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)

Estradiol (E2) facilitates the lordosis reflex that occurs in response to flank stimulation in female rats. Lordosis appears to be regulated in part by the synthesis of proteins in the ventral medial hypothalamus (VMH) that are transported to the midbrain central gray (MCG). We developed a strategy involving microinfusion of radioactive amino acids, followed by 2-dimensional gel electrophoresis, to identify proteins that may be regulated by E2 in the VMH and transported to the MCG. A mixture of 35S-methionine and 35S-cysteine (2:1, total 500-1000 microCi), suspended in 1 microliter PBS, was infused bilaterally into the VMH over a period of 2 hr into matched pairs of ovariectomized female rats, one of which was given a Silastic implant containing E2 at the beginning of infusion or 1 week earlier. The rats were sacrificed 12 hr after the end of infusion, and several brain regions were obtained by microdissection. Samples were analyzed by 2-dimensional gel electrophoresis, entailing isoelectric focusing in the first dimension and SDS-PAGE (molecular-weight separation) in the second dimension, followed by fluorography. We could routinely separate at least 250 spots. We consistently found a protein spot with an apparent molecular weight of 70 kDa, pI of about 5.9, which almost always appeared in the VMH and MCG of rats given E2 replacement but very rarely in samples from ovariectomized rats given no E2 replacement. A spot immediately acidic to this protein (70 kDa, pl about 5.8) appeared to vary inversely with this E2-induced protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:An estradiol-induced protein synthesized in the ventral medial hypothalamus and transported to the midbrain central gray. 282 60

Proliferating cell nuclear antigen (PCNA), also called cyclin, was purified from PBS extract of rabbit thymus by using a combination of ammonium sulfate fractionation, DEAE-Sephacel, HPLC ion exchange, and HPLC gel filtration column chromatography. PCNA was purified more than 600 times and was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. SDS-PAGE showed that a 36 kD protein was selectively isolated in this purification process, and this protein was identified as PCNA by immunoblotting. Other previously identified nuclear antigens, Sm, nRNP, SS-A/Ro, SS-B/La, histone, and DNA, were not detected in this preparation by counterimmunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). Purified PCNA was used as an antigen to develop ELISA for rapid and specific detection of anti-PCNA in human sera. For further purification, the 36 kD band was electrophoretically eluted from SDS gel slices. The amino acid composition and the first 25 residues from the N-terminus of the protein were determined by using electroeluted PCNA. This amino acid sequence was found to be unique and showed little sequence homology with existent proteins in the protein identification resources databank.
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PMID:Purification and N-terminal amino acid sequence of proliferating cell nuclear antigen (PCNA)/cyclin and development of ELISA for anti-PCNA antibodies. 286 7

Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 X 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45,000 X g, 4 degrees C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4 degrees C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70,000 molecule which upon reduction electrophoresed with an apparent Mr of 85,000-90,000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.
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PMID:Reversible biotinylation of C1q with a cleavable biotinyl derivative. Application in C1q receptor (C1qR) purification. 325 72

The overall composition of the Clostridium tyrobutyricum cell envelope did not vary significantly during cell growth and was characterized by a high protein content (about 40% dry weight). Teichoic and teichuronic acids were absent and the neutral sugar content low. Insoluble peptidoglycan represented only 10-12% of the cell envelope (dry weight basis); it contained glucosamine, muramic acid, alanine, diaminopimelic acid and glutamic acid (molecular ratio 1/1/2/1/1). SDS-PAGE revealed the presence of about 50 proteins in this cell envelope; however, one high molecular weight protein was largely predominant. They were not covalently bound to the peptidoglycan and their relative amounts were practically constant through cell growth and with various extraction treatments. A brief heat treatment of whole cells in PBS caused selective release of the major cell envelope proteins together with flagellin; this method was used to characterize these proteins in 37 strains of C. tyrobutyricum and some other clostridia. The major envelope proteins had molecular weights ranging from 96 to 145 Kd and the flagellins from 32 to 72 Kd.
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PMID:Major protein components in the cell envelope of Clostridium tyrobutyricum. 331 69

A hybrid cell line (DCH-5) constructed from an adherent cell of mouse spleen and the thymoma BW5147, and selected for adherence to hydrophobic surfaces, secretes a factor which augments the mitogenic response of thymocytes. Properties of the factor were compared with those of a P388D1 and J774.1 macrophage-derived interleukin 1 (IL-1). On polyacrylamide gel electrophoresis in Tris-glycinate buffer the IL-1-like factor of DCH-5 cells was heterogeneous and its components were more negatively charged than components of macrophage-derived IL-1. Charge differences between these factors were also confirmed by isoelectric focusing (IEF) (pI of IL-1 was 5.4, pI of the IL-1-like factor was 3.5). SDS-PAGE and gel filtration demonstrated that both factors consisted of several components of near molar masses (approximately 17 kg/mol). Gel filtration showed that in PBS the IL-1-like factor of DCH-5 cells was partially aggregated. Antibodies specific to IL-1 inhibited the activity of the IL-1-like factor of DCH-5 cells. Thus, mouse DCH-5 cells provide a new source of IL-1-like factor which might be useful for further elucidation of the heterogeneity of interleukins.
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PMID:Interleukin 1-like factor produced by a hybrid of an adherent mouse spleen cell and a thymoma cell. 348 55

The glycoproteins of porcine zonae pellucidae were fractionated into three families (PZP1-3) by high-performance liquid chromatography on a gel filtration column. Their molecular weights were estimated to be 164K for PZP1, 99K for PZP2, and 55K for PZP3; and they were present in an approximate weight ratio of protein moiety of 2:3:18, respectively. Differences in carbohydrate composition position between them were found, although their amino acid compositions were similar to each other. CD spectra showed that the three families had an almost identical secondary structure of the helical form in the presence of SDS. In PBS without SDS, they had different secondary structures, predominantly in the beta-form, and probably different tertiary structures.
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PMID:Fractionation of glycoproteins from porcine zonae pellucidae into three families by high-performance liquid chromatography. 359 85

