Immunochemical Study Research Articles

The fate of Crotalus atrox venom in the lung of the mouse: an electron microscopic immunochemical study: Rolf I. Schiff and Joseph F. Gennaro Jr. (Department of Biology, New York University, New York, NY 10003, U.S.A.)

The fate of Crotalus atrox venom in the lung of the mouse: an electron microscopic immunochemical study: Rolf I. Schiff and Joseph F. Gennaro Jr. (Department of Biology, New York University, New York, NY 10003, U.S.A.)

An immunochemical study of the diapause-associated protein of the southwestern corn borer, Diatraea grandiosella

The distribution and titre of the diapause-associated protein (DAP), which accumulates in the fat body of diapausing larvae of the southwestern corn borer, Diatraea grandiosella, were determined using an antibody raised to the purified protein. Immunoblotting and immunodiffusion showed that the highest concentration of DAP was present in the fat body. Small amounts of the protein were present in the haemolymph and even lower levels were detected in other tissues. Rocket immunoelectrophoresis showed that DAP accumulated in the fat body at the beginning of diapause, reached a plateau, and gradually declined towards the end of diapause. Similarly, the titre of DAP in last instar non-diapausing larvae treated with a JH analogue increased, reached a plateau and then declined. Double immunodiffusion and immunoblotting using the DAP-antibody and extracts of the fat body of D. grandiosella and the southern cornstalk borer, Diatraea crambidoides, and the sugarcane borer, Diatraea saccharalis, revealed the partial immunochemical identity of the DAP of D. grandiosella and a protein present in these two species. Denaturing electrophoresis with immunoblotting showed that the DAP-related proteins of D. crambidoides and D. saccharalis have apparent molecular weights of 33,000 and 36,000, respectively, as compared to a molecular weight of 35,000 for DAP of D. grandiosella. A dot blot analysis showed that no cross reaction occurred between the “diapause proteins” present in the haemolymph of adults of the Colorado potato beetle, Leptinotarsa decemlineata, and the DAP of D. grandiosella.

In vivo regulation of δ-aminolevulinate dehydratase activity

The interrelationships among purified δ-aminolevulinate dehydratase (ALA-D; EC 4.2.1.24) activity, Pb, and ALA-D concentrations were studied in vitro. The ratio of Pb to the ALA-D subunit, but not the Pb concentration, determined the relative activity of ALA-D, indicating the significance of the amount of ALA-D in studying the mechanism of enzyme inhibition by Pb. To elucidate the in vivo mechanisms of ALA-D regulation, male Wistar rats, 6 months old, were treated with Pb, Zn, and glutathione (GSH) separately or in combination for 130 days. After Pb administration, the amount of ALA-D, as determined by radioimmunoassay, increased in the presence and in the absence of the Zn-GSH pretreatment, even if the enzyme activity were higher (the Zn-GSH-Pb-treated than that of the control. Zn and GSH restored the enzyme activity in vivo synergistically. Since immunochemical study showed the identity of the liver ALA-D with the erythroid ALA-D, the liver and erythroid data were pooled to quantify the interrelationship among ALA-D activity, and Pb, Zn, SH, and the enzyme concentrations. The equation was the relative activity of ALA-D(%)=0.256·[Pb/ALA-D subunit] 2 − 9.56·[Pb/ALA-d subunit] − 0.000281·[ Zn·SH /ALA-D subunit] 2+0.0898·[ Zn·SH /ALA-D subunit]+33.6 (multiple correlation coefficient = 0.909, n = 108, p < 0.01). The result indicated that 83% of the in vivo regulation of ALA-D activity is explained when the four factors, Pb, Zn, SH, and ALA-D concentrations, are considered in combination.

Myosin changes in hypertrophied human atrial and ventricular myocardium. A correlated immunofluorescence and quantitative immunochemical study on serial cryosections.

Two antigenically distinct types of myosin heavy chain, referred to as alpha and beta, have been identified in autoptic and bioptic specimens of human heart using specific antimyosin antibodies. By immunofluorescence heavy chain alpha was present in all atrial myocytes and in a variable number of ventricular myocytes. Heavy chain beta was present in all ventricular myocytes and in a number of atrial myocytes. Ventricular hypertrophy in patients with aortic stenosis, systemic hypertension or tetralogy of Fallot was characterized by an almost complete absence of fibres reactive with anti-alpha. A striking decrease in alpha chain reactivity and a parallel increase in beta chain reactivity was apparent in the hypertrophied left atria of patients with mitral stenosis. To quantify these myosin changes a novel procedure was developed whereby myosin was extracted from single cryosections serial to those processed for immunofluorescence and the relative amount of alpha and beta heavy chain was determined by enzyme immunoassay. Heavy chain alpha was less than 5% in most normal ventricular specimens and disappeared completely under the effect of pressure overload. On the other hand heavy chain beta was generally undetectable in the left atrial myocardium but increased up to 90% in biopsies of hypertrophied atria.

