Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amino acid metabolism of 23 different Brucella strains was investigated for differentiation purposes. The results were evaluated by thin layer chromatography, after enzymatic incubation. The organisms (Tab. 1) were grown on Tryptose blood agar at 37 degree C for 24 or 48h. Two mg wet weight of bacteria in 0.2 ml PBS, 0.01 M, were incubated with 12.5 microng (0.025 ml) amino acid in small tubes for 16h at 37 degree C, and centrifuged for 15 min at 7500 X g. For controls, bacterial suspensions were heated for 15 min at 100 degree C to destroy enzymatic activity, and also contrifuged for 15 min at 7500 X g. Usually 4 micronl of the supernatant fluids (6 micronl for L-asparagine, and 10 micronl for L-proline) were pipetted on the thin layer plate. The tests were run in n-butanol acetic acid water, 20:5:5, with a distance of 8 cm. Amino acids were stained with ninhydrine. The tests were repeated 3-5 times with identical results. Amino acid metabolism was indicated by different staining intesities (+ to ++) in comparison to control preparations. All species could be exactly differentiated from each other, with the exception of B. suis, biotype 2, and B canis, which could not be differentiated by their amino acid metabolism. Biotyps of the same species were mostly identical. The results of these investigations could be reproduced qualitatively as well as quantitatively. The method described is recommended for routine investigations.
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PMID:[Investigations of amino acid metabolism by thin layer chromatography for differentiation of Brucella (author's transl)]. 86 72

Encouraged by recent reports of extraction of diagnostically useful DNA from formalin-fixed tissues, we devised a study to determine which fixatives are suitable for DNA hybridization studies. Seven lymph node specimens (6 lymphomas and one reactive hyperplasia) were studied. Specimens were divided into portions, each of which was processed separately. Processing protocols followed were: 1) snap freezing; 2) immersion in formalin, ethanol, glutaraldehyde, B5 and methanol acetic acid (MAA) Michel's transport medium or phosphate buffered saline for 2, 24, 48 and 120 hrs.; 3) paraffin embedding following fixation. DNA was obtained by proteinase K digestion followed by phenolchloroform extraction. DNA was cleaved with restriction endonucleases (Eco RI and Bam HI), separated by agarose gel electrophoresis, after which Southern blot hybridization with radio-labelled probes for J-heavy and T-beta genes was performed. Fixation with formalin, glutaraldehyde and B5 resulted in poor yields of DNA. MAA produced degradation of DNA and Michel's medium and PBS gave low quantities and purity of extracted DNA. Ethanol fixation consistently permitted extraction of large amounts of high molecular weight DNA suitable for hybridization studies and compared favourably with the fresh-frozen 'gold standard'. We conclude that ethanol may be the fixative of choice when transport or storage conditions limit the availability of fresh frozen tissue for DNA hybridization studies.
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PMID:The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue. 313 29

Searching for the best procedure for simultaneous estimation of the anterior pituitary hormones, extraction efficiencies of various media, additives such as urea and triton X-100, and physical treatments such as freezing-thawing (F-T) and sonication, were examined by measuring prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) in the extracts. Ethanolic media (60% EtOH) gave high yields of PRL at neutral to alkaline pH, but poor extraction of GH accompanied by a marked loss of its immunoreactivity during storage. Ethanolic media also gave a poor yield of LH even at high pH. Aqueous media like PBS at various pH, 0.1 M acetic acid and distilled water were considerably effective in the extraction of GH, LH, FSH and TSH if they were coupled with F-T and sonication. However, high yields of PRL could not be obtained with these aqueous media even with F-T and sonication. Hartree's 40% EtOH-6% ammonium acetate, pH 5.1, solubilized considerable amounts of glycoprotein hormones, but yielded almost no GH and only a small amount of PRL. The addition of triton X-100 to PBS (pH 7) at 0.1% resulted in the maximum extraction of glycoprotein hormones with homogenization and F-T, but further sonication was necessary for GH and PRL. When the anterior pituitaries were homogenized and frozen-thawed in PBS (pH 7) containing 1 M urea, yields of PRL, GH, LH, FSH, and TSH were maximum, and sonication did not cause any additional extraction, indicating that this procedure, i.e. homogenization and F-T in 1 M urea-PBS, would be the best for the simultaneous estimation of these anterior pituitary hormones.
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PMID:Choice of extraction procedure for estimation of anterior pituitary hormone content. 343 4

