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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several methods of coating whole cells of Mycobacterium tuberculosis H37 RV to ELISA microtitre plates were compared with the aim of developing an ELISA screening assay for murine monoclonal antibodies in culture supernatants and human antibodies in patient sera. Undercoats of nylon or poly-L-lysine were compared to polystyrene as adsorptive surfaces for the bacteria, the effect of increased ionic strength and iclusion of SDS in the coating buffer measured, and methanol (70%) and glutaraldehyde (5%) investigated for their efficiency as fixatives of the bacterial monolayers. The results suggest PBS as a satisfactory coating buffer for the bacterial cells on polystyrene, and 70% methanol the preferred fixative for the dried antigen-coated plates.
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PMID:Polystyrene, poly-L-lysine and nylon as adsorptive surfaces for the binding of whole cells of Mycobacterium tuberculosis H37 RV to ELISA plates. 212 47

A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg2+. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. The maximal difference in fluorescence between untreated and HN2-treated cells was observed after heating at 100 degrees C for 5 min in PBS containing 1.25 mM MgCl2. Higher concentrations of MgCl2 inhibited MOAB binding to HN2-treated cells and heating at lower concentrations induced binding to control cells. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl2. Thus, the presence of phosphates and MgCl2 during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S1 nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. Specific reactivity of MOAB F7-26 with deoxycytidine was established by avidin-biotin ELISA. Single-stranded conformation was necessary for the binding of MOAB to deoxycytidine on the DNA molecule. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.
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PMID:Decreased stability of DNA in cells treated with alkylating agents. 225 76

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

Immunohistochemical and histochemical methods are increasingly used and their application in surgical pathology is obvious. Especially we used these methods on bone marrow core biopsies. Optimal and comparable results have been obtained by using different methods after halving the biopsy cores longitudinally and/or transversally. The two halves were used for cytologic imprints. Two parts of the biopsy cores were embedded in polymethacrylate at low temperature (-20 degrees C). The methacrylate-embedded biopsy part for routine histology was fixed in Schaffer's solution (methanol-formalin-fixative). The methacrylate-embedded undecalcified section of 4 microns may be stained by most stains commonly employed in routine histopathology after removal of the plastic. The sections are virtually free of artefacts such as shrinkage and swelling in the light microscope. The second methacrylate-embedded part of biopsy cores was fixed in 2% paraformaldehyde with 5% sucrose in 0.02 M phosphate buffer (pH 7.4) and dehydrated in ethyleneglycolmonobutylether. All procedures were carried out at 4 degrees C. This method permits the use of immunohistochemical and histochemical procedures. The immunohistochemistry was carried out at sections of 4 microns after removal of the plastic with methoxide and use of proteolytic enzyme (0.1% alpha-chymotrypsin) to unmask antigens in sections. Surface and intracellular immunoglobulins were very well detected with the indirect FITC method. The histochemical procedures are carried out at sections of 7-8 microns after removal of plastic with xylene and toluol. The sections were incubated for specific esterase and nonspecific esterases, acid and alkaline phosphatase and then examined by light microscopy. A third part of biopsy cores may be immediately frozen, and cryostat sections are stained and evaluated for rapid diagnosis and used for immunohistologic analysis with mono- and polyclonal antibodies (FITC method) and/or histochemical investigations. Imprints of biopsy cores are evaluated for cytological, cytochemical and/or immunocytological analysis with mono- and polyclonal antibodies (FITC method). The cryostat sections and the imprints are fixed for all methods with 2% paraformaldehyde and 5% sucrose in PBS (0.02 M, pH 7.4) at 4 degrees C for 30 minutes. The best diagnostic results were obtained in the myelo- and lymphoproliferative disorders using the combination of methods described here. Examples were demonstrated.
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PMID:[Immuno- and enzymehistochemical studies of methacrylate-embedded biopsy material, especially iliac crest biopsies]. 313 12

Several concentrations of trehalose (0.0, 0.04, 0.1, 0.25 M) in combination with three concentrations of glycerol (1.0, 1.5, 2.0 M) were evaluated for the cryopreservation of murine embryos. Embryos were transferred through increasing concentrations of glycerol in Dulbecco's phosphate-buffered saline with 10% fetal calf serum (PBS + FCS) to reach the final glycerol concentrations. They were then randomly assigned to one of the concentrations of trehalose. A total of 506 morulae were packaged individually in 0.25-ml plastic straws and cooled from ambient temperature at 1.0 degrees C/min in a programmable methanol freezer. Embryos were seeded at -7 degrees C and then cooled to -25 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. After thawing and a one-step dilution of glycerol, embryos were cultured for 48 hr and viability was determined by blastocoel formation. Highest viability (70.0%) after 48 hr in culture was obtained for embryos frozen in 1.5 M glycerol plus 0.10 M trehalose as compared to 31% viability for embryos frozen with glycerol alone. These observations suggest that trehalose can be used in combination with glycerol as a cryoprotectant and that a high rate of viability can be achieved after a one-step dilution of the cryoprotectants.
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PMID:Cryopreservation of murine embryos with trehalose and glycerol. 340 7

