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Salvador Luria in 1942 showed that viruses are visible only by electron microscopy anxiety lightheadedness buy discount atarax 10mg on line. Alfred Hershey proved that nucleic acids anxiety disorder 3000 cheap atarax 25 mg free shipping, but not proteins anxiety xanax and copd 25mg atarax sale, are the genetic material in viruses anxiety symptoms while driving order atarax 10mg otc. Binding of viruses involve specialized microdomains on the host cell membranes anxiety 12 step groups 10mg atarax otc, the lipid rafts and the glycans anxiety symptoms menopause order atarax 25mg line. The neuraminidase (N) present in the virus plays an important role in budding of new virions. There are 16 types of H and 9 types of N antigens that make the different strains of influenza virus. After entry into the host cell, the viruses utilize the host cell machinery for growth and replication. For example, Herpes simplex virus, if introduced into a culture of Vero cell line (monkey kidney cells), the virus produces lysis of cells. Viruses were first grown in tissue culture by John Enders (Poliomyelitis), Frederick Robbins (Herpes simplex virus) and Thomas Weller (Varicella virus); all the three were awarded Nobel Prize in 1954. Lytic Cycle and Lysogeny Normally virus is replicated inside the cell, then the new virus particles get out from the cell. Instead of the lytic process, some viruses remain dormant for months or years inside the host cells. It enters the host bacteria, multiplied quickly, then the host cell is lysed and free viral particles come out, to infect neighboring cells. Thus the repressor gene is turned "off", so that all other lambda genes are turned "on". Subsequent infection of a new host by such a virus may introduce new genes to the new host. This process was studied by Joshua Lederberg (Nobel Prize 1958) and Max Delbruck (Nobel Prize 1969). This can be considered as genetic engineering by nature, or horizontal transmission of genes. There are only a handful of antiviral drugs available, when compared to the vast array of antibacterial agents. This is because viruses are intracellular and secondly the virus utilizes the host cell machinery for its replication. Site 1: Adsorption and penetration of virus into host cell is inhibited by antibodies, either passive or active. Neuraminidase inhibitors can be used in the treatment of influenza virus infections. Mutations in the virus at the neuraminidase binding site can lead to resistance to the drug. It was developed by Gertrude Elion and George Hitchings, who were awarded Nobel Prize in 1988. In the host cell, it is first phosphorylated by the virus-specific thymidine kinase; then triphosphate form is produced by the cellular enzymes. Site 5: Late protein synthesis is controlled by protease inhibitors, such as Ritonavir, Saquinavir and Indinavir. Chapter 42; Inheritance, Mutations and Control of Gene Expression 511 Viruses as Jumping Genes Viruses may be considered as genes escaped from the existing cells of a lower evolutionary scale; they are jumping genes (see also oncogenes and viral integration in Chapter 51). Fossil records show that exoskeleton appeared in different species more or less simultaneously. About 160 million years ago, ammonites with exoskeletons appeared all over the world. These organisms are very slow moving; but the gene moved rapidly throughout the world. It may be that the gene for alkaline phosphatase has originated in few organisms, which was then transferred horizontally within a short period of paleobiology. In that case, cancer is the deferred payment by the individual for the benefit availed by the species as a whole. Epigenetic Modifications this is a mechanism that results in stable propagation of gene activity states from one generation of cells to the next. Epigenetic states can be modified by environmental factors which may result in the expression of abnormal phenotypes. These epigenetic modifications control gene expression and changes are also inherited. Genetic code is comparable to writing in indelible ink using the sequence of 4 nucleotides. This information is normally transferred from generation to generation with high fidelity. Information provided by epigenome is like a code written in pencil which can be erased and rewritten. Unlike genetic information that distinguishes one person from another (molecular fingerprint) the epigenome distinguishes one cell type from another. However, at times pencil writing leaves smudges even after erasing, similarly, the epimutations may be transmitted to the next generation. But all the offsprings are not affected and even affected persons may not transmit the defect to their progeny, since the genetic imprints are erased and rewritten. Epigenetic modifications referred to as genomic imprinting occurs very early in the embryo. In Prader Willi Syndrome, a mutant gene is derived from father and in Angelman syndrome, the mutant gene is from mother. In some other cases, mutation produces an abnormal protein, which is non-functional; but interferes with the functions of a normal gene of a normal allele. For example, in Osteogenesis imperfecta type I, the abnormal protein interferes with normal triple helix formation of collagen. Methylation occurs naturally on cytosine bases at CpG sequences and is involved in controlling the correct expression of genes. Differentially methylated cytosines give rise to distinct patterns specific for each tissue type and disease state. Environmental stressors including toxins, as well as microbial and viral exposures, can change epigenetic patterns and thereby effect changes in gene activation and cell phenotype. The occurence of "large offspring syndrome" in cattle is attributed to exposure of embryos in vitro to environmental changes. A similar condition, "Beckwith-Weidmann syndrome", which is an imprinted disorder was found to occur with increased frequency in children born by "assisted reproductive technologies". Alterations in gene expression are implicated in the pathogenesis of several neuropsychiatric disorders, including drug addiction and depression. Changes in gene expression in neurons, in the context of animal models of addiction