Immunochemical study and acinar localization of AM 1, a murine submandibular glycoprotein with esterolytic activity

Glycoprotein AM 1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 × g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM 1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM 1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM 1 was found in the murine brain, heart, lung, liver, spleen, kidney, pacreas, spinal cord and testis. In addition, glycoprotein AM 1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pacreatic kallikrein. The cellular localization of glycoprotein AM 1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM 1 could be demonstrated in the extract of the SM C secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM 1 was hardly detectable in the SM B secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Consomitantly, glycoprotein AM 1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.

Immunochemical study of chromatin non-histone proteins. II. Localization of immunogenic tissue-specific proteins in nuclease-hypersensitive sites of chromatin.

Localization of immunogenic tissue-specific and tissue-non-specific non-histone proteins in thymocyte chromatin has been investigated using antibodies against rat thymus and liver chromatin. After chromatin digestion by DNAase II and subsequent fractionation with 2 mM MgCl2, the Mg2+-soluble fraction interacts with both types of antibodies 5-6 times more effectively than Mg2+-insoluble chromatin. The experiments on chromatin digestion with DNAase I indicate that tissue-specific and tissue-non-specific proteins, reacting with antibodies, are released only upon hydrolysis of the first 1-3% of DNA. Further digestion with DNAase I causes no additional solubilization of these proteins. The chromatin fraction enriched in immunogenic proteins is also released upon autolytic digestion of chromatin with endogenous nuclease. The data obtained suggest that by their function tissue-specific and tissue-non-specific antigenic determinants belong to the same class of non-histone proteins localized in the chromatin sites hypersensitive to nucleases. The possible role of these proteins in regulation of the transcription is discussed.

Immunochemical study of chromatin non-histone proteins. I. Production of antibodies against rat thymus and liver chromatin; analysis of tissue specificity and intranuclear distribution of immunogenic proteins.

Rabbit antibodies against rat thymus and liver chromatin are obtained. Antithymus IgG are found to interact only with homologous chromatin, while antiliver IgG interact with both liver and thymus chromatin. After preincubation of antiliver IgG with thymus chromatin the antibodies interact with homologous chromatin only. Thus chromatin of both organs contains tissue-specific proteins. Antiliver and antithymus IgG are used to investigate a distribution of tissue-specific and tissue-non-specific immunogenic proteins in thymocyte nuclei. It is shown that these proteins are practically lacking in nuclear membranes, matrix, nuclear sap, nucleous chromatin and do not participate in attaching DNA to the nuclear matrix.

Increase of choline acetyltransferase by colchicine in primary cell cultures of spinal cord.

Colchicine (5-10 microM) increased choline acetyltransferase (ChAT) activity 5-10-fold and suppressed acetylcholinesterase (AChE) and glutamate decarboxylase (GAD) activities to 30% and 50%, respectively, of the levels of control cells in mouse spinal cord cells cultured for several days. The synthesis of radiolabeled acetylcholine (ACh) from [14C]choline was also enhanced 4.6-fold, although the uptake of [14C]choline into cells was decreased to 80% of control level. Neither the incorporation of [3H]leucine into protein nor the total amount of protein was increased by colchicine. Vinblastine also increased ChAT activity while cytochalasin B was not effective. Immunochemical titration study revealed that the increase of ChAT activity by colchicine was due to the accumulation of ChAT molecules. Co-culture of spinal cord cells with skeletal muscle markedly stimulated ChAT activity, and the addition of colchicine to the cocultures showed greater than additive effect. These observations indicate that colchicine increases ChAT molecules in a specific manner, that the stimulatory effect of colchicine on ChAT activity is possibly mediated via the interaction with microtubules, and that the increase of ChAT activity is based on a mechanism different from that of co-cultures with skeletal muscle cells.

Immunochemical study of 20 monoclonal antibodies to human plasma low density lipoproteins and their use for apolipoprotein B determination.

Immunochemical study of 20 monoclonal antibodies to human plasma low density lipoproteins and their use for apolipoprotein B determination.

