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)

Pulmonary macrophages of mice in the steady state were isolated by lavage with PBS containing EDTA and subsequent enzymatic digestion of tissue with pronase and DNA-ase. By this method, the total pulmonary macrophage population was obtained in two cell suspensions, one with a pure population of pulmonary alveolar macrophages (PAM) and the other with a mixed population of pulmonary alveolar and pulmonary tissue macrophages (PTM). The morphological, cytochemical, and functional characteristics of both PAM and PTM were like those of mature tissue macrophages except for the presence of C3 receptors. These receptors were almost absent on PAM and present on a larger number of cells in the mixed population of PAM and PTM. The total pulmonary macrophage population of mice in the steady state is approximately equal to 2 x 10(6), of which about 93% are PAM and about 7% are PTM. In labeling experiments with 3H-thymidine, the low in vitro labeling indices (less than 3%) for both PAM and the mixture of PAM and PTM, showed that both are essentially nondividing cells. In vivo labeling studies showed an increase in the number of labeled macrophages that can only be attributed to labeled monocytes migrating into the lungs. Additional evidence was provided by a decrease in the labeling indices of pulmonary macrophages when mice were treated with hydrocortisone acetate, which causes a severe monocytopenia, thus preventing monocyte influx into the lungs. Confirmation of the bone marrow origin was obtained in mice labeled after x-irradiation with partial bone marrow shielding: labeled pulmonary macrophages were found in the exposed lungs. In all experiments, the labeling indices were identical in the two macrophage populations isolated. These results show that the influx of monocytes is the source of cell renewal for the pulmonary macrophages. No indications for an interstitial division or maturation compartment in the lung were found. Quantitation of the efflux of labeled monocytes from the blood, and the number of labeled pulmonary macrophages, showed that in the steady state about 15% of the monocytes leaving the circulation become pulmonary macrophages and that the turnover time of pulmonary macrophages is approximately equal to 27 d.
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PMID:Origin, Kinetics, and characteristics of pulmonary macrophages in the normal steady state. 44 91

It has previously been shown experimentally using S. faecalis that the relative number of blue-staining Gram-positive bacteria was reduced by demineralizing agents as well as by disinfection (Wijnbergen & van Mullem 1987, van Mullem & Wijnbergen 1989). In this study the cumulative effect of disinfection, experimental period, fixation and demineralization was investigated. The use of additives to the fixative and the demineralizing agents to limit the loss of blue-staining bacteria was also studied. The numbers of bacteria were determined using a haemocytometer, and the percentage of blue-staining organisms were ascertained from smears. When compared with the start of the experiment, the sequence of disinfection with chlorhexidine, storage in PBS, fixation with neutral formaldehyde and demineralization with formic acid or EDTA appeared to reduce the relative numbers of Gram-positive staining bacteria. For storage periods of 0 and 4 days the reduction factors were 100 and 600, respectively, using formic acid. These factors were 50 and 95, respectively, using EDTA. Addition of cetyl pyridinium chloride to the fixative and the demineralizing agents, and addition of neutral formaldehyde to the demineralizing agents lowered these reduction factors to 80 and 200, respectively, for formic acid, and 40 and 85, respectively, for EDTA. If the results are extrapolated to animal experiments where disinfection, experimental period, fixation and demineralization form part of the experimental framework, even the lowest reduction in the number of blue-staining bacteria could lead to false interpretation of tissue sections.
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PMID:The cumulative effect of disinfection, storage, histological fixation and demineralization on number and staining ability of gram-positive bacteria. 172 82