and depression, are mediated in part by epigenetic mechanisms that alter chromatin structure on specific gene promoters. Aptamers Aptamers are oligonucleotides that exhibit specificity against amino acids, drugs, proteins and other biomolecules. Aptamers, first reported in 1990, are attracting interest in the areas of therapeutics and diagnostics. A vaccine is prepared from the Hepatitis B virus surface proteins, which will give protection from infection. Originally, the virus was isolated from pooled blood of patients, and the specific protein was isolated. It is absolutely essential to make sure that the preparations of vaccines or clotting factors are free from contaminants such as hepatitis B particles. Thus normal, heterozygous and homozygous individuals in the family could be identified. Gene Therapy An important application of recombinant technology is in gene therapy. Normal genes could be introduced into the patient so that genetic diseases can be cured. Biotechnology may be defined as "the method by which a living organism or its parts are used to change or to incorporate a particular character to another living organism". Biotechnology involves the application of scientific principles to the processing of materials by biological agents. The use of new varieties of microorganisms to breakdown pollutants in soil or water to harmless end products is known as bioremediation. Quantitative Preparation of Biomolecules If molecules are isolated from higher organisms, the availability will be greatly limited. For example, to get 1 unit of growth hormone, more than 1000 pituitaries from cadavers are required. Risk of Contamination is Eliminated It is now possible to produce a biological substance without any contamination. This results in the sticky ends cuts will generally have overlapping or sticky ends. This is usually achieved by restriction endonucleases which are referred to as "molecular scissors". Werner Arber showed that certain enzymes of bacteria restrict the entry of phages into host bacteria. The Restriction endonucleases are named after the species and strains of bacteria and the order of discovery. The Roman numeral "one" indicates the order of discovery of an enzyme from that species. This feature has profound significance in clinical practice because, antibiotic resistance property is exchanged between bacteria. The numbers denote the length of restriction fragment in kbp 514 Textbook of Biochemistry; Section E: Molecular Biology. Homopolymer Tailing the sticky ends of the vectors usually reconnect themselves without taking up the foreign molecule (recircularization). To circumvent this problem, homopolymer tailing is done in both vector and insert molecules. After the transfection, the bacteria are cultured in a medium containing ampicillin and tetracycline. These colonies are replica-plated onto another agar plate containing chloramphenicol. Colonies in the original plate, corresponding to the dead colonies in the replica-plate are selected. Such a vector carrying the foreign gene, which is translated into a protein, is called expression vector. A clone is a large population of identical bacteria or cells that arise from a common ancestor molecule. Transfection of Vector into the Host the process by which plasmid is introduced into the host is called transfection. Then calcium ion channels are opened, through which the plasmid is imbibed into the host cell. Now the host cells are 516 Textbook of Biochemistry; Section E: Molecular Biology Human Recombinant Proteins Hundreds of human proteins are now being synthesized by the recombinant technology. Other useful products thus produced are interleukins, interferons, anti-hemophilic globulin, hepatitis B surface antigen (for vaccination) and growth hormone. A collection of these different recombinant clones, is called a gene library. Linkage Analysis If two genes are close together on the same chromosome, they do not assort independently during meiosis. When two genes are far apart, they are not linked even though they are on the same chromosome. The 2 alleles are co-inherited with greater frequency if they are physically located close to each other. By 1997, Celera Genomics, headed by Craig Venter, a private enterprise funded by Perkin-Elmer Company independently embarked on a similar project, which hastened the work. These areas were further broken into small pieces and sequenced (shotgun technique). Overlapping sequences were arranged, and fragments were re-written with the help of computer programs. Pharmacogenomics is a recently emerged science from the genome project; it is the use of genetic information towards the development of new drugs and their targets of action. By means of linkage analysis, human genetic mapping (location of important genes) was completed by 1994. By December 1998, human chromosome 5 (about 6% of human genome) was sequenced completely. The final version of the sequence of the entire human genome was completed in 2003. Some of the important ones are, Hemophilus influenzae (1995), yeast (1996), Escherichia coli (1997), Caenorhabditis elegans (1998), Mycobacterium tuberculosis (1998), rat (2004) and chimpanzee (2005). The consortium of research workers aim to identify all the functional elements of the genome. In situ strategy when the expression cassette is injected to the patient either intravenously or directly to the tissue. A great leap in medical science has taken place on the 14th September 1990, when a girl suffering from Adenosine deaminase deficiency (severe Immunodeficiency) was treated by transferring the normal gene for adenosine deaminase. It is intracellular delivery of genes to generate a therapeutic effect by correcting an existing abnormality. Only somatic gene therapy, by inserting the new gene into somatic cell of the patient is under trial. Gene transfer by retroviral vector 518 Textbook of Biochemistry; Section E: Molecular Biology Table 43. This is introduced into a culture containing packaging cells having gag, pol and env genes. The replication-deficient, but infective, retrovirus vector carrying the human gene, now comes out of the cultured cells. This strategy is very suitable for treatment of all diseases produced by single gene mutations.