Using a monospecific, monoclonal antibody against the glucocorticoid receptor (GR), an immunocytochemical study was performed to investigate the intracellular localization of GR both in the presence or absence of ligand. With all fixation methods tested (paraformaldehyde, acetic acid in ethanol, Bouin's fixative, and bensochinone in PBS), it was possible to obtain specific GR staining. Fixation with paraformaldehyde was chosen for further studies on the effect of permeabilization, using several concentrations of Triton X-100 or saponin. A rat Rueber hepatoma (H-4-II-E) and a human uterus carcinoma (NHIK 3025) cell line were used as well as cultured hepatocytes from normal rat. The accessibility of the different cell compartments after fixation and permeabilization was tested for by using antibodies against cellular constituents with known locations (i.e. core-nucleosome proteins and tubulin), in combination with the anti-GR antibody in double immunofluorescence staining experiments. The specific GR stain obtained with the indirect peroxidase antiperoxidase technique or with fluorescein isothiocyanate-labeled second antibodies was shown to be present both in the cytoplasm and in the nucleus. Staining of all cellular compartments was abolished (peroxidase antiperoxidase) or diminished (fluorescein isothiocyanate) if the monoclonal antibody was preincubated with a 90% pure GR preparation. These findings are in contrast to recently reported immunocytochemical studies, where a strict nuclear existence of the estrogen and progestin receptors has been reported. Consequently, generalizations with regard to steroid receptor localization cannot be made. Furthermore, an in vitro model is described, where the effect of dexamethasone administration upon the localization of receptor staining in H-4-II-E cells can be studied.
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PMID:Intracellular localization of the glucocorticoid receptor: evidence for cytoplasmic and nuclear localization. 354 55

Different fixatives and immunohistochemical methods were tested for detection of fibronectin in various paraffin embedded tissues: rat kidney, spleen, gastro-intestinal tract, muscle, normal and fibrotic liver and human skin. Using cryostat sections, localisation with immunofluorescence and peroxidase technics comparable to those obtained in unfixed tissue sections, could be obtained with the following fixatives: 10% formalin in PBS containing 4% sucrose; 96% ethanol; 96% ethanol + 1% acetic acid; a series of ethanol solutions of increasing strength: 70-80-96%. These fixatives also proved to be the best for paraffin embedding. Without enzyme digestion, however, satisfactory results could not be obtained with either indirect peroxidase or immunofluorescence methods in paraffin embedded tissues. Following digestions with the enzymes at the concentrations described in the literature, the alteration of tissues made the morphological localization of fibronectin difficult. The self-sandwich peroxidase method following a gentle pepsin digestion gave results closest to those of unfixed cryostat sections; however a slight increase in background staining was observed but without interfering with the evaluation of results.
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PMID:Immunohistochemical detection of fibronectin using different fixatives in paraffin embedded sections. 635 96

Various types of extraction were tested to increase the immunological yield of BCA, a CEA-like primary breast cancer associated carcinoma antigen. To allow a comparison, the different extraction techniques were applied to only one breast tumour. The comparison of the various systems was based on two parameters: protein yield and immunological activity, assayed in a RIA 125I CEA-anti CEA system. The following extraction methods were described and compared in this paper: 3M KCl; 1N HClO4; neutral pH extraction (PBS) in the absence and presence of various detergents (anionic, neutral and cationic), basic pH extraction (1N NaOH) and acid pH extraction (1.5M acetic acid) in the presence of urea and various detergents. The more significant systems were applied also to the extraction of CEA, from colonic adenocarcinoma liver metastases. The best results for both the antigens studied were obtained by using neutral detergents (1% NP 40) at neutral pH.
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PMID:BCA (breast cancer antigens): different purification extraction methods. 712 28