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

Potent and novel fibrinolytic enzymes (lumbrokinase [LK]) were extracted from the earthworm, Lumbricus rubellus. These enzymes were very stable and showed greater antithrombotic activity than other currently used fibrinolytic proteins. An LK fraction showing the most potent fibrinolytic activity was immobilized onto a polyurethane (PU) surface to investigate its enzymatic activity and antithrombotic activity. A methanol-extracted PU surface was coated with 3% (wt/vol) maleic anhydride methylvinyl ether copolymer (MAMEC)/tetrahydrofuran (THF) solution, and the surface was incubated in an LK solution/phosphate-buffered saline (PBS, pH 7.4). The surface properties were characterized by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), electron spectroscopy for chemical analysis (ESCA), and dynamic contact angle. The stability of immobilized LK was determined by caseinolytic activity assay and the specificity of immobilized LK on fibrinogen/fibrin was observed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The antithrombotic activity of immobilized LK was evaluated using an ex vivo rabbit A-A shunt experiment. LK immobilization was confirmed by ATR-FTIR and ESCA. Immobilized LK demonstrated stable proteolytic activity during various incubation periods. Immobilized LK proteolyzed fibrinogen and fibrin almost specifically, while it hardly hydrolyzed other plasma proteins including plasminogen and albumin. In the ex vivo A-A shunt experiment, the LK-immobilized surface significantly prolonged occlusion time over control surfaces. This is primarily due to the high thrombolytic activity of immobilized LK. In this work, a highly efficient surface modification method on the PU surface was developed, and this LK immobilization technique will be very useful in improving the blood compatibility of blood-contacting devices.
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PMID:Surface characteristics and properties of lumbrokinase-immobilized polyurethane. 761 90

Normal skin was cryoprotected by submerging it in a mixture of 30% dimethylformamide (DMF) in PBS or RPMI. Subsequently it was frozen in liquid propane gas. Cryosubstitution was carried out at -90 degrees C by using methanol to which uranyl acetate or osmium tetroxide were added. The tissue was embedded in either Lowicryl K4M at -40 degrees C or in Epon at +60 degrees C. The tissue was evaluated by its overall preservation of ultrastructural details and by its labeling intensity after incubation with either anti-desmoglein or anti-type VII collagen monoclonal antibodies. The mixture of DMF and PBS caused an electron-dense precipitate within the cell. The overall morphology was better in Epon-embedded material than in K4M-embedded material. However, the labeling was best in K4M material. Regardless of whether the tissue was embedded in Epon or K4M, the addition of osmium tetroxide markedly reduced the degree of labeling.
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PMID:Techniques in immuno-electron microscopy. I. Cryosubstitution. 779 89

Six fractions of strong and novel fibrinolytic enzymes (lumbrokinase, LK) were extracted from the earthworm Lumbricus rubellus. The enzymes in these fractions appeared to be very stable and showed greater antithrombotic activity than other currently used antithrombotics. The authors immobilized an LK fraction that shows the most potent fibrinolytic activity on a polyurethane (PU) surface to investigate its enzymatic and antithrombotic activity. The methanol extracted PU surface was treated with a 3% (wt/vol) maleic anhydride methylvinyl ether copolymer (MAMEC) solution and finally incubated in an LK solution in PBS (pH 7.4). The immobilized LK activity was estimated by the fibrin plate method and caseinolytic activity assay. The antithrombotic activity was evaluated by in vitro 125I-fibrinogen adsorption in fresh whole blood and 99mTc platelet adhesion tests. In addition, the occlusion time was determined through ex vivo rabbit A-A shunt experiments. The content and unit activity of immobilized LK were found to be 24 micrograms/cm2 and 18 IU/cm2, respectively. The relative activity ratio of immobilized LK to soluble LK was found to be approximately 34%. Immobilized LK was stable within a various pH range and resistant to inhibitors and thermal inactivation. Less fibrinogen was adsorbed and fewer platelets adhered on an LK-immobilized surface than on PU and PU-MAMEC controls. The ex vivo occlusion time of untreated PU and PU-MAMEC surfaces were only 32 and 42 minutes, respectively. But that of LK-immobilized PU was extended to 140 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antithrombotic activity of a lumbrokinase immobilized polyurethane surface. 826 50

Mature mammalian spermatozoa have a compact and stable nuclear structure conferred by protamines instead of histones, which are present in all other cellular types. Chromomycin A3 (CMA3) is a useful tool for the detection of protamine deficiency in sperm chromatin. The purpose of this study was to correlate the percentage of spermatozoa staining positively for CMA3 with sperm parameters and in-vitro fertilization of human oocytes. Spermatozoa were collected from 56 fertile and 18 infertile men, and washed twice in PBS, fixed in two changes of methanol : acetic acid (3 : 1 v : v) spread on rinsed slides treated with APES and dried. Twenty-four of the semen samples were subjected to both Percoll and swim-up, and were stained subsequently with CMA3. CMA3-stained spermatozoa were expressed as a percentage in a count of 200 spermatozoa. A substantial variation in the percentage of CMA3-stained cells was observed in ejaculated human spermatozoa, varying between 8% and 77%. A strong negative correlation (r = -0.64, p < 0.001) was found between sperm count and the percentage of CMA3-stained spermatozoa. No correlation was found between CMA3-stained spermatozoa and their motility, while excessive sperm morphological abnormalities were related positively to CMA3-staining. Spermatozoa in samples exhibiting low (8-62%) CMA3-staining had significantly higher fertilizing rates in vitro than did samples exhibiting high (49-77%) CMA3-staining. The mean percentage of CMA3-stained spermatozoa after swim-up or Percoll preparation (26% vs 31%) did not differ significantly. These results demonstrate a close relationship between CMA3-staining, fertilization and sperm count, and suggest potential application of this marker for the prediction of sperm quality and fertilizing capacity.
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PMID:Chromomycin A3-staining as an indicator of protamine deficiency and fertilization. 869 34


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