The mixture of substances to be separated is made volatile at one end of the column and the vapors are swept over the column by an inert carrier gas like argon or nitrogen anxiety symptoms on kids buy generic atarax 10mg. The fractions emerging from the column are detected and quantitated by a detecting device anxiety symptoms on dogs generic 25mg atarax fast delivery. Gel Filtration (Size Exclusion) Chromatography It is also called molecular sieving anxiety symptoms skin rash 10 mg atarax sale. Hydrophilic cross linked gels like acrylamide (Sephacryl) anxiety symptoms zenkers diverticulum discount atarax 10mg online, agarose (Sepharose) and Chapter 54; General Techniques for Separation anxiety 8 year old son order 10mg atarax amex, Purification and Quantitation 603 anxiety 1 week before period order atarax 10mg mastercard. Sephadex (gel filtration) chromatography A = protein solution is added on the top of the column; B = small proteins get inside the beads, and so takes a longer time to reach the bottom; C = larger molecules cannot enter into the beads, so travels quickly, and reaches the bottom faster. Gas liquid chromatography dextran (Sephadex) are used for separation of molecules based on their size. Sephadex is widely used and the range of separation is based on pore size designated by the symbols G-10 to G-200. The small molecules can enter the gel particles, then come out, re-enter into another particle. But the large immunoglobulin molecules cannot enter the pores and sidetrack the gel particles; so they move in the column rapidly. In short, larger molecules will come out first, while smaller molecules are retained in the column. The gel filtration technique is used for (a) separation of protein molecules; (b) purification of proteins; and (c) molecular weight determination. The liquid phase passes through this column under high pressure (1000 times atmospheric pressure). The column may be packed with materials for adsorption, partition or ion exchange. The method is therefore based on the same principle as for those types already described, but separation is achieved with better resolution and high speed (within minutes). Ion Exchange Chromatography In this method, the separation is based on electrostatic attraction between charged biological molecules to oppositely charged groups on the ion exchange resins. These resins are cross linked polymers containing ionic groups as part of their structure. The polymer must be sufficiently cross linked to have negligible solubility, but porous enough for the ions to diffuse freely through it. The ionic groups in cation exchange resins are sulphonic and carboxylic groups, whereas anion exchange resins have a quaternary nitrogen (N+). The separation is based on the ionic character of proteins and amino acids (iso-electric point). When cations (C+) are passed through the column, Na+ in the resin 604 Textbook of Biochemistry; Section G: Advanced Biochemistry. Ion-exchange chromatography; A = negatively charged molecules attach with the beads and so move slowly; B= positively charged molecules repel with the beads, so move faster in the column no radioactivity in the supernatant. In B, equal quantity of unlabelled hormone is added, when labelled and unlabelled antigen molecules compete for the antibody. The displacement of labelled antigen is proportional to the unlabelled antigen in the system (Table 54. A series of test tubes are taken, in which constant quantity of antibody, constant quantity of labelled antigen and different but known quantities of unlabelled antigen are added. Affinity Chromatography the technique is based on the high affinity of specific proteins for specific chemical groups. Conversely, antibodies can be purified by passing through a column containing the antigen. The specificity of antibody and the sensitivity of radioactivity are combined in this technique. In tube A, the labelled hormone molecules are combined with the antibody molecule; so there is. Affinity chromatography Chapter 54; General Techniques for Separation, Purification and Quantitation 605 Table 54. The radioactivity in the precipitate is inversely related to the unlabelled antigen added. The values of the radioactivity in the precipitate (last column) are shown as a graph in Figure 54. The radioactivity in the precipitate is plotted in this graph at the Y-axis, when the corresponding value in the X-axis will give the actual quantity of hormone present in that sample. There is a competition between the unlabelled hormone (antigen) present in the biological specimens and the added labelled antigen to combine with the antibody. The more the unlabelled antigen, less of the labelled antigen will combine with the antibody. The antigen-antibody reaction is allowed to take place for a definite period of time. At the end of the incubation period, the tube will contain free and bound antigen (labelled or unlabelled), as shown in Box. Principle of Radio Immuno Assay Ag + Ag* + Ab [Ag-Ab] + [Ag*-Ab] + Ag + Ag* (bound (free radioradioactivity) activity). A series of standard tubes containing known but varying concentration of the pure antigen are taken along with the unknown biological specimen. The level of the hormone in the specimen can be obtained from a calibration curve prepared from the measured radioactivity of the known standards. The radioisotope commonly used for labelling the antigen is 125I (radioactive iodine). Half life of 125I isotope is about 60 days; iodinated antigen should be used within a few months. This test is commonly employed to detect antigens or antibodies present in very small quantities in tissues or blood. Then color reagent, containing hydrogen peroxide and diamino benzidine (as described below, under antigen detection) is poured over. Therefore from the color intensity, the concentration of the antibody can be calculated. Color of filter and color of solution are complementary Color of filter Wavelength Violet Blue Green Yellow Red 420 470 520 580 680 Color of solution Brown Yellowish brown Pink Purple Green/blue. Immunofluorescennce By this time, antigen (T4 in this example) present in the serum is fixed on the antibody. Therefore intensity of the color may be measured, from which the