Gustavo Blanco

Molecular Basis of Immunity

The immune system is actively involved in the protection of the body against invading foreign agents. It consists of two different systems, innate and adaptive. The innate system is unspecific and it does not require prior exposure with the foreign agent to respond. Phagocytic cells with TLR ( toll-like receptors ) recognize molecules in microorganisms and initiate an inflammatory process or promote the release of interferons. Adaptive system is specific and its response is enhanced by new encounters with the invading agent; it can develop memory against the aggressor. It is mediated by B and T lymphocytes , located in lymphoid organs and circulating blood. The B cells are involved in the production of immunoglobulins (Ig) or antibodies ( humoral immunity ); the T lymphocytes are effectors of the cell-mediated immunity ( cellular immunity ). Antigens (Ag) are agents (microorganisms, particles, cells, or molecules) that can trigger the immune response. Small portions of the antigen (antigenic determinants or epitopes ), recognized as foreign by the immune system, are responsible for the immune reaction. Humoral immunity depends on the production of antibodies by B lymphocytes. Immunoglobulins are composed of a structural unit of four polypeptide chains linked together by disulfide bridges. Two are the heavy or H chains and the other two are the light or L chains . Variable segments of L and H form the complementarity determinant regions which recognize an epitope in the antigen. Five classes of Ig exist that differ in their heavy chain. IgG, IgA, IgM, IgD, and IgE contain γ, α, μ, δ, and ɛ chains, respectively. Light chains can be either of two types, κ or λ. IgG, IgD, and IgE are monomers, IgM are pentamers and IgA are dimers of the basic Ig structure. An individual is capable of synthesizing more than 10 7 Igs with different specificities. This heterogeneity in Ig synthesis is achieved through rearrangement of the genes encoding the variable portions of H and L chains. Monoclonal antibodies have identical antigen specificity and are synthesized by plasma cells derived from the same clone. The complement includes a series of plasma proteins that are involved in body defense, promoting the lysis or phagocytosis of foreign cells, bacteria, and viruses. The complement proteins circulate as zymogens, which are activated by a sequential cascade of reactions. Two pathways for complement activation are known. The classical pathway is activated by antigen–antibody complexes. The a lternative pathway is activated by macromolecules on the surface of microorganisms. Cellular immunity is carried out by effector T cells , of which there are several types: helper (T H ), killer (T K ), and suppressor (T S ) cells. T-cell receptors recognize the Ag only if it is bound to the major histocompatibility complex (MHC or HLA in humans) on the surface of accessory cells. T cells recognize foreign MHC proteins in organ grafts and are responsible for transplant rejection. After T cell recognition, the cells carrying the foreign MHC proteins are eliminated. Cytokines are proteins secreted by cells of the immune system with paracrine or autocrine action. Their general role is to regulate the immune response and to promote inflammation. Interferons are low–molecular mass glycoproteins of the cytokine family, which are synthesized by cells that have been invaded by viruses; they try to counteract the infectious agent.

The Genetic Information (II)

