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Showing papers in "Methods in Enzymology in 1985"


Book ChapterDOI
TL;DR: 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)-GSSG reductase recycling assay for total glutathione is a specific, sensitive, rapid, and reliable procedure, however, because the method depends on an accurate standard curve, appropriate standards containing the protein precipitating agent are essential.
Abstract: Publisher Summary There are a number of procedures, for example, chemical, enzymatic, and chromatographic for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in biological samples. Enzymatic and chromatographic methods for the determination of glutathione in biological samples are described in this chapter. Because GSH readily oxidizes nonenzymatically and because it is a good substrate of γ-glutamyl transpeptidase, the biological samples are acidified quickly to reduce oxidation of GSH to GSSG and to mixed disulfides, and to inactivate γ-glutamyl transpeptidase. Glutathione oxidizes rapidly at pH values greater than 7. Acid treatment inactivates γ-glutamyl transpeptidase, which catalyzes the reactions that decrease the levels of both GSH and GSSG. The optimum method for treating biological samples depends upon the tissue and the experimental system. The discussed 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)-GSSG reductase recycling assay for total glutathione is a specific, sensitive, rapid, and reliable procedure. However, because the method depends on an accurate standard curve, appropriate standards containing the protein precipitating agent are essential.

2,623 citations


Book ChapterDOI
TL;DR: A purification method of glutathione reductase from calf liver and rat liver is described and it is shown that this enzyme has a major role as a reductant in oxidation–reduction processes, and serves in detoxication and several other cellular functions of great importance.
Abstract: Publisher Summary Glutathione reductase is a flavoprotein catalyzing the NADPH-dependent reduction of glutathione disulfide (GSSG) to glutathione (GSH). The reaction is essential for the maintenance of glutathione levels. Glutathione has a major role as a reductant in oxidation–reduction processes, and serves in detoxication and several other cellular functions of great importance. A purification method of this enzyme from calf liver and rat liver is described in this chapter. Similar methods are used for the purification of the enzyme from yeast, porcine, and human erythrocytes. All the steps are carried out at about 5°. The purification method from calf liver consists of various steps including preparation of cytosol fraction, chromatography on DEAE-sephadex, precipitation with ammonium sulfate, and chromatography on hydroxyapatite. The purification of glutathione reductase from rat liver is usually combined with the preparation of glutathione transferases, thioltransferase, and glyoxalase I.

2,366 citations


Book ChapterDOI
TL;DR: The method demonstrates a remarkable effectiveness for overcoming the phase ambiguity problem that has the major obstacle in the use of SIR or SAS data alone for macromolecular studies.
Abstract: Publisher Summary The chapter presents a procedure that is applicable to single isomorphous replacement (SIR) data without anomalous scattering information as well as to pure single-wavelength anomalous scattering (SAS) data alone, and it does not require the presence of noncrystallographic symmetry. Test results shows that it is very effective; not only can it remove the phase ambiguity but it will also refine the phases at the same time. In actual application to SIR data, the method has even produced results that appear to be superior to those obtained from the multiple isomorphous replacement method. One of the situations in which the phase ambiguity problem occurs is when the isomorphous replacement method is applied to a noncentrosymmetric structure using only one isomorphous data set. The method demonstrates a remarkable effectiveness for overcoming the phase ambiguity problem that has the major obstacle in the use of SIR or SAS data alone for macromolecular studies. The computer programs used in the studies has been assembled into a program package.

