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 actions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are thought to be mediated through receptor proteins which have been described in a variety of avian and mammalian tissues, but not in the liver. To determine if a binding protein for 1,25-(OH)2D3 is present in this tissue, rat liver was homogenized in a low ionic strength buffer containing 10 mM Tris (pH 7.4), 2.2 m sucrose, 3 mM calcium chloride, 0.2% Triton X-100, and 0.04% Trasylol (sucrose buffer) and centrifuged over a 10-ml cushion of sucrose buffer at 61,000 x g for 80 min at 4 C. The resultant nuclear pellet was extracted in a 26 mM Tris (pH 7.4) buffer containing 0.3 M potassium chloride, 5 mM dithiothreitol, 1 mM EDTA, and 10 mM sodium molybdate. Saturable 1,25-(OH)2D3 binding was identified in high salt extracts of rat liver nuclei and was eliminated by treatment with trypsin. This liver binding protein cosediments on high salt 5-20% sucrose density gradients with the 1,25-(OH)2D3 receptor protein from intestine and is distinct from the 6.OS tissue binding protein for 25-hydroxyvitamin D3. Perfusion of rat liver with PBS to remove receptor-positive blood cells before isolation of the nuclei did not change 1,25-(OH)2D3 binding. The nuclear protein bound 1,25-(OH)2D3 more avidly than either 24,25-(OH)2 D3 or 25-hydroxyvitamin D3. Saturation analysis of 1,25-(OH)2D3 binding revealed an apparent equilibrium dissociation constant of 20.6 +/- 2.2 pM (mean +/- SEM) at 4 C and a maximum binding capacity of 49.0 +/- 14.6 fmol/extract from 1.0 mg DNA. The 1,25-(OH)2D3-binding binding protein was present in liver nuclei isolated from mice, rabbits, and chicks and in nuclei isolated from cultured rat hepatocytes. The ligand specificity, sedimentation coefficient, limited binding capacity, trypsin sensitivity, and nuclear location of the hepatic 1,25-(OH)2D3-binding protein are similar to those of 1,25-(OH)2D3 receptors described in other tissues and suggest that the liver may be a target organ for [1,25-(OH)2D3] action.
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PMID:A 1,25-dihydroxyvitamin D3 receptor-like protein in mammalian and avian liver nuclei. 283 67

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

The effects of separate lipoproteins or of serum with high or low lipoprotein concentrations on formation of lipophilic carcinogen adducts with DNA and on mutagenicity of the carcinogen was investigated using V79 Chinese hamster lung cells. Binding of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to DNA and BPDE induction of 6-thioguanine (6-TG)-resistant mutants in V79 cells was significantly lower after 1 or 4 h when the medium was supplemented with purified HDL, and was lower after 1 h but not 4 h when the medium was supplemented with serum containing a high concentration of mixed lipoproteins (LP). Cells grown in medium without serum or LP supplementation exhibited the highest levels of both BPDE-DNA adduct formation and mutagenesis after 1 h. At 1 h, cells exposed to BPDE in LDL-supplemented medium showed decreased adduct formation and mutagenesis when compared to cells treated with BPDE in PBS-supplemented medium. After 4 h, cells treated with BPDE in LDL-supplemented medium gave the highest levels of adduct formation and the highest mutation frequency. These results suggest that both LDL and HDL effectively decrease the concentration of BPDE available to V79 cells exposed to the mutagen for short periods of time, resulting in decreased interaction of BPDE with DNA and decreased BPDE-associated mutagenesis, but that both BPDE-DNA adduct formation and mutagenesis increased as a function of increased exposure time in the presence of LDL. The results suggest that LDL, but not HDL, uptake by adsorptive endocytosis may be associated with potentiated entry of BPDE into V79 cells as a function of time.
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PMID:High-density lipoproteins decrease both DNA binding and mutagenicity of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in V79 Chinese hamster cells. 307 76

The (NZB X NZW)F1 mouse is recognized as an important animal model of the human disease systemic lupus erythematosus (SLE). Groups of NZB/W F1 mice were treated either with IFN-gamma or with PBS. The results demonstrate that IFN-treated animals have accelerated development of fatal immune complex glomerulonephritis relative to age-matched controls. On the other hand, administration of mAbs specific for IFN-gamma to such mice from 4 to 7 mo of age resulted in significant remission. Development of both proteinuria and anti-DNA antibodies were delayed and survival at 11 mo was increased from less than 20% to 95% in treated mice relative to controls (p less than or equal to 0.001). These findings may have therapeutic implications for the treatment of SLE.
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PMID:In vivo treatment of (NZB X NZW)F1 lupus-like nephritis with monoclonal antibody to gamma interferon. 311 9