One of the more recently discovered collagens, type V (or A-B) collagen, in its native fibillar form mediates human platelet aggregation and the release of serotonin. In agreement with a recent report, it has no detectable effect on human platelets in the soluble or amorphous form. The possibility that the observed results might be due to contaminating interstitial collagens was eliminated by taking advantage of unusual solubility properties of type V collagen. Type V collagen dissolved in 0.1M acetic acid formed native-type fibrils when dialyzed against PBS and amorphous fibrils when dislyzed against 0.05M Tris/0.13M NaCl, pH 7.4, at 4 degrees C. Interstitial collagens remained in solution under both of these conditions. In addition, type V collagen treated with sufficient, purified synovial collagenase to digest all contaminating interstitial collagen retained its platelet-aggregating properties. The purity of type V collagen was confirmed by SDS-PAGE of CNBr digests. These data indicate that the quaternary structure of type V collagen is important in its recognition by platelet membranes.
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PMID:Collagen-platelet interaction. Type V(A-B) collagen induced platelet aggregation. 735 Feb 45

Spontaneously existing and chemically induced micronuclei were isolated from mouse blood. 50 microliters of cardiac blood was diluted with PBS and centrifuged. After this, the cell pellet was subjected to hypotonic treatment, fixed with acetic acid-methanol (1:3), and the lysate was filtrated through a 2-microns polycarbonate nucleopore membrane. Isolated micronuclei were air-dried on a glass slide and subjected to fluorescence in situ hybridization (FISH) using a mouse centromeric gamma satellite probe. Approximately half of the micronuclei isolated from vehicle control mice showed centromere signal(s). In these preliminary studies, the proportion of centromere-positive micronuclei was increased by treatment with spindle poisons (colchicine and vinblastine sulfate), decreased only slightly by 1-beta-D-arabinofuranosylcytosine, and was generally unaffected by mitomycin C.
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PMID:Isolation of micronuclei from mouse blood and fluorescence in situ hybridization with a mouse centromeric DNA probe. 751 3

Glucose-6-phosphate dehydrogenase (G6PD) was purified from rabbit reticulocytes by using a single immunoaffinity chromatographic step. Antibodies against rabbit erythrocyte G6PD were raised in a goat, purified near to homogeneity and immobilized on CarboLink gel (Pierce). Nonspecific binding sites on the matrix were saturated with myokinase from rabbit muscle. Reticulocyte lysate was directly loaded onto the column, allowed to enter the gel bed and incubated for 15 min at room temperature. The column was washed first with PBS and then with 1 M NaCl. The enzyme was eluted with 0.1M acetic acid in 1M NaCl. Two protein bands of about 66.2 kDa were co-eluted with the G6PD, which was however well separated in SDS-PAGE. The eluent destroyed the enzyme activity but the G6PD yield was higher than 90%.
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PMID:Purification of glucose-6-phosphate dehydrogenase from rabbit reticulocytes by immunoaffinity chromatography. 805 Aug 74

In this survey, lipid metabolism and activities of the lipolytic enzymes diglyceride lipase (DLase) and phospholipase A (PLase A) in the oyster digestive glands (ODGs), with 4% added acetic acid (the same acid concentration as vinegar) and incubated at 37 degrees C for 3 h, were investigated. Significant decreases in triglyceride, phosphatidycholine, and phosphatidylethanolamine and increases in monoglyceride, lysophosphatidylcholine, and lysophosphatidylethanolamine were observed in the acetic acid-treated ODGs. Changes in ODGs treated with PBS were smaller than in the acetic acid-treated ones but larger than in the nontreated ones. Both PLase A1, and PLase A2 in ODGs were activated by addition of acetic acid and incubated at 37 degrees C for 3 h. PLase A1 activities were higher than those of PLase A2, in all experimental ODGs. Addition of formic acid also induced activation of PLase A at pH 2. On the other hand, DLase in ODGs decreased remarkably with acetic acid treatment. These data showed that the increase in lipid metabolites such as free fatty acids and lysophospholipids in the acetic acid-treated ODGs might be due to catabolism of PLase A, which was activated by the acid treatment at 37 degrees C.
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PMID:Is phospholipase in vinegared oysters a casual agent for human poisoning? 860 47


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