977 citations


Book ChapterDOI
TL;DR: The flexible coordinate data set and the absence of any connectivity dictionary mean that any sort of molecular fragment can be displayed, and the fragment rotation/translation with neighbor calculations can be used to dock substrates into active sites and this has recently been enhanced to include local energy minimization.
Abstract: Publisher Summary The program FRODO is implemented on the Vector General 3400 (with DEC, VAX, or PDP-11 computers), MMS-X, Evans & Sutherland PS 2 (PDP-11), MPS (VAX or PDP-11), and PS300. The advantages of building a model with FRODO rather than in a Richards box including speed, accuracy, control and volume. FRODO has extensive model-making features to produce coordinates from a given sequence which have standard bond lengths and angles and preferred torsion angles. FRODO is designed as a tool for the practicing protein crystallographer. However, some of its features make it of more use: the flexible coordinate data set and the absence of any connectivity dictionary mean that any sort of molecular fragment can be displayed, the fragment rotation/translation with neighbor calculations can be used to dock substrates into active sites and this has recently been enhanced to include local energy minimization, and the edit features in the SAM option allow one to change residue types and delete or insert new residues.

792 citations


Book ChapterDOI
TL;DR: Some extensions of the current export versions of the programs that have been implemented or are envisioned are described, expected to be important in realizing the goal of producing refined structural models that reproduce the diffraction patterns to within the accuracy of the measured data and which are compatible with prior stereochemical knowledge of macromolecules.
Abstract: Publisher Summary The chapter focuses on the practical application of stereochemically-restrained refinement to macromolecular crystals. Details of computational procedures and minimization algorithms are treated and need not be considered in routine applications. However, it is important to understand the nature of the function being minimized. Thorough structural refinement has become an integral part of macromolecular crystallography. The chapter describes some extensions of the current export versions of the programs that have been implemented or are envisioned. Atomic motion and conformational heterogeneity (or disorder) is major impediments to successful refinement. The use of Fourier transformations to compute structure factors and gradient vectors might greatly improve speed for large problems. There are numerous other improvements that can be envisioned including a method for modeling the fluid solvent, an appropriate treatment of the correlation between occupancy and thermal parameters of discrete solvent molecules, restraints for nonbonded contacts from crystal packing, inclusion of attractive potentials for nonbonded contacts, provision for refining partial structures, and proper estimation of standard deviations. Extensions such as these are expected to be important in realizing the goal of producing refined structural models that reproduce the diffraction patterns to within the accuracy of the measured data and which are compatible with prior stereochemical knowledge of macromolecules.

558 citations


Book ChapterDOI
TL;DR: This chapter highlights one of the methods for the analysis of experimental data, along with the assumptions, advantages, and disadvantages of the method.
Abstract: Publisher Summary This chapter highlights one of the methods for the analysis of experimental data, along with the assumptions, advantages, and disadvantages of the method. The most important experimental detail to understand for any parameter estimation procedure is the sources and magnitudes of the random and nonrandom experimental errors superimposed on the data. Moreover, computers are not oracles. The investigator needs to be continually aware that the output of any computer program is no better than what goes into it. It is important to realize that a computer is in essence the same as any other instrument in a laboratory. To obtain optimal results it must be used correctly. To use it correctly, the user must understand the assumptions which went into the development of the computer programs and their limitations.

436 citations



Book ChapterDOI
TL;DR: This chapter focuses on mammalian γ-glutamyl transpeptidase, emphasizing the rat kidney enzyme, the most extensively studied transpepticase, the key pathway for the synthesis and degradation of glutathione.
Abstract: Publisher Summary This chapter focuses on mammalian γ-glutamyl transpeptidase, emphasizing the rat kidney enzyme, the most extensively studied transpeptidase. γ-glutamyl transpeptidase plays a key role in the γ-glutamyl cycle, a pathway for the synthesis and degradation of glutathione. Glutathione is synthesized from its constituent amino acids within the cells by the successive actions of γ-glutamylcysteine and glutathione synthetases. The reactions allow the storage of cysteine as glutathione and γ-glutamyl transpeptidase catalyzes the first step in the pathway that leads to release of the cysteine moiety from the tripeptide. Homogenates of animal tissues exhibit a wide range of transpeptidase activity. Low transpeptidase activity in a tissue homogenate is often misleading because specific localization studies indicate that there are specific regions of intense enzyme activity in many tissues. In most mammals, the kidney exhibits by far the highest activity.