concentration of the antigen is calculated. Instead of enzyme directly fixed over the antibody; biotin is labelled on the first antibody. The advantage here is that for each biotin fixed, 4 avidin molecules, and so 4 enzyme molecules are fixed. When antigen-antibody complex is formed, the active site of enzyme is not available for substrate binding. Such a system will eliminate the separation of antigenantibody complex, or the washing procedure. Immunofluorescence Antibody tagged with fluorescein isothiocyanate is incubated with cells. Immunocytochemistry Histopathology sections may be layered with antibody tagged with Horse radish peroxidase, and then hydrogen peroxide and chromogen are added; color is thus produced. For example, a slide from colon cancer is reacted with specific antibody against an oncogene. The amount of light absorbed or transmitted by a colored solution is in accordance with the Beer-Lambert law. In the colorimeter, the length of the column through which the light passed is kept constant, by using test tubes or cuvettes of the same diameter for both test and standard, so that the only variable is the concentration. The ratio of intensity of emergent light to intensity of incident light (E/i) is termed as transmittance (T). Since it is in logarithmic scale, values too low or too high are not acceptable for accurate results (sensitive range is between 0. The method of estimation is arranged to give readings within this sensitive range. Filter, used for selecting the monochromatic light (mono = single; chrome = color). Filters will absorb light of unwanted wavelength and allow only monochromatic light to pass through. This light will have maximum absorbance when passed through a particular colored solution. The solution is taken in the cuvettes of fixed diameter to keep the path length common to the test as well as to the standard. The solution absorbs part of the light and the remaining transmitted light is allowed to fall on the photocells. In clinical laboratory, serum sample and reagents are mixed and incubated at 37oC for a fixed time, say 10 minutes, to develop the color optimally. Here the optimum color is not developed; but is quicker and hence is often used in autoanalysers. Spectrophotometer A spectrophotometer has all the basic components of photoelectric colorimeter with more sophistication. Wave lengths in the ultraviolet region are also utilized in the spectrophotometer. Light is separated into a continuous spectrum of wavelengths and passed through the solution. The advantage of the spectrophotometer over the colorimeter, is that the former is 1000 times more sensitive. Therefore even minute quantities of the substance (very dilute solution) can be assessed in the spectrophotometer. To take an example, protein solutions with high concentration (mg/ ml) may be measured by colorimeter. If the protein concentration is only microgram/dl, then colorimeter is ineffective, where spectrophotometer can be used. Further, the electrical power source operating the light must be carefully stabilized. The characteristic absorption maximum (in wavelength) of some important substances are; protein, peptide linkage. Luminiscence Chemiluminiscence, bioluminiscence and electro-luminescence are types of luminescences where the excitation caused by chemical, biological or electrical reaction respectively. In luminiscence, the light emission occurs from an excited singlet state and the light emitted when the electron returns to the ground state (higher energy to lower energy level). The excitation event is caused by a chemical reaction like oxidation of luminal, acridine esters or luciferin by an oxidant. Bioluminiscence is a biological reaction, luciferase being a common biocatalyst used for increasing the efficiency of the reaction. Electroluminiscence is generated from stable precursors at the surface of an electrode; ruthenium tris chelate can be used to label haptens or large molecules like proteins. Compared to conventional electrodes, optodes have certain advantages like miniaturization, long term stability and does not need a reference electrode. Biosensors these are chemical sensors having an optical device or transducer and a biological recognition element. The concentration of the analyte is recognized by an enzyme based biosensor (catalytic reaction) or an affinity based sensor (binding specificity). When the recognition element interacts with the analyte, there is product formation or reactant consumption on the surface of the sensor. This change in property is converted by a transducer to an electrical signal and quantified. Implantable subcutaneous glucose sensors are also being used to adjust the dose of insulin. Intravascular sensors that release nitric oxide have been developed to decrease the possibility of thrombosis. Flame Photometer this is an analytical instrument used for quantitative analysis of sodium, potassium, calcium and lithium in biological fluids like blood, serum and urine. In a colorimeter the optical absorption property is employed, while in a flame photometer the property of emission spectroscopy is utilized. Sodium, potassium, calcium and lithium have the property of emitting a light of the characteristic wavelength of that particular element, when sprayed into a flame (incandescence). The equipment consists of an atomiser, which draws sample solution; and a compressor which pumps air at high pressure. The electric charge given out by the photosensor is detected, amplified and displayed. Ion Selective Electrodes Nowadays, more sensitive but costly equipment, using ionselective electrodes are available to detect sodium, potassium, calcium and lithium. Glass electrode, made up of very thin glass membrane, allows ions to permeate through. The potential difference across the glass membrane of the electrode is quantitated by the instrument. An electrode potential is generated across a selectively permeable membrane separating two different concentrations of an ion.
