant DNA techniques.  Proteins that can result from the expression of recombinant DNA within living cells are termed recombinant proteins. Applications of recombinant DNA technology 1. Specific Probes for Diagnosis of genetic Diseases: 2. DNA cloning: Quantitative Preparation of Biomolecules - Insulin for diabetic Mellitus. - Factor VIII for males suffering from hemophilia A. - Human growth hormone for the treatment of dwarfism. - Hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus. 83 Molecular Biology 3. Gene therapy:  Gene therapy may be either somatic cell gene therapy which targets the patient’s somatic cells; or germline gene therapy which targets the germline cells. However, at present only somatic gene therapy is permitted in humans.  It involves the following approaches: - Gene replacement, which involves the removal of a defective gene and its replacement with a normal one. - Gene correction, wherein a pathological change in the nucleotide sequence is repaired. - Gene augmentation defined as an introduction of genetic material into cells without any attempt to delete or modify the endogenous genetic material. 84 Molecular Biology Human genome project Definition: It is an international co-operative effort that will provide a huge genetic information's essential to study health and disease. Project goals were to: 1- Identify all genes in human DNA. 2- Determine the sequences of the base pairs that make up human DNA. 3- Store this information in databases. Potential applications of genome research include: a. Molecular Medicine Diagnosis of disease. Detection of genetic predispositions to disease. Gene therapy. b. Risk Assessment: Assess health damage and risks caused by radiation exposure, mutagenic chemicals, and cancer-causing toxins c. DNA Forensics (Identification): Identify suspects whose DNA may match evidence left at crime scenes. Identify crime and catastrophe victims. Establish paternity and other family relationships. Match organ donors with recipients in transplant programs. 85 Molecular Biology Stem cells Stem cells are immature, unspecialized cells that have the potential to develop into many different cell lineages via differentiation. Sources of adult stem cells in the oral and maxillofacial region: 1. Bone marrow derived from orofacial bone. 2. Dental pulp stem cells. 3. Stem cells from human exfoliated deciduous teeth. 4. Salivary gland-derived stem cells. Medical application of stem cells: - Corneal repair. - Treatment of liver disease. - Treatment of Diabetes. - Spinal cord regeneration. - Cardiac repair after myocardial infarction. Dental application of stem cells: - Pulpal regeneration. - Craniofacial reconstruction. - Engineering of new teeth. 86  References Lippincott s review of biochemistry (6 th edition): Richard A. Harvey, Denise R. Ferrier. Lippincott William & Wilkins, London. Harper s Biochemistry: Robert K Murray, Peter A Mauers, Drayl K Grammer and Victor W Rodwell. Published by Appleton and Lang, California. Topics in Dental Biochemistry (2011): Martin Levine. Springer-Verlag Berlin Heidelberg. Published by Springer. Textbook of Medical Biochemistry (Eighth Edition): MN Chatterjea, Rana Shinde. Published by Jitendar P Vij Jaypee Brothers Medical Publishers (P) Ltd. Textbook of Biochemistry for Medical Students (Sixth Edition): D M Vasudevan, Sreekumari S, Kannan Vaidyanathan. Published by Jitendar P Vij Jaypee Brothers Medical Publishers (P) Ltd