276 citations


Book ChapterDOI
TL;DR: This chapter investigates glutathione transferases derived from human liver, a group of related enzymes that catalyze the conjugation of glutATHione with a variety of hydrophobic compounds bearing an electrophilic center.
Abstract: Publisher Summary This chapter investigates glutathione transferases derived from human liver. The glutathione transferases are a group of related enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds bearing an electrophilic center. The proteins also act as intracellular binding proteins for a large number of lipophilic substances, including bilirubin. Human glutathione transferases have been purified from liver, erythrocytes, placenta, and lung. A simple and rapid procedure for the purification of basic (α-ɛ) and neutral (μ) glutathione transferases from human liver cytosol is described in the chapter. The enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The steps of the purification procedure include (1) preparation of cytosol fraction, (2) chromatography on Sephadex G-25, (3) chromatography on DEAE-cellulose, and (4) chromatography on Sephadex G-25.

259 citations


Book ChapterDOI

255 citations


Book ChapterDOI
TL;DR: This chapter presents the isolation procedure of the γ-glutamylcysteine synthetase enzyme and the physical properties of the enzyme are reviewed.
Abstract: Publisher Summary Glutathione is synthesized by the consecutive actions of γ-glutamylcysteine synthetase and glutathione synthetase. The most highly purified preparations of the γ-glutamylcysteine synthetase enzyme have been obtained from rat kidney and rat and sheep erythrocytes. This chapter presents the isolation procedure of this enzyme. The assay method (ADP formation) is discussed. The enzyme activity is measured in reaction mixtures containing L-glutamate, L-α-aminobutyrate, and ATP by a coupled enzyme procedure in which the rate of formation of ADP, in the presence of pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, and NADH, is obtained from the decrease in the absorbance of NADH at 340 nm. The physical properties of the enzyme are reviewed in the chapter. The molecular weight of the rat kidney enzyme is about 104,000. The enzyme dissociates under denaturing conditions to yield two subunits of molecular weights about 73,000 and 27,700. Treatment of the enzyme with dithiothreitol followed by gel electrophoresis under nondenaturing conditions leads to partial dissociation into subunits.

Book ChapterDOI
TL;DR: Primary cultures of perinatal mouse or rat brain consist of many cell types, such as astroblast, oligodendroblasts, ependymal cells, capillary endothelial cells, phagocytic cells, and mesenchymal Cells, but are devoid of neurons; one cell type—the astroblast—appears to dominate quantitatively.
Abstract: Publisher Summary Primary cultures of perinatal mouse or rat brain consist of many cell types, such as astroblasts, oligodendroblasts, ependymal cells, capillary endothelial cells, phagocytic cells, and mesenchymal cells, but are devoid of neurons In spite of cellular heterogeneity, one cell type—the astroblast—appears to dominate quantitatively Most of these cells have been identified immunocyto-chemically by using antibodies against cellular markers, in most cases, cell-type specific proteins From the size of most of the hormonal stimulations, it has been concluded that the action must be on the most abundant cells—the astroblasts The fact that a hormone listed as inhibitory interferes with the action of a stimulating hormone has been interpreted as evidence for the copresence of the corresponding receptors in the same cell surface Thus, the interference of one group of hormones with the action of another group of hormones has been used to allocate a set of receptors to one cell type within a heterogeneous population of cells