The genetic information is contained in DNA. DNA replication ensures that all cells of an individual, by successive divisions, receive DNA of equal structure and in the same quantity. The entire DNA in chromosomes constitutes the genome. Genes are portions of DNA encoding for proteins and RNAs. The sequence of nucleotides in DNA is a “coded message” (genetic code) that indicates the amino acid sequence of the protein to be synthesized; the unit of information is a triplet of bases (codon). Different combinations of the 4 bases in triplets can form 64 codons; each encodes an amino acid. Most amino acids have several synonymous codons. Three of the codons indicate protein termination. In eukaryote genes, coding segments in DNA ( exons ) are separated by introns . During transcription, RNA is synthesized from a DNA template, called the antisense strand . The other DNA strand, not transcribed, is the sense or coding strand and has the same sequence as the synthesized RNA. Splicing is the process by which the precursor mRNA undergoes changes in the nucleus to form mature mRNA. Protein biosynthesis occurs in the cytoplasm via a process called translation . This requires first the activation of amino acids , which are transferred to specific tRNAs. Initiation of protein synthesis involves binding of initiation factors to ribosome. This complex moves on the mature mRNA, in the 5′ to 3′ direction. The anticodons of tRNAs loaded with amino acids successively bind to the corresponding codons in the mRNA. Peptide bonds are formed and protein elongation takes place in successive cycles that are repeated adding one amino acid at a time. Once the chain is completed, termination is identified by a stop codon (UAA, UAG, or UGA) and the polypeptide chain is released. One mRNA can simultaneously direct the synthesis of several polypeptide molecules (polysome). The proteome refers to the total number of proteins synthesized by an organism. Mutations are changes in the DNA sequence, some of which result in absence of expression of a particular protein, or in the production of abnormal proteins. Transposons are DNA sequences inserted in different locations of the genome. Oncogenes are genes responsible for the carcinogenic transformation of cells. Epigenetics includes the modification of gene expression which does not involve changes in the DNA sequence.

Purine and Pyrimidine Metabolism

Humans can produce nitrogenous bases endogenously. For the biosynthesis of purines, the purine ring is built from residues of different origins, including glycine, glutamine, aspartate, formyl tetrahydrofolate, and CO 2 . The molecular assembly is performed with ribose-5-P bound to it. First, 5-phosphoribosyl-1-pyrophosphate (PRPP) is formed by phosphoribosylpyrophosphate synthetase . Finally a nucleotide is obtained. There is also a salvage pathway that utilizes purines from degraded nucleotides. Purine catabolism starts with the degradation of nucleic acids into nucleotides and nucleosides. Adenosine is deaminated by adenosine deaminase . Inosine formed is cleaved by nucleoside phosphorylase to produce hypoxanthine and ribose-P. Then, hypoxanthine is oxidized to xanthine by xanthine oxidase . Guanosine is hydrolyzed to guanine and ribose. Guanine is deaminated to xanthine by guanase . Xanthine, formed from both adenine and guanine, is oxidized into the final product, uric acid , which is excreted in the urine. Elevated levels of urate in the blood can determine its precipitation in joints and cartilages (gout). Pyrimidine biosynthesis requires the binding of aspartate and carbamoyl phosphate to form carbamoylaspartate, catalyzed by aspartate transcarbamoylase . This product is cyclized into orotic acid and finally renders pyrimidines. Pyrimidine catabolism produces soluble compounds, which can be easily removed or used. Degradation of cytosine produces β-alanine, CO 2 , and NH 3 . Thymine produces β-aminoisobutyrate, CO 2 , and NH 3 ; β-aminoisobutyrate is converted into succinyl-CoA.

Biochemical Bases of Endocrinology (II) Hormones and Other Chemical Intermediates