Ocular mucin, the major product of conjunctival goblet cells, constitutes the innermost layer of preocular tear film. Ocular mucin is known for its limited amount and inaccessibility. Using impression cytology, mucus strands collected from the inferior fornix of either rabbit or human eyes were found to contain inflammatory cellular debris. In order to circumvent these difficulties and to isolate native mucin molecule(s), we bathed rabbit eyes in fluid containing isotonic PBS and 5.5 X 10(-4) M acetylcholine for 4 or 12 hr. Bathing fluids containing rabbit ocular mucin (ROM), 1 ml per eye, were pooled and combined with 1M guanidine HCl and protease inhibitors containing EDTA, PMSF, and sodium azide to avoid any possible enzymatic degradation, and then separated under the same conditions by Sepharose CL-4B. In parallel, commercial porcine stomach mucin (PSM) was purified and used to compare with ROM. We also developed nitrocellulose-based dot semi-quantitative assays for nucleic acid, protein, and glycoprotein. PAS-positive fractions monitored by such a dot assay were collected at CL-4B void volume and then separated from nucleic acid contaminants by CsCl-gradient ultracentrifugation. A protein fraction, 65K, poorly-glycosylated, with high contents of Asx, Glx, and Gly was found strongly associated with both ROM and PSM, and was only separable by ultracentrifugation in 4M guanidine HCl and CsCl. Purification of the ROM was verified by SDS-polyacrylamide gel electrophoresis, amino acid analysis, and carbohydrate analysis. These results will allow future exploration of the molecular mechanism by which tear film is achieved.
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PMID:Purification and characterization of rabbit ocular mucin. 362 33

Serum from humans infected with Schistosoma mansoni, when reacted with a Biomphalaria glabrata soluble hepatopancreas antigen extract (BgSHA) yields 2 lines of precipitation by gel diffusion and 1 by immunoelectrophoresis. The IgG from the serum of a human infected with S. mansoni was coupled to CNBr-activated Sepharose 4B. BgSHA (8.0 mg) was then filtered through the gel and the bound antigens, denoted BgSm, eluted with HCl-glycine, pH 2.6. These bound antigens comprised 2.8% of the total BgSHA. BgSm was then applied to an anti-Fasciola hepatica column as above. The drop through in PBS (38% yield) and containing the BgSm antigens depleted of cross-reactivity with F. hepatica was then tested by the ELISA to evaluate its serodiagnostic potential. These antigens detected a primary S. mansoni infection by 4 wk but were less sensitive than SmSEA in the detection of a primary infection with S. mansoni. However, the BgSm-specific antigens were more specific than SmSEA and showed less cross-reactivity with the serum of mice infected with F. hepatica. At least 16 peptides were seen by silver staining following SDS-PAGE with 5-20% gradient gels. The 2 more prominent bands obtained were estimated to have molecular weights of 62 and 66 kd. Nitrocellulose strips blotted with BgSHA were incubated with the serum of mice infected with S. mansoni for 12 wk and developed 6 bands with molecular weights of 66, 57, 55, 50, 48 and 32 kd.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and partial characterization of shared antigens of Biomphalaria glabrata and Schistosoma mansoni and their evaluation by the ELISA and the EITB. 393 33

A spectrometric assay for assessing erythrophagocytosis by mononuclear phagocytes is described. It is based on the haemoglobin-catalyzed conversion of a benzidine derivative into a coloured product in the presence of H2O2 (pseudoperoxidase activity). The assay is set up in microtitre plates, and following an uptake phase and removal of non-ingested erythrocytes, pseudoperoxidase activity is measured in detergent lysates of phagocytes, using an ELISA reader photometer. Various detergents and substrates were evaluated. SDS was found to be the most suitable detergent. Diaminobenzidine (in PBS, pH 7.4) was the substrate of choice for enumerating ingested erythrocytes in a range from 10(4) to 5-8X10(5) sheep erythrocytes. Ortho-tolidine (in acetate buffer, pH 5.5) could be used in a range from 2X10(3) to 2X10(5) sheep erythrocytes. The results obtained with human peripheral blood monocytes or monocyte-derived macrophages and IgG-sensitized sheep erythrocytes correlated well with those obtained using 51Cr-labelled, IgG-sensitized erythrocytes.
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PMID:A rapid and sensitive method allowing photometric determination of erythrophagocytosis by mononuclear phagocytes. 403 2

In order to investigate whether defective phages of Bacillus subtilis killed sensitive bacteria by a lysis from without mechanism, the minimal number of phages required for killing was determined. This figure was found to vary with the m.o.i., giving a value of 1 on extrapolation to an m.o.i. of 0. This excluded lysis from without as the only killing mechanism, although it might play a role at high m.o.i.s. This was confirmed by experiments on leakage of ATP and u.v.-absorbing material, the uptake of oxygen and the effect of the phages on the membrane potential. Apart from a short, initial leakage of ATP, the cell membrane was not affected at low m.o.i.s. These results lead to the conclusion that at low m.o.i.s. the phages acted on a cytoplasmic component. Treatment of defective phages for 10 min at pH 2.5 resulted in breakdown of the phages without complete abolition of the killing activity. The active component, which was shown not to be DNA, could not be isolated from the mixture, but SDS gel electrophoresis of PBS X and a non-killing mutant of this phage suggested that a protein with a mol. wt. of 85000 was involved in killing.
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PMID:Effect of defective phages on the cell membrane of Bacillus subtilis and partial characterization of the phage protein involved in killing. 679 49


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