Book ChapterDOI
TL;DR: This chapter provides an overview of the assay methods of glutathione peroxidase enzyme, a selenium-dependent enzyme that is active with both organic hydroperoxides and H 2 0 2.
Abstract: Publisher Summary Glutathione peroxidases catalyze the reduction of hydroperoxides (ROOH) by glutathione (GSH). “R” may be an aliphatic or aromatic organic group or, simply, hydrogen. The products are H 2 O, an alcohol (ROH), and glutathione disulfide (GSSG). Regeneration of GSH from GSSG in the cell is effected by the enzyme glutathione reductase. Assays of glutathione peroxidase activity are based on the measurement of ROOH or GSH consumption. Alternatively, GSSG production is monitored by coupling to the reaction catalyzed by glutathione reductase. Oxidation of NADPH is recorded spectrophotometrically or fluorometrically. This chapter provides an overview of the assay methods of glutathione peroxidase enzyme. There are two major types of glutathione peroxidase. One type is distinguished by containing selenium in the form of covalently bound selenocysteine in its active site. This selenium-dependent enzyme is active with both organic hydroperoxides and H 2 0 2 . The second type of glutathione peroxidase consists of proteins that do not depend on selenium for catalysis and have negligible activity with H 2 0 2 . This class is constituted by glutathione transferases.

Book ChapterDOI
TL;DR: The CSF bioassay is based on the CSF-dependent formation of colonies of granulocytes and/or macrophages by bone marrow cells cultured in semisolid agar or methyl cellulose medium and is suitable for large numbers of assays.
Abstract: Publisher Summary This chapter discusses the macrophage colony-stimulating factor—that is, CSF The production of granulocytes and macrophages from immature hemopoietic progenitor cells in tissue culture is dependent on the presence of a group of specific growth factors These factors are termed the CSFs because in semisolid culture media they stimulate individual hemopoietic progenitor cells to form colonies of granulocytes and macrophages They are believed to regulate granulocyte and macrophage production in intact animals although their in vivo effects have not been elucidated At least 4 CSF subclasses can be defined by the kind of mature cells they produce in culture The CSF bioassay is based on the CSF-dependent formation of colonies of granulocytes and/or macrophages by bone marrow cells cultured in semisolid agar or methyl cellulose medium For large numbers of assays the agar culture method is more rapid and less expensive However, good cellular morphology of stained colony cells is more easily obtained with colonies from methyl cellulose cultures Bioassays may be carried out in a number of species including chickens or man Methods for the bioassay of murine and human CSFs are also described further in the chapter

Book ChapterDOI
TL;DR: Many of the problems associated with highly reactive allylic pyrophosphates are circumvented by introduction of the labile carbon-oxygen bond in the final step by a direct displacement with inorganic pyroph phosphate and by a chromatographic purification on cellulose.
Abstract: Publisher Summary This chapter highlights the synthesis and purification of commonly used compounds–isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, and farnesyl pyrophosphate. Along with isopentenyl pyrophosphate, these compounds are substrates and products for the prenyltransferases that catalyze the basic chain elongation reactions in the pathway. The procedure commonly used to synthesize allylic pyrophosphates was first reported in 1959 and has not been altered significantly since then. The reaction involves treatment of a mixture of the alcohol and inorganic pyrophosphate with trichloro-acetonitrile to generate a complex mixture of organic and inorganic mono-, di-, and triphosphates. Yields of the desired products rarely exceed 30%, and further losses are usually encountered during purification. In addition, the procedure becomes difficult to manage if more than 5-10 mg of material is involved. Therefore, many of the problems associated with highly reactive allylic pyrophosphates are circumvented by introduction of the labile carbon-oxygen bond in the final step by a direct displacement with inorganic pyrophosphate and by a chromatographic purification on cellulose. The procedure has been used to prepare a variety of pyrophosphates in addition to those listed above and can be scaled-up to produce useful quantities of material.

Book ChapterDOI
TL;DR: This chapter presents a set of protocols, as developed in the laboratory, that can be used to study simultaneously and comparatively the susceptibility of Ns and Ni to be ADP-ribosylated by cholera and pertussis toxin.
Abstract: Publisher Summary Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (Ns) and inhibitory (Ni) regulatory components of adenylyl cyclise. Cholera toxin acts on Ns by ADP-ribosylating its αs subunit. It uses NAD+ as a cosubstrate. ADP-ribosylation of Ns alters its properties so that its guanosine triphosphate (GTP) hydrolyzing capacity is inhibited and the action of GTP is potentiated. In intact cells, this leads to the increases in cyclic adenosine monophosphate (AMP) levels. Both pertussis and cholera toxin are hexameric multisubunit molecules. Cholera toxin is composed of one A and 5 B subunits; pertussis toxin is formed of one S1, one S2, one S3, two S4, and one S5 subunits. This chapter presents a set of protocols, as developed in the laboratory, that can be used to study simultaneously and comparatively the susceptibility of Ns and Ni to be ADP-ribosylated by cholera and pertussis toxin.