Purification of cytochrome P-450 catalyzing 25-hydroxylation of vitamin D 3 from rat liver microsomes

Cytochrome P-450 catalyzing 25-hydroxylation of cholecalciferol (cytochrome P-450 cc25) was purified from rat liver microsomes based on its catalytic activity at each purification step. The specific cytochrome P-450 content of the final preparation was 15.1 nmol/mg of protein. Reconstituted activity of 25-hydroxylation of cholecalciferol with the purified enzyme was 2.3 nmol/min/mg of protein, which was 4,300 times as high as that in microsomes. The minimum molecular weight of the enzyme was 50,000 based on SDS-polyacrylamide gel electrophoretogram. Amino terminal sequence of the P-450 cc25 was H 2N-Met-Asp-Pro-Val-Leu-Val-. Immunochemical study showed that the purified P-450 cc25 was homogeneous and the cytochrome was immunochemically different from either cytochrome P-450(PB-1) or cytochrome P-448(MC-1).

Immunochemical study on PHI/PHM with use of synthetic peptides

We have synthesized PHI and PHM (human PHI) as well as their fragments, PHI (1–6), PHI (1–15), PHI (14–19), PHI (14–27), PHI (20–27), PHM (1–15) and PHM (13–27), by the solution or solid-phase method for peptide synthesis. Using the highly purified synthetic peptides as immunogens or haptenic immunogens, five kinds of PHI/PHM specific antisera were produced. The major antibody-recgonition sites of the five antisera were located respectively in the PHI C-terminal (R8201), in the PHI N-terminal (R8403), in the PHM C-terminal (R8502), and in the PHM whole molecule (R8702 and R8703). Radioimmunoassays (RIAs) with antisera R8201, R8403 and R8502, respectively, showed a wide distribution of immunoreactive (IR) PHI/PHM in porcine and human gastrointestinal and brain tissues. The concentrations of IR-PHI in the porcine gastrointestinal tissues, however, differed between the R8201 and R8403 RIAs employed for measurement. By using these two different PHI RIAs, the IR-PHI in the porcine brain tissue extract was shown to be almost a single component coeluting with synthetic PHI in gel filtration. The IR-PHI in the extract of porcine lower intestine on the other hand, contained, besides a PHI-like component, unidentified component(s) eluting immediately after synthetic PHI in gel filtration; this crossreacted with the PHI C-terminal specific R8201 antiserum but not with the N-terminal specific R8403 antiserum, suggesting the presence of the C-terminal-related fragment(s) of PHI in the tissues.

Immunochemical and acarologic study of mites

The data derived in the study of Dermatophagoides pteronyssinus by microscopic and immunological assays (double radial immunodiffusion in 2 modifications and indirect rat mast cell degranulation) are provided. The immunochemical assays permitting the detection of mites in 93.3% of cases have been shown to compare very favourably with the microscopic techniques that make it possible to detect mites only in 41.7% of cases.

Evolution of butyrylcholinesterase in higher primates: An immunochemical study

1. Serum butyrylcholinesterase (BuChE; EC 3.1.1.8) of man and the higher primates was tested enzymatically and immunochemically, with the aid of monoclonal antibodies (McAb) developed against the enzyme isolated from human blood. 2. Enzyme activities showed great differences across species and among individuals, but all samples tested were dibucaine-sensitive. 3. One McAb showed similar affinities for BuChE of each species, but another showed marked differences in affinity, preferring species in the order: man > chimpanzee = pygmy chimpanzee > gorilla ⪢ orangutan > gibbon. 4. We conclude that at least one epitope of BuChE underwent progressive modification during the later stages of primate evolution.

Immunochemical study of host proteins in human supragingival compared with denture plaque

The contribution of proteins from saliva and gingival crevicular fluid (GCF) to plaque was determined by comparing extracts of supragingival plaque of unknown age formed on normal teeth with plaque formed on artificial teeth in complete or partial dentures where crevicular fluid is absent. There was a total absence of albumin and a virtual absence of IgG from denture plaque samples, confirming their crevicular origin. The concentration of lactoferrin was much higher than that of lysozyme in all supragingival but not in the denture plaque samples, suggesting that GCF provided more lactoferrin than lysozyme to plaque. Amylase was a component in both denture and supragingival plaque, present in similar amounts in both deposits. Cysteine-containing phosphoproteins from saliva were in low concentration but present in all plaque samples; proline-rich proteins were virtually absent, reflecting the high vulnerability to proteolysis of these proteins. Salivary proteins in plaque extracts do not correspond with their relative concentrations in saliva.

Microsomal NADH-Cytochrome &lt;italic&gt;b&lt;/italic&gt;&lt;sub&gt;5&lt;/sub&gt; Reductase of Bovine Brain: Purification and Properties&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of P. aeruginosa O:3(a),c and O:3a,d,e lipopolysaccharides.