Chloride shift; reactions in tissues 258 Textbook of Biochemistry; Section B: General Metabolism spectrum has only a single broad band at 565 nm anxiety 7 year old son buy 25 mg atarax. On re-oxygenation by vigorous shaking of the test tube anxiety symptoms in children checklist order atarax 25 mg with mastercard, the absorption spectrum changes to the original anxiety symptoms dizziness generic 10 mg atarax. Laboratory Diagnosis When hemoglobin is examined spectroscopically anxiety 7 months pregnant discount 10mg atarax, oxyHb has 2 absorption bands at 540 and 580 nm anxiety symptoms and treatments atarax 10mg with visa. When sodium hydrosulphite is added anxiety symptoms feeling cold purchase 10 mg atarax with amex, de-oxygenation occurs, the color changes to purple, and the absorption 2: Isohydric Transport of Carbon Dioxide i. Oxy-Hb is More Negatively Charged Than Deoxy-Hb: the iso-electric point of oxyhemoglobin is 6. In the lungs: In lung capillaries, where the pO2 is high, oxygenation of hemoglobin occurs. When 4 molecules of O2 are bound and one molecule of hemoglobin is fully oxygenated, hydrogen ions are released. In anemia, where the total concentration of Hb is reduced, increased oxygen unloading alone will ensure proper oxygenation of tissues. This increase in O2 affinity is physiologically advantageous in facilitating transplacental oxygen transport. The synthesis of HbF starts by 7th week of gestation; it becomes the predominant Hb by 28th week. However, HbF level may remain elevated in children with anemia and beta thalassemia, as a compensatory measure. Embryonic Hemoglobins Several hemoglobins are found during fetal life, but absent in adult life. Examples are Hb Gover-1 (2 zeta and 2 epsilon chains); Gover-2 (2 alpha chains and 2 epsilon chains); Hb Portland (2 gamma and 2 delta chains). Embryonic hemoglobins are produced from 3rd to 8th weeks of gestation, when the site of erythropoiesis is in liver. Clinical Manifestations: Clinical symptoms manifest when carboxy-Hb levels exceed 20%. But on adding a reducing agent like sodium dithionite, it fails to convert carboxy-Hb to deoxy-Hb; whereas oxy-Hb is converted. When the ferrous (Fe++) iron is oxidized to ferric (Fe+++) state, met-Hb is formed. An increase in methemoglobin in blood, (methemoglobinemia) is manifested as cyanosis. Congenital Methemoglobinemia Presence of Hb variants like HbM can cause congenital methemoglobinemia. Oral administration of methylene blue, 100-300 mg/day or ascorbic acid 200-500 mg/day decreases met-Hb level to 5-10% and reverses the cyanosis. Met-hemoglobinemia may develop by intake of water containing nitrates or due to absorption of aniline dyes. Drugs which produce met-hemoglobinemia are: acetaminophen, phenacetin, sulphanilamide, amyl nitrite, and sodium nitroprusside. Glucose-6-phosphate dehydrogenase deficiency: In persons with this enzyme deficiency, the condition may be manifested even with small doses of drugs. The color changes to dark brown and absorption spectra show a band in the red with its center at 633 nm, while the bands for Oxy-Hb persist. It can combine with negatively charged chloride, to form hemin or hematin chloride. Sickle cell trait protects from malaria crystals can be prepared from even very old blood stains in medicolegal cases. Sulf-hemoglobinemia When hydrogen sulfide acts on oxy-hemoglobin, sulf-hemoglobin is produced. It can occur in people taking drugs like sulphonamides, phenacetin, acetanilide, dapsone, etc. Hemoglobinopathies Hundreds of hemoglobin variants leading to hemoglobinopathies have been discovered (Box 22. In addition, although rare, gamma and delta chain variants have also been described (see Table 22. The hemoglobin variants may be classified into 5 major types, based on their clinical manifestations. Hemoglobinopathy and Thalassemia Abnormalities in the primary sequence of globin chains lead to hemoglobinopathies. In other words, normal globin chains in abnormal concentrations result in thalassemias. Unstable Hb variants have an increased tendency to denature and hence tend to form molecular aggregates within cells which lead to increased hemolysis. The membrane bound Heinz bodies alter the surface of the red cells, creating indentations. Hemoglobin S (HbS) (Sickle Cell Hemoglobin) Of the hemoglobin variants, HbS constitutes the most common variety worldwide. In 1949 Linus Pauling (Nobel prize, 1954) established that a hemoglobin with abnormal electrophoretic mobility is responsible for the sickling disease. The glutamic acid in the 6th position of beta chain of HbA is changed to valine in HbS. The substitution of hydrophilic glutamic acid by hydrophobic valine causes a localized stickiness on the surface of the molecule. The deoxygenated HbS may be depicted with a protrusion on one side and a cavity on the other side, so that many molecules can adhere and polymerize. The heterozygous state is very common in Central and West Africa as well as in East and Central parts of India. The slave trade has played an important role in spreading the gene from Africa to different parts of America. In the electrophoresis, the abnormal HbS can be detected along with normal Hb in persons with HbS trait. HbS gives protection against malaria: the high incidence of the sickle cell gene in Chapter 22; Hemoglobin 263 population coincides with the area endemic for malaria. Electrophoresis Electrophoresis at alkaline pH shows a slower moving band than HbA. Lack of this charge on HbS makes it less negatively charged, and decreases the electrophoretic mobility towards positive pole. Management of Sickle Cell Disease Repeated blood transfusions may be required in severe anemia. Treatment with anti-sickling agents like hydroxyurea, cyanate and aspirin, that interfere with polymerization are tried. It is primarily seen in orientals of South-East Asia (Thailand, Myanmar, Bangladesh, etc). Hemoglobin C In normals, the 6th amino acid in beta chain is glutamic acid; it is replaced by lysine in HbC (Table 22. HbD Punjab results from replacement of beta 121 glutamic acid by glutamine (HbD Punjab) (Table 22. M-Hemoglobins (Hb M) these are a group of variants, where the substitution occurs in the proximal or distal histidine residues of alpha or beta chains. Alpha 58 His Tyr (Hb M Boston) Beta 92 His Tyr (Hb M Hyde Park) As a result, the heme has a tendency to get oxidized to hemin, forming methemoglobin. If one parent is heterozygous for HbS and another for C or beta thalassemia, 