Book ChapterDOI
TL;DR: Although the expression is not accurate for a specific atom, averages taken over all, or large parts, of a structure become very good approximations of total surface area.
Abstract: Publisher Summary This chapter focuses on the calculations of certain geometrical volumes and areas that can be derived from high-resolution structural data In the presently used procedures no allowance is made for errors or fluctuations in the coordinates The structure is simply a collection of points in three-dimensional space Sensitivity to error, if required, is estimated by repeating the calculation with appropriately altered coordinate lists and comparing the results None of the algorithms places any restrictions on the position of the atom centers The chapter describes an approximate analytical expression for the accessible area rather than the numerical calculation The equations are differentiable and can be used directly as a factor to incorporate solvent influences in energy minimization procedures The derivation assumes a random distribution of spheres surrounding the target atom and includes a correction for excluded volumes Although the expression is not accurate for a specific atom, averages taken over all, or large parts, of a structure become very good approximations of total surface area

Book ChapterDOI
TL;DR: This chapter focuses on biochemical assays for Ca 2+ -selective channels in electrically excitable membranes, which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydro-pyridines, diltiazem (and various other drugs), as well as inorganic di- or trivalent cations.
Abstract: Publisher Summary This chapter focuses on biochemical assays for Ca 2+ -selective channels in electrically excitable membranes, which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydro-pyridines, diltiazem (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives, which block calcium channels with ED 50 values in the nanomolar range. The affinity for their drug receptor site within the calcium channel can be very high. Their interaction with other membrane components, including sodium channels or neurotransmitter receptors, is negligible, which makes the choice of tissue preparation and purity of the respective membrane fraction less critical. A spectrum of compounds exists that regulate calcium channel function from blockade to opening. The chapter also discusses the tissue specificity of channel labeling, the complex interactions of divalent cations with the nimodipine-labeled calcium channels, and the allosteric regulation of nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers.

Book ChapterDOI
TL;DR: This chapter describes methods that can be used to calculate the binding constants and stoichiometry of particular protein-ligand interactions from the dependence of the observed fluorescence of a protein ligand mixture upon total ligand concentration.
Abstract: Publisher Summary This chapter illustrates that fluorescence spectroscopy has developed into a routine experimental procedure for the study of protein-ligand interactions. To study ligand binding by this method, one only requires that a change in quantum yield be consequent upon ligand binding, whether one is observing ligand fluorescence, intrinsic protein fluorescence, or fluorescence of a covalently or noncovalently bound fluorescent probe which is sensitive to ligand binding. Because of the indirect observation of ligand binding by such changes in fluorescent intensity, methods of data analysis differ from those which are used routinely to analyze binding data obtained using methods such as equilibrium dialysis, where the free ligand concentration is measured directly. This chapter describes methods that can be used to calculate the binding constants and stoichiometry of particular protein-ligand interactions from the dependence of the observed fluorescence of a protein ligand mixture upon total ligand concentration. The relevant theory, approach, and possible pitfalls associated with the fluorescent measurement of interactions is presented with respect to both perturbation of ligand fluorescence on binding, along with the perturbation of the intrinsic fluorescence of acceptor on interacting with ligand.