On mild acid degradation of Pseudomonas aeruginosa O:3(a),c, O:3a,d,e and O:3(a),d,f lipopolysaccharides O-specific polysaccharides were isolated. The trisaccharide repeating units of O:3(a),c and O:3a,d,e polysaccharides contained 2-acetamido-2,6-dideoxy-D-galactose and 2,3-(1-acetyl-2-methyl-2-imidazolino-5, 4)-2,3-dideoxy-D-mannuronic acid, which were identified previously as the constituents of P. aeruginosa O:3a,b and O:3a,d O-specific polysaccharides, as well as 2,3-diacetamido-2, 3-dideoxyl-L-guluronic acid, which has never before been found in nature. The last monosaccharide was identified without being isolated in the free state, by means of 13C nuclear magnetic resonance spectroscopy. The structures of O:3(a),c and O:3a,d,e polysaccharides were established by selective hydrogen fluoride solvolysis followed by modification of the trisaccharides obtained and analysis of the 13C nuclear magnetic resonance spectra at each modification stage. The polysaccharides possessed similar structures of repeating units differing from each other in the anomeric configuration of the N-acetylfucosamine residue only: leads to 4)DManImU-(beta 1 leads to 4) LGul(NAc)2U(alpha 1 leads to 3)DFucNAc(beta 1-and leads to 4)DManImU(beta 1 leads to 4)LGul(NAc)2U(alpha 1 leads to 3)DFucNAc(alpha 1-, where DManImU = 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, LGul(NAc)2U = 2,3-diacetamido-2,3-dideoxy-L-guluronic acid, DFucNAc = 2-acetamido-2,6-dideoxy-D-galactose. The same components were detected in the O:3(a),d,f polysaccharide but not in the same stoichiometric ratio; this polysaccharide possessed no regular structure. The immunochemical study of lipopolysaccharides of all five P. aeruginosa O:3 serotypes showed a definite interrelation between their serological properties and structures of the corresponding O-specific polysaccharides.

Comparative physicochemical and immunochemical study of three soluble antigens of human leukocytes

Immunochemical assay was used to identify 3 soluble leukocyte antigens in leukolysates. According to the common classification these antigens were denoted as RAL-3, RAL-4, and RAL-5. The physiochemical properties of these antigens were studied as was their content in extracts of different tissues and in biological fluids.

Timing of stages and changes in numbers of multiply-infected erythrocytes during the asexual cycle of Plasmodium cynomolgi bastianellii.

Previous evaluations of the timing of developing stages in the erythrocytic cycle of the simian malaria parasite, Plasmodium cynomolgi, a frequently used model for relapsing malaria in man, have depended on procurement of blood samples at 4- or 6-hourly intervals. This brief report describes the blood cycle as determined from hourly bleedings, thus providing a more discriminating sequence usable for immunochemical or biochemical study of specific stages. The approximate timing of development for five defined stages in the asexual cycle was determined to be 12-14 hours for young trophozoites, 22-24 hours for developing trophozoites, 5-7 hours for mature trophozoites, 5-6 hours for early schizonts, and 3-5 hours for late schizonts. It was also demonstrated that the rate of erythrocytic multiple infection declined in proportion to the maturity of the parasite. Young and developing trophozoites presented the most numerous cases of multiple infection whereas very few schizonts were seen multiply infecting a host cell.

Degradation of transmembrane proteins in Ca2+-enriched human erythrocytes. An immunochemical study.

Apart from causing the formation of gamma-glutamyl-epsilon-lysine cross-linked polymers, exposure of human erythrocytes to Ca2+ and ionophore A23187 leads to a breakdown of the two major transmembrane proteins, i.e. the anion-transporting band 3 and glycophorin. This apparently proteolytic phenomenon was examined by crossed immunoelectrophoretic techniques. The main product of the cleavage of band 3 had a chain weight of about 55,000 and showed good precipitation with the antibody raised against the intact protein. The degradation of glycophorin was more rapid and, when complete, gave rise to small fragments which were barely precipitated with antiglycophorin antibody. Incubation of the cells with pepstatin or N-ethylmaleimide prior to and during Ca2+ loading prevented the breakdown of both transmembrane proteins. Histamine, a competitive inhibitor of the transglutaminase-catalyzed formation of gamma-glutamyl-epsilon-lysine cross-links in Ca2+-enriched erythrocytes, also provided some protection, suggesting that the breakdown of the two transmembrane proteins might in some manner be related to the transglutaminase-dependent polymerization process. Pathophysiological implications of the proteolytic event, which would distort the normal interaction of membrane proteins with the cytoskeleton, are discussed.

Immunochemical and immunocytochemical study of S-100 protein in rat adipocytes

S-100 is a protein originally believed to be unique to the nervous system. We report here on the presence of S-100 in rat adipocytes, using immunohistochemical and immunochemical methods. We demonstrate that the protein in adipose tissue is present at a concentration comparable to that measured in the nervous tissue and is immunologically identical to brain S-100, indicating that the protein can no longer be regarded as being specific to the nervous system.