25% chances are that the child will be a double heterozygote. An individual inherits only 2 beta chain genes; but 4 alpha chain genes are inherited. So, the alpha chain variants constitute only 25% of the total hemoglobin in circulation and are less likely to produce impairment of red cell function (codominant inheritance). Inheritance of HbC trait and generation of HbS-C disease 264 Textbook of Biochemistry; Section B: General Metabolism ii. Thalassemia may be defined as the normal globin chains in abnormal proportions (Box 22. The gene function is abnormal, but there is no abnormality in the polypeptide chains. Reduction in alpha chain synthesis is called alpha thalassemia, while deficient beta chain synthesis is the beta thalassemia. Inheritance Beta thalassemias are phenotypically described as beta (+) or beta (o) depending on whether there is beta chain synthesis or not. Since there are 2 pairs of alpha genes per cell, a single gene deletion in one chromosome or a pair of genes in the chromosomes does not have much effect on a chain production. Alpha thalassemia is rarer because alpha chain deficiency is incompatible with life. These syndromes are mainly seen in people of Asian, African and Mediterranean origin. In homozygous state, clinical manifestations are severe, and hence called Thalassemia major. In heterozygous conditions, the clinical signs and symptoms are minimal; they are called Thalassemia minor. Defect in heme synthesis: Nutritional deficiency of iron, copper, pyridoxal phosphate, folic acid, vitamin B12 or vitamin C. Defect in regulators: Erythropoietin synthesis is reduced in chronic renal failure. Hemorrhage Hematuria, hematemesis, hemoptysis, peptic ulcer metrorrhagia and hemorrhoides are the usual causes for hemorrhage. These precipitates or inclusion bodies lead to membrane damage and destruction of red cells. Homozygous beta thalassemia is characterized by severe anemia, hypersplenism and. The marrow in the skull bones expand producing the "hair-on-end appearance" described in X-ray. Hemoglobin Lepore It is composed of 2 alpha chains and 2 delta-beta chimeric chains. The Hb carries oxygen from lungs to tissue capillaries, from where oxygen diffuses into tissues. In severe physical exercise, pO2 in muscles lowers to 5 mm Hg, when myoglobin releases all the bound oxygen. Myoglobin in Urine and Blood Severe crush injury causes release of myoglobin from the damaged muscles. Being a small molecular weight protein, Mb is excreted through urine (myoglobinuria). Serum myoglobin estimation is useful in early detection of myocardial infarction (Chapter 23). Perhaps about 75% of patients attending a primary health center may have signs and symptoms directly or indirectly related to anemia. The most common cause for anemia in India, is iron deficiency which is described in Chapter 35. Enzymes used as therapeutic agents Clinical Enzymology and Biomarkers biomarkers are used to detect cardiac diseases, which may be a. Commonly used biomarkers for early detection of acute myocardial infarction are: 1. Markers for Cardiac Diseases Serial testing of the following cardiac enzymes is usually done to guide the prognosis (Box 23. On the other hand, there are a few nonfunctional enzymes in plasma, which are coming out from cells of various tissues due to normal wear and tear. Their normal levels in blood are very low; but are drastically increased during cell death (necrosis) or disease. The reference ranges for enzymes in plasma depends on the method of assay used; and therefore will vary from laboratory to laboratory. Hence, the values given in this book are only for a general guidance, and should not be taken as absolute. Cardiac Biomarkers A biomarker is a clinical laboratory test which is useful in detecting dysfunction of an organ. The area under the peak and slope of initial rise are proportional to the size of infarct. However, Troponins are now accepted as reliable markers for myocardial infarction, and hence discussed here. Troponin I (TnI) is encoded by 3 different genes, giving rise to 3 isoforms; the "slow" and "fast" moving forms are skeletal variety. Cardiac isoform is specific for cardiac muscle; the amino acid sequence is different in skeletal muscle isoform. Troponin I is released into the blood within 4 hours after the onset of symptoms of myocardial ischemia; peaks at 14-24 hours and remains elevated for 3-5 days post-infarction. The initial increase is due to liberation of the cytoplasmic fraction and sustained elevation is due to the release from myofibrils. Serum level of Troponin T (TnT) increases within 6 hrs of myocardial infarction, peaks at 72 hours and then remains elevated up to 7-14 days. The magnitude of the peak value as well as the area under the graph will be roughly proportional to the size of the myocardial infarct. Although both of them have the same molecular weight (32 kD), there are minor amino acid variations. So 5 combinations of H and M chains are possible; H4, H3M, H2M2, M3H and M4 varieties, forming 5 iso-enzymes. The isoenzymes are usually separated by cellulose acetate electrophoresis at pH 8. It is a marker of liver injury and shows moderate to drastic increase in parenchymal liver diseases like hepatitis and malignancies of liver.
Syndromes
Most of the riboflavin-linked dehydrogenases are concerned with electron transport in (or to) the respiratory chain (Chapter 13) anxiety symptoms body buy atarax 25 mg line. Cytochromes May Also Be Regarded as Dehydrogenases the cytochromes are iron-containing hemoproteins in which the iron atom oscillates between Fe3+ and Fe2+ during oxidation and reduction anxiety symptoms brain zaps order 10 mg atarax visa. Except for cytochrome oxidase (previously described) anxiety symptoms zika discount 10mg atarax mastercard, they are classified as dehydrogenases anxiety symptoms jaw pain generic atarax 10mg with mastercard. Several identifiable cytochromes occur in the respiratory chain anxiety symptoms keep coming back buy atarax 10mg without prescription, ie anxiety chat rooms atarax 25 mg otc, cytochromes b, c 1, c, and cytochrome oxidase. Cytochromes are also found in other locations, eg, the endoplasmic reticulum (cytochromes P450 and b 5), and in plant cells, bacteria, and