Book ChapterDOI
TL;DR: By somatic cell hybridization, several hybrid cell lines have been generated from mouse neuroblastoma and rat glioma cells, some of which display a long list of neuronal properties and in which these differentiated functions are well expressed, especially suited for studying such functions among which are susceptibilities to peptide hormones.
Abstract: Publisher Summary Due to the complexity of the mammalian nervous system, results from biochemical or pharmacological experiments with pieces or homogenates of nervous tissue are difficult to interpret. Only recently, a few cases have been reported of apparently relatively homogeneous populations of certain cell types from nervous tissue. This chapter studies molecular mechanisms of nervous tissue functions that have been resting completely on model systems derived from tumors of the nervous system and describes the methodology for handling tumor cell lines of neuronal character. By somatic cell hybridization, several hybrid cell lines have been generated from mouse neuroblastoma and rat glioma cells, some of which display a long list of neuronal properties and in which these differentiated functions are well expressed. Therefore, these cells are especially suited for studying such functions among which are susceptibilities to peptide hormones.




Book ChapterDOI
TL;DR: It appears that glutathione serves as a storage form as well as a transport form of cysteine within cells and in the metabolic processing of certain endogenous compounds such as estrogens, prostaglandins, and leukotrienes.
Abstract: Publisher Summary Glutathione participates in the reduction of the disulfide linkages of proteins and of other molecules. It functions in the synthesis of the deoxyribonucleotide precursors of DNA. Glutathione is transported out of many cells; this process is connected with a transport system for γ-glutamyl amino acids, reactions that involve the cell membrane and its immediate environment, and the interorgan transport of amino acid sulfur. Glutathione plays a role in the inactivation of a number of drugs and in the metabolic processing of certain endogenous compounds such as estrogens, prostaglandins, and leukotrienes. Glutathione is a coenzyme for a number of enzymes. The concentration of glutathione within cells is generally much higher than that of cysteine and it appears that glutathione serves as a storage form as well as a transport form of cysteine. The available procedures for the modification of glutathione metabolism have developed from studies on particular enzymes that are involved in glutathione synthesis and utilization. For example, it is possible to decrease tissue concentrations of glutathione by inhibition of its synthesis by use of selective inhibitors of γ-glutamylcysteine synthetase.

Book ChapterDOI
TL;DR: This chapter provides an overview of the glutamine synthetase from mammalian tissues, which closely resemble each other with respect to amino acid composition, subunit structure, and molecular weight.
Abstract: Publisher Summary This chapter provides an overview of the glutamine synthetase from mammalian tissues. The glutamine synthesis reaction is freely reversible. When the enzyme is incubated with 10 m M concentrations each of L-glutamate, ammonium ions, and ATP in the presence of Mg 2+ at pH 7.0 and 37 ° , equilibrium is attained when about 90% of the L-glutamate is converted to L-glutamine. Substitution of hydroxylamine for ammonia in this system leads to a reaction that goes to greater than 99% of completion. The glutamine synthetases of mammalian origin closely resemble each other with respect to amino acid composition, subunit structure, and molecular weight. They differ substantially in these respects from bacterial glutamine synthetases, some of which exist in adenylylated forms. Glutamine synthetase activity may be followed by measuring the rate of formation of inorganic phosphate, ADP, or glutamine. ADP may be determined by coupling the glutamine synthetase reaction with those catalyzed by pyruvate kinase and lactate dehydrogenase. By use of reaction mixtures containing labeled glutamate, the disappearance of glutamate and the formation of glutamine may be determined. A commonly used procedure for determining glutamine synthetase activity involves replacement of ammonia by hydroxylamine. In this reaction, γ-glutamylhydroxamate is formed, which gives a characteristic color reaction on addition of ferric chloride.