Hormones, growth factors, cytokines, and neurotransmitters are chemical mediators, which communicate and integrate biological systems. Hormones include multiple different substances, which according to their structure could be steroidal, amino acid derivatives, eicosanoids, peptides, and proteins. They operate in very low concentrations, exhibit high specificity, have half-lives of seconds to days, and their secretion is regulated by a variety of stimuli that depend on the hormone type. Hormones bind to specific receptors in the target cells, forming a hormone-receptor complex. Hypothalamic hormones include various factors that regulate the synthesis and secretion of all hormones produced in the pituitary gland. They include (1) corticotropin-releasing hormone , (2) thyrotropin-releasing hormone , (3) luteinizing and follicle-stimulating releasing hormones , (4) prolactin-releasing hormones , (5) growth hormone–releasing hormone , (6) melanocyte stimulant hormones , (7) prolactin inhibitory hormone , (8) growth hormone – inhibitory hormone , or somatostatin , and (9) melanocyte inhibitory hormone . The anterior pituitary or adenohypophysis secretes (1) Adrenocorticotropin, a polypeptide which stimulates the synthesis of adrenal cortex gland hormones, primarily glucocorticoids. (2) Thyroid-stimulating hormone, a glycoprotein that activates thyroid hormones synthesis and release. (3) Gonadotropins include follicle-stimulating hormone and luteinizing hormone (LH). Follicle-stimulating hormone induces Graafian follicle maturation and stimulates ovarian estrogen production. LH controls the development of the corpus luteum and ovarian estrogen and progesterone secretion. In testis, LH stimulates the development of the seminiferous tubules and the production of testosterone. (4) Prolactin is a protein which stimulates corpus luteum formation, progesterone production, and mammary gland development. (5) Melanocyte-stimulating hormone plays a role on melanocyte pigmentation. (6) Somatotropin or growth hormone (GH), a protein which stimulates cell proliferation and growth. Neurohypophysis produces oxytocin, a nonapeptide, which stimulates uterus contraction; and vasopressin or antidiuretic hormone, a nonapeptide with vasopressor effects and involved in water reabsorption in the kidney. Placenta produces chorionic gonadotropins (GH) and placental lactogen with actions similar to those secreted by the adenohypophysis. Thyroid gland produces thyroxine (3,5,3’,5’-tetraiodothyronine or T 4 ) and 3,5,3’-triiodothyronine ( T 3 ). T 3 and T 4 stimulate RNA and protein synthesis, increase oxygen consumption, and promote glucose, lipid, and amino acid utilization in tissues. At physiological concentrations they have anabolic effects, while at high doses they have catabolic action. Calcitonin is also synthesized in the thyroid gland; it decreases Ca 2+ and phosphate levels in plasma. Adrenal gland cortex hormones include glucocorticoids (cortisol–cortisone), mineralocorticoids (aldosterone–deoxycorticosterone), and androgens (androstenedione–dehydroepiandrosterone). These are all steroidal hormones. Corticosteroids have important effects on glucose, lipids, and amino acids metabolism. Also, they have antiinflammatory actions. Mineralocorticoids increase Na + and Cl − reabsorption and K + excretion in renal tubules. Androgenic steroids exert anabolic effects on proteins. The adrenal gland medulla produces adrenaline or epinephrin and noradrenalin, which have effects in glucose metabolism and prepare the body to cope with stress conditions. Pancreas hormones include insulin, glucagon, and somatostatin. Insulin is a protein that stimulates uptake of glucose in cells. Insulin activates all pathways of glucose utilization; it is the main hypoglycemic hormone. It also influences fatty acid and triacylglycerol synthesis, reduces lipolysis in adipose tissue, and activates RNA and protein synthesis. Glucagon favors pathways that increase glycemia. Somatostatin has inhibitory action on the synthesis and secretion of GH, insulin and glucagon, thyroid-stimulating hormone, and gastrin and secretin. Testis produces testosterone, a steroid that favors the development of reproductive organs, accessory glands, and secondary sex characteristics of males. Ovary produces estradiol and estrone, steroidal hormones with anabolic action on the female genital organs, and progesterone, a progestational hormone . Parathyroid glands produce parathyroid hormone, a protein that is involved in Ca 2+ homeostasis. Other tissues, such as kidney, heart, and the gastrointestinal tract, release substances that have local and systemic effects.