yeasts. Accumulation of peroxides can lead to generation of free radicals, which in turn can disrupt membranes and perhaps cause diseases including cancer and atherosclerosis (see Chapters 15 & 44). Peroxidases Reduce Peroxides Using Various Electron Acceptors Peroxidases are found in milk and in leukocytes, platelets, and other tissues involved in eicosanoid metabolism (Chapter 23). In the reaction catalyzed by peroxidase, hydrogen peroxide is reduced at the expense of several substances that will act as electron acceptors, such as ascorbate, quinones, and cytochrome c. The reaction catalyzed by peroxidase is complex, but the overall reaction is as follows: In erythrocytes and other tissues, the enzymeglutathione peroxidase, containing selenium as a prosthetic group, catalyzes the destruction of H2 O2 and lipid hydroperoxides through the conversion of reduced glutathione to its oxidized form, protecting membrane lipids and hemoglobin against oxidation by peroxides (Chapter 21). Catalase Uses Hydrogen Peroxide as Electron Donor & Electron Acceptor Catalase is a hemoprotein containing four heme groups. In addition to possessing peroxidase activity, it is able to use one molecule of H2 O2 as a substrate electron donor and another molecule of H2 O2 as an oxidant or electron acceptor. Under most conditions in vivo, the peroxidase activity of catalase seems to be favored. Thus, the enzymes that produce H 2 O2 are grouped with the enzyme that destroys it. However, mitochondrial and microsomal electron transport systems as well as xanthine oxidase must be considered as additional sources of H2 O2. They catalyze the incorporation of oxygen into a substrate molecule in two steps: (1) oxygen is bound to the enzyme at the active site and (2) the bound oxygen is reduced or transferred to the substrate. Dioxygenases Incorporate Both Atoms of Molecular Oxygen into the Substrate the basic reaction is shown below: Examples include the liver enzymes, homogentisate dioxygenase (oxidase) and 3-hydroxyanthranilate dioxygenase (oxidase), which contain iron; and L-tryptophan dioxygenase (tryptophan pyrolase) (Chapter 29), which utilizes heme. Monooxygenases (Mixed-Function Oxidases, Hydroxylases) Incorporate Only One Atom of Molecular Oxygen into the Substrate the other oxygen atom is reduced to water, an additional electron donor or cosubstrate (Z) being necessary for this purpose. Cytochromes P450 Are Monooxygenases Important for the Detoxification of Many Drugs & for the Hydroxylation of Steroids Cytochromes P450 are an important superfamily of heme-containing monooxgenases, and more than 50 such enzymes have been found in the human genome. These cytochromes are located mainly in the endoplasmic reticulum in the liver and intestine, but are also found in the mitochondria in some tissues. In the endoplasmic reticulum of the liver, cytochromes P450 are found together with cytochrome b 5 and have an important role in detoxification. The rate of detoxification of many medicinal drugs by cytochromes P450 determines the duration of their action. Benzpyrene, aminopyrine, aniline, morphine, and benzphetamine are hydroxylated, increasing their solubility and aiding their excretion. Many drugs such as phenobarbital have the ability to induce the synthesis of cytochromes P450. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein Fe 2 S2. Mitochondrial cytochrome P450 systems are found in steroidogenic tissues such as adrenal cortex, testis, ovary, and placenta and are concerned with the biosynthesis of steroid hormones from cholesterol (hydroxylation at C2 2 and C2 0 in side-chain cleavage and at the 11 and 18 positions). In addition, renal systems catalyzing 1 - and 24hydroxylations of 25-hydroxycholecalciferol in vitamin D metabolism-and cholesterol 7 -hydroxylase and sterol 27-hydroxylase involved in bile acid biosynthesis in the liver (Chapter 26)-are P450 enzymes. The ease with which superoxide can be formed from oxygen in tissues and the occurrence of superoxide dismutase, the enzyme responsible for its removal in all aerobic organisms (although not in obligate anaerobes) indicate that the potential toxicity of oxygen is due to its conversion to superoxide. Superoxide is formed when reduced flavins-present, for example, in xanthine oxidase-are reoxidized univalently by molecular oxygen. Superoxide can reduce oxidized cytochrome c or be removed by superoxide dismutase: In this reaction, superoxide acts as both oxidant and reductant. Thus, superoxide dismutase protects aerobic organisms against the potential deleterious effects of superoxide. The enzyme occurs in all major aerobic tissues in the mitochondria and the cytosol. Although exposure of animals to an atmosphere of 100%oxygen causes an adaptive increase in superoxide dismutase, particularly in the lungs, prolonged exposure leads to lung damage and death. Antioxidants, eg, -tocopherol (vitamin E), act as scavengers of free radicals and reduce the toxicity of oxygen (Chapter 44). Oxidoreductases have a variety of functions in metabolism; oxidases and dehydrogenases play major roles in respiration; hydroperoxidases protect the body against damage by free radicals; and oxygenases mediate the hydroxylation of drugs and steroids. Tissues are protected from oxygen toxicity caused by the superoxide free radical by the specific enzyme superoxide dismutase. Most of this takes place inside mitochondria, which have been termed the "powerhouses" of the cell. A number of drugs (eg, amobarbital) and poisons (eg, cyanide, carbon monoxide) inhibit oxidative phosphorylation, usually with fatal consequences. Several inherited defects of mitochondria involving components of the respiratory chain and oxidative phosphorylation have been reported. The outer membrane is characterized by the presence of various enzymes, including acyl-CoA synthetase and glycerolphosphate acyltransferase. Note that the enzymes of the citric acid cycle and -oxidation (Chapters 22 & 17) are contained in mitochondria, together with the respiratory chain, which collects and transports reducing equivalents, directing them to their final reaction with oxygen to form water, and the machinery for oxidative phosphorylation, the process by which the liberated