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90 degree light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90 degree light scatter and protein content. M-phase nuclei can be distinguished from G2-phase nuclei on cytograms of 90 degree light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single-parameter histogram.
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PMID:Flow cytometric discrimination of mitotic nuclei by right-angle light scatter. 313 55

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

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and sieving. Immediately after dissociation the cells were preincubated in a PBS solution at -6 to +54 degrees C for 3 and 20 min. After this preincubation cells were rotated for 60 min at 37 degrees C in the PBS solution. Cellular adhesivity was estimated during this time period and EM pictures of organized in vitro aggregates after 24-28 h were taken. In a separate series of experiments, freshly dissociated were treated with DNAase before the rotation procedure. Preincubation in a cold or a warm medium did not alter the inhibition of cellular adhesivity significantly. Distinct inhibition of cellular adhesion was observed in cells preincubated above 53 degrees C. Adhesion was also inhibited below -5 degrees C, however, this effect was mainly dependent on the rate of freezing and thawing. Digestion of dissociated cells with DNAase (20 micrograms/ml) decreased cell adhesion. At 37 degrees C the adhesivity decreased by about 20%. Aggregates of cells preincubated at 0 degrees C for 20 min did not exhibit marked EM changes after 24-28 h in vitro. The present results have shown the rather high resistance of molecules responsible for cellular adhesion and its reversibility to temperature changes. Furthermore, non-specific cellular adhesion was shown on physically active DNA molecules.
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PMID:The effect of short-term temperature changes on the adhesivity of nerve cells. Participation of DNA molecules on non-specific cellular adhesion. 315 10

It is well documented that injury to bone marrow is followed by an osteogenic phase that precedes the complete tissue regeneration. We have recently shown that postablation healing of bone marrow in rat tibiae is associated with a systemic increase in osteogenesis. It was hypothesized that a growth factor(s) with an effect on osteogenic cells is produced in the healing limb, is transferred to the blood circulation, and enhances osteogenesis systemically. To test growth factor production, healing bone marrow-conditioned medium was prepared with tissue separated from rat tibias during the osteogenic phase and assayed for enhancement of mitogenic activity in culture of osteogenic rat osteosarcoma cells (ROS 17/2). Partial purification of healing bone marrow-conditioned medium-derived growth factor(s) consisted of gel filtration on Sephadex G-25, boiling, chromatography on heparin-Sepharose, and gel filtration on Sephadex G-75. Mitogenic activity eluted in the void volume of the Sephadex G-25 column (mol wt greater than 5,000). Potent activity resolved from heparin-Sepharose with PBS, and on filtration by Sephadex G-75 this activity recovered in 3 peaks with mol wt estimates of 35,000, 19,000, and less than 10,000. The partially purified factor also showed considerable stimulatory effect on DNA synthesis in osteoblastic fetal rat calvarial cells and on in vitro elongation of fetal long bone; it had only a small effect on nonosteoblastic ROS and fetal rat calvarial cells. These data indicate that healing bone marrow produces growth factor activity with a preferential effect on osteogenic cells. It is suggested that local growth factors have a role as mediators in the sequence of events whereby bone marrow expresses its osteogenic potential. During postablation healing of bone marrow these factors may also function as systemic promoters to osteogenic cells.
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PMID:Regenerating bone marrow produces a potent growth-promoting activity to osteogenic cells. 316 64

The cytoplasmic enzyme ribonucleotide reductase is essential for DNA synthesis, and its activity is strongly correlated with cellular proliferation. This paper describes a flow cytometric technique for the simultaneous measurement of DNA content and the M1 subunit of ribonucleotide reductase. Data are presented for cycling cultured human leukemic lymphoblasts in which M1 is constitutively expressed, and peripheral blood lymphocytes in which it is only detectable with certainty after mitogen stimulation. The choice of fixation procedure strongly influenced the amount of M1 subunit detected. Paraformaldehyde (PF) at concentrations of 2% (w/v in PBS) or greater provided optimal results. Fixation at 37 degrees C was significantly more effective in preserving M1 than fixation at room temperature or 4 degrees C. These variables are shown to have affected cytoplasmic retention during postfixation processing. Their relevance to the flow cytometric measurement of other intracellular components by this procedure are discussed.
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PMID:Immunofluorescent quantification of ribonucleotide reductase M1 subunit and correlation with DNA content by flow cytometry. 331 59

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation less than 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.
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PMID:Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. 350 96


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