Metabolism in Some Tissues

Different tissues present some differences in their metabolism. Liver : metabolizes glucose by converting it to G-6-P by glucokinase when glycemia increases. All pathways of glucose, triacylglycerols, phospholipid, cholesterol, ketone bodies, and protein synthesis are active. Fatty acids, subjected to β-oxidation, constitute the main energy source. The liver synthesizes triacylglycerols, phospholipids, and cholesterol; it also forms ketone bodies but cannot use them. Liver is rich in aminotransferases and glutamate dehydrogenase; it is the organ where urea is formed. In addition, gluconeogenesis is high. The liver is important in ethanol metabolism , which is converted to active acetate that follows several pathways, including final oxidation to CO 2 and H 2 O in the citric acid cycle. The liver is also involved in the biotransformation or detoxification of xenobiotics. In skeletal muscle , metabolism varies depending on the type of work performed. Intense exercise can be carried out thanks to anaerobic metabolism , by degrading muscle glycogen and producing lactate. During a short time of low intensity exercise, muscle increases the oxidation of fatty acids. Muscle also uses ketoacids from branched chain amino acids as energy source. The heart has continuous activity and generates energy aerobically. It uses fatty acids as main fuel, but glucose, ketone bodies, and lactate are also oxidized. Adipose tissue represents the major energy reserve of the body; it synthesizes triacylglycerols (TAG). Main pathways for G-6-P are glycolysis and pentose phosphate. Lipolysis is catalyzed by lipase. The central nervous system uses only glucose to obtain energy; it consumes 20% of the total O 2 used by the whole body at rest. Under conditions of prolonged fasting, the brain can also oxidize ketone bodies.

Integration and Regulation of Metabolism

Compounds of different origin and nature can produce common metabolites and products. Hexoses, glycerol, fatty acids, and amino acids render acetyl-CoA, which is oxidized in the citric acid cycle. The respiratory chain is the final common destination of electrons from different substrates. Carbohydrates generate fatty acids and triacylglycerols. Amino acids form α-ketoacids by transamination. Glucose also produces α-ketoacids. After deamination, amino acids can form carbohydrates (glucogenic amino acids) or ketone bodies (ketogenic amino acids). Some metabolites (glucose-6-P, pyruvate, acetyl-CoA) are “crossroads” compounds of several metabolic pathways. Metabolic regulation is achieved by targeting key enzymes on a pathway, either by modifying the activity of preexistent enzymes (changes in substrate level, allosteric effectors, covalent modification) or changing the amount of enzyme (synthesis or degradation). Glycogenolysis is regulated through the control of glycogen phosphorylase and phosphorylase kinase . Glycogenesis is regulated by modulating the activity of glycogen synthase . Glycolysis is controlled by targeting hexokinase and phosphofructokinase . Gluconeogenesis is modulated at the level of g lucose-6-P phosphatase , fructose-l,6-bisP phosphatase , and pyruvate carboxylase. Oxidative decarboxylation of pyruvate is modulated via the pyruvate dehydrogenase (PDH) multienzyme complex. The citric acid cycle is regulated at various levels, including citrate synthase , isocitrate dehydrogenase , α-ketoglutarate dehydrogenase , and glutamate dehydrogenase . The Pasteur effect describes a phenomenon consisting of the decrease in glucose consumption in the presence of oxygen. Allosteric regulation of phosphofructokinase is responsible for this effect. Triacylglycerols in adipose tissue are hydrolyzed by lipase , a hormone-regulated enzyme. Fatty acid biosynthesis is mainly regulated at the level of acetyl CoA carboxylase . Cholesterol biosynthesis is regulated by controlling 3-OH-3-methylglutaryl-CoA reductase . Metabolism of nitrogenous compounds, such as the synthesis of some amino acids, of purines, and pyrimidines is regulated by the final product. Cellular oxidations are adjusted by the content of nucleotides in the cell. The energy charge of the cell depends on the relative concentration of ATP, ADP, and AMP of cells. When the energy charge is high, energy-consuming metabolic pathways are stimulated, whereas those producing ATP are inhibited.