Book ChapterDOI
TL;DR: This chapter addresses long-acting, drug-delivery systems comprising steroidal drugs microencapsulated in aliphatic polyester resins, which are needed to produce injectable controlled-release doses.
Abstract: Publisher Summary This chapter addresses long-acting, drug-delivery systems comprising steroidal drugs microencapsulated in aliphatic polyester resins Because the aliphatic polyesters slowly degrade, owing to hydrolysis of ester linkages, when they are exposed to water they disappear from the tissues soon after (or during) drug release Many drug and biologic substances, other than the steroids, can be microencapsulated in the biodegradable polyesters to produce injectable controlled-release doses The specific copolymer resins and the appropriate microencapsulation processes must be chosen carefully, however, to achieve the rates and durations of drug release desired When steroid hormones are microencapsulated in the biodegradable copolyesters in which the drug is homogeneously dispersed within the resin matrix to form a monolithic microcapsule and the microcapsules are injected either intramuscularly or subcutaneously, the drugs are slowly released into the tissues principally by a diffusion mechanism The microcapsules are designed to release norethisterone at a nearly constant rate for 3 months The 85:15 poly(DL-lactide-co-glycolide) resin is selected as the biodegradable excipient because the onset of resin fragmentation occurs at about 50 days postinjection Total resorption of the resin by the tissues is complete in about 180 days

Book ChapterDOI
TL;DR: Two subclasses (or isotypes) of IgA, IgA l and IgA 2 , are identified in human serum and secretions, and determination of subclasses and allotypes are described in the chapter.
Abstract: Publisher Summary This chapter provides an overview of immunoglobulin A (IgA). IgA is found in adult human serum in concentrations that range from 66 to 344 mg/dl. In secretions IgA represents, under normal conditions, the predominant immunoglobulin isotype. Serum IgA occurs mostly in the form of monomeric (6.5-7 S) molecules composed of two H (MW = 53,000) and two L (MW = 22,500) chains. A minor portion of the IgA present in sera of normal individuals is found in polymeric form with monomers connected by disulfide bonds and linked to an additional polypeptide called J chain. In external secretions monomeric IgA constitutes only a minor component; most of the IgA occurs in a polymeric form. The chapter further discusses subclasses and allotypes of IgA. Two subclasses (or isotypes) of IgA, IgA l and IgA 2 , are identified in human serum and secretions. IgA 1 accounts for 80-90% of serum IgA, whereas in external secretions IgA 1 constitutes only 50 to 74% of the total IgA. Determination of subclasses and allotypes are also described in the chapter.

Book ChapterDOI
TL;DR: This chapter focuses on the poly(lactic/glycolic acid) biodegradable drug–polymers (PLGA) matrix delivery systems, which have applications in subcutaneous, rumenal, and oral delivery.
Abstract: Publisher Summary This chapter focuses on the poly(lactic/glycolic acid) biodegradable drug–polymers (PLGA) matrix delivery systems. Many types of drug compounds have been incorporated in PLGA matrices, including steroid hormones, quinazoline antimalarials, and proteins. These systems have applications in subcutaneous, rumenal, and oral delivery. The duration of the drug release is primarily limited by the retention time of the device at the site; short-term delivery is easily accomplished in any route of application; however, subcutaneous systems are required for lifetimes greater than 1 year. The rate and duration of release are controlled by system design, and the system design parameters are selected by the choice of manufacturing processes. Polymer preparation and purification control the composition, size, and dispersity; matrix preparation is a function of the drug loading; and the shaping into the dosage forms determines the density, size, and shape. The two largest areas of growth for PLGA matrix delivery applications are expected to be in protein hormone and enzyme therapy and in single dose immunizations. Other applications of PLGA matrix systems are in vitro systems such as nutrient delivery to cell growth media and pesticide delivery.

Book ChapterDOI
TL;DR: The continued use over many years of mass spectrometry for bile acid analysis can be attributed to its value as a method for providing definitive qualitative and quantitative information.
Abstract: The continued use over many years of mass spectrometry (MS) for bile acid analysis can be attributed to its value as a method for providing definitive qualitative and quantitative information. Its combination with gas chromatography (GC—MS) has been particularly important for investigating the stereochemical variety of the structure of bile acids in relation to their biosynthesis, transport, and metabolism.