free energy is trapped as high-energy phosphate. Components of the Respiratory Chain Are Contained in Four Large Protein Complexes Embedded in the Inner Mitochondrial Membrane Electrons flow through the respiratory chain through a redox span of 1. The four complexes are embedded in the inner mitochondrial membrane, but Q and cytochrome c are mobile. The Fe-S take part in single electron transfer reactions in which one Fe atom undergoes oxidoreduction between Fe2+ and Fe3+. Flow of electrons through the respiratory chain complexes, showing the entry points for reducing equivalents from important substrates. Of the eight H+ removed from the matrix, four are used to form two water molecules and four are pumped into the intermembrane space. Since the inner mitochondrial membrane is impermeable to ions in general and particularly to protons, these accumulate in the intermembrane space, creating the proton motive force predicted by the chemiosmotic theory. F1 is attached to a membrane protein complex known as F0, which also consists of several protein subunits. The enzyme complex consists of an F 0 subcomplex which is a disk of "C" protein subunits. The subunit fits inside the F1 subcomplex of three and three subunits, which are fixed to the membrane and do not rotate. For clarity, not all the subunits that have been identified are shown-eg, the "axle" also contains an subunit. Two more high-energy phosphates per mole of glucose are captured in the citric acid cycle during the conversion of succinyl CoA to succinate. These reactions are known as oxidative phosphorylation at the respiratory chain level. Under certain conditions, the concentration of inorganic phosphate can also affect the rate of functioning of the respiratory chain. The remaining free energy that is not captured as high-energy phosphate is liberated as heat. They may be classified as inhibitors of the respiratory chain, inhibitors of oxidative phosphorylation, and uncouplers of oxidative phosphorylation. Sites of inhibition of the respiratory chain by specific drugs, chemicals, and antibiotics. N - Ethylmaleimide, hydroxycinnamate, and atractyloside inhibit the indicated systems. The uncoupler that has been used most frequently is 2,4-dinitrophenol, but other compounds act in a similar manner. Thermogenin (or the uncoupling protein) is a physiological uncoupler found in brown adipose tissue that functions to generate body heat, particularly for the newborn and during hibernation in animals (Chapter 25). Such systems are necessary for uptake and output of ionized metabolites while preserving electrical and osmotic equilibrium. However, dicarboxylate and tricarboxylate anions and amino acids require specific transporter or carrier systems to facilitate their passage across the membrane. Monocarboxylic acids penetrate more readily in their undissociated and more lipid-soluble form. The net uptake of malate by the dicarboxylate transporter -Ketoglutarate transport also requires an requires inorganic phosphate for exchange in the opposite direction. The net uptake of citrate, isocitrate, or cis aconitate by the tricarboxylate transporter requires malate in exchange. It is believed that active uptake of Ca2+ by mitochondria occurs with a net charge transfer of 1 (Ca+ uniport), possibly through a Ca2+ /H+ antiport. The H+ /Pi symport Ionophores Permit Specific Cations to Penetrate Membranes Ionophores are lipophilic molecules that complex specific cations and facilitate their transport through biologic membranes, eg, valinomycin (K+). The transfer of reducing equivalents through the mitochondrial membrane requires substrate pairs, linked by suitable dehydrogenases on each side of the mitochondrial membrane. Although this shuttle is present in some tissues (eg, brain, white muscle), in others (eg, heart muscle) it is deficient. The complexity of this system is due to the impermeability of the mitochondrial membrane to oxaloacetate, which must react with glutamate to form aspartate and cytosol. Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. Malate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. The shuttle allows rapid transport of high-energy phosphate from the mitochondrial matrix into the cytosol. These are funneled into the respiratory chain, where they are passed down a redox gradient of carriers to their final reaction with oxygen to form water. The redox carriers are grouped into four respiratory chain complexes in the inner mitochondrial membrane. Three of the four complexes are able to use the energy released in the redox gradient to pump protons to the outside of the membrane, creating an electrochemical potential between the matrix and the inner membrane space. In this way, oxidation is closely coupled to phosphorylation to meet the energy needs of the cell. Many well-known poisons such as cyanide arrest respiration by inhibition of the respiratory chain. In plants, glucose is synthesized from carbon dioxide and water by photosynthesis and stored as starch or used to synthesize the cellulose of the plant cell walls. Animals can synthesize carbohydrates from amino acids, but most are derived ultimately from plants. Glucose is the most important carbohydrate; most dietary carbohydrate is absorbed into the bloodstream as glucose formed by hydrolysis of dietary starch and disaccharides, and other sugars are converted to glucose in the liver. Glucose is the major metabolic fuel of mammals (except ruminants) and a universal fuel of the fetus. It is the precursor for synthesis of all the other carbohydrates in the body, including glycogen for storage; ribose and deoxyribose in nucleic acids; galactose in lactose of milk, in glycolipids, and in combination with protein in glycoproteins and proteoglycans. Diseases associated with carbohydrate metabolism include diabetes mellitus, galactosemia, glycogen storage diseases, and lactose intolerance. Monosaccharides are those sugars that cannot be hydrolyzed into simpler carbohydrates. They may be classified as trioses, tetroses, pentoses, hexoses, or heptoses, depending upon the number of carbon atoms, and as aldoses or ketoses, depending upon whether they have an aldehyde or ketone group.
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