Biological Oxidations: Bioenergetics

Biological oxidations do not take place by direct transfer of electrons (e − ) from substrate to oxygen. They are carried out in successive stages by different e − acceptors with increasing reduction potential. This allows for a stepwise release of energy and its best utilization by the cell. The respiratory chain , located in the mitochondrial inner membrane, comprises a series of H or electrons (e − ) acceptors arranged according to increasing reduction potential, associated with enzymes that catalyze e − transfer. It is composed of complex I or NADH–ubiquinone reductase, complex II or succinate–ubiquinone reductase, ubiquinone or coenzyme Q, complex III or ubiquinone–cytochrome c reductase, cytochrome c , located on the outer face of inner membrane, complex IV or cytochrome oxidase. Finally 4 e − are transferred to 2 O atoms, which with 4 H + form 2 H 2 O. The energy produced by the flow of e − is coupled to phosphoryl transfer, synthesizing adenosine triphosphate (ATP) from ADP in a process known as oxidative phosphorylation . Each e − pair from substrates of NAD-linked dehydrogenases generates three molecules of ATP, while substrates oxidized by FAD-dependent enzymes produce two ATP. The chemio-osmotic hypothesis explains the mechanism underlying oxidative phosphorylation. The energy generated by the flow of reducing equivalents is used to pump protons from the mitochondrial matrix outward into the inner membrane, at the site of complexes I, III, and IV. The proton gradient created across the mitochondrial inner membrane drives proton flux through the F 1 F 0 or ATP synthase complex , which couples proton transport to phosphate addition to ADP. Compounds that reduce or eliminate the proton gradient inhibit phosphorylation. Inhibitors can block e − transfer at different levels of the respiratory chain. Rotenone, amytal, and other barbiturates act at the level of complex I; antimycin A, at complex III; and cyanide, carbon monoxide, and azide, on complex IV. Inhibitors of oxidative phosphorylation include proton and K + ionophores, which suppress the mitochondrial electrical potential gradient, acting as uncoupling agents, and compounds, such as oligomycin, which interfere with the function of the F 1 F 0 ATPase. Brown fat present in infants and hibernating animals has thermogenin, a protein that inhibits ATP synthesis, uncoupling mitocondrial function and contributing to maintain body temperature. Oxidative phosphorylation is mainly controlled by ADP levels. Phosphorylation at substrate level is another way to generate ATP by phosphoryl transfer from high energy metabolites.

Chemical Composition of Living Beings

Biogenic elements are essential components of living organisms. They include: (1) primary elements (O, C, H, N, Ca, and P), which comprise ∼98% of the total body mass of an adult human and participate in the composition of essential body molecules; (2) secondary elements (Na, K, Cl, S, Mg), which exist as salts and inorganic ions and Fe, which is part of important molecules, as hemoglobin; and (3) trace elements or oligoelements (I, Cu, Zn, Mo, Se, and Co), which are present in very scarce amounts, but are key to body function. The biogenic elements combine to form biological compounds . These include inorganic and organic substances. Among the inorganic compounds is water, the solvent present in body fluids and tissues. It comprises 65% of the total body weight of an adult individual. Other inorganic compounds are nonsoluble, such as calcium phosphate, which is an essential component of bone. The organic biological compounds include proteins, carbohydrates, lipids, and nucleic acids. Others, such as vitamins, hormones, and pigments have carbon as a key component and perform essential roles.

Posttranslational Protein Modifications

Abstract After translation, different modifications occur to proteins to give them their final structure. Protein folding is essential for the proper conformation and function of proteins. It is assisted by heat shock proteins called chaperones and multimeric complexes known as chaperonins. Protein misfolding can lead to the formation of intra- and extracellular protein aggregates of fibrillar structure, which have damaging effects on the cell (as occurs in amyloidosis and neurodegenerative diseases). Prion proteins can change to a misfolded conformation and behave as infectious agents, which can cause different alterations, such as “mad cow” disease. Other posttranslational modifications, critical to protein function, include elimination of the N-terminal formyl methionine residue, formation of disulfide bonds between cysteines, covalent modifications, hydroxylation, carboxylation, acetylation, methylation, amidation, deamidation, phosphorylation, ADP-ribosylation, addition of oligosaccharides (glycosylation), addition of prosthetic groups, sumoylation, and ubiquitination.