The epithelial-mesenchymal transition of cardiac endothelium is a critical developmental event in the formation of valvular and septal anlagen. We have demonstrated previously that this event can be mimicked in culture by treating atrioventricular canal (AV) endothelium with EDTA-soluble proteins extracted from embryonic heart tissue. This activity was fractionated by ultracentrifugation of the EDTA extract, indicating that the critical proteins existed as a multicomponent complex. Based on these results we propose that: (1) the in vitro particulates in EDTA extracts correspond to an observed particulate form of extracellular matrix within the myocardial basement membrane (MBM) of mesenchyme-forming regions and (2) one or more of the proteins in the MBM particulates function to elicit the epithelial-mesenchymal transition. To test these hypotheses we utilized an antiserum, termed ES1, prepared against EDTA-extractable particulates from embryonic chick hearts. Both ES1 and an anti-fibronectin monoclonal antibody (M3H) co-localized in situ to particles within the MBM; however, no ES1 reactivity towards fibronectin could be detected by ELISA or immunoblot analysis. The ES1-positive MBM particulates were removed by extraction with EDTA, but not with PBS, indicating a divalent cation-mediated association of the constituent proteins. ES1 antibodies recognized two major (28 and 46 kDa) and three minor (93, 109, and 180 kDa) proteins on immunoblots of EDTA-extractable proteins. When tested in culture, ES1 antiserum inhibited the formation of mesenchyme from AV endothelium in a dose-dependent manner, while M3H did not. These results are consistent with an active role for one or more of the ES1 antigens in initiating the formation of AV mesenchyme. The localization of ES1 antigens to the extracellular matrix at other dynamic interfaces, e.g., ectoderm/neural tube and limb bud ectoderm/mesoderm, point to a potentially general importance of ES1 antigens in mediating similar developmental interactions.
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PMID:An antiserum (ES1) against a particulate form of extracellular matrix blocks the transition of cardiac endothelium into mesenchyme in culture. 204 Mar 70

Osteocalcin or bone GLA protein (BGP) is found at high levels in only two tissues, the extracellular matrix of bone and dentine. Tissue culture experiments have demonstrated that BGP is synthesized by two osteoblastic osteosarcoma cell lines (ROS 2/3 and 17/2) and by normal osteoblastic cells in primary culture. BGP was not found in rat cartilage nor in liver, kidney, lung, spleen, brain, heart, thymus, skeletal muscle. In this study secretion of BGP was assayed by RIA in the supernatants of 48-hour cultures of peripheral blood lymphocytes or monocytes. Lymphocyte cultures were carried out using RPMI-1640 supplemented with L-glutamine and antibiotics at the concentration of 1 x 10(6) cells/ml and activated by PHA (10 ng/ml). Peripheral blood monocytes were purified by adherence to plastic Petri dishes and treated with cold PBS supplemented with EDTA. Monocytes were cultured as previously described and stimulated with LPS (50 micrograms/ml). Cell-free supernatants were obtained by centrifugation and stored at -20 degrees C, until the BGP assay was performed. The authors did not observe secretion of detectable amounts of BGP in the supernatants of short-term lymphocyte or monocyte cultures. These data indicate that circulating mononuclear cells are not involved in BGP synthesis and secretion.
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PMID:Mononuclear cells are not involved in BGP synthesis and secretion. 206 4

Serine proteases, such as alpha-chymotrypsin or elastase, caused an aggregation of rat ascites tumour cell lines, AH-130, AH-109A and YS, in a protein free medium which preserved the cell viability. This aggregation, which was monitored spectrophotometrically, was dependent upon the protease activities and was resistant to treatment with either a calcium chelating reagent (EDTA) or neuraminidase. However, the tumour cell aggregates were redispersed by treatment with deoxyribonuclease I (DNase I). This dispersal effect was dependent upon the DNase activity. A possible relationship between the tumour cell aggregation and development of blood-borne metastasis was studied. An intravenous inoculation in rats of tumour cell aggregates performed by the alpha-chymotrypsin treatment resulted in significantly higher numbers of lung metastatic foci than an injection of single cells. When the re-separated single cells, prepared in vitro by treatment with DNase I following alpha-chymotrypsin treatment, were injected instead of the aggregates, the enhancement of metastasis was reversed. These enhancement and reversal effects were mimicked in vivo by intravenous injections of protease and nuclease following inoculation of a single cell suspension. That is, the number of metastatic foci caused by single cell inoculation followed by an intravenous alpha-chymotrypsin injection, was higher than that in a control group receiving PBS instead of alpha-chymotrypsin. Again, this augmentation was reversed by an injection of DNase I following alpha-chymotrypsin injection. Furthermore, an injection of DNase I alone itself reduced the starting number of metastases resulting from injection of the single tumour cell suspension. These data suggest that the metastatic behaviour of tumour cells may be increased by protease inducible DNA dependent cell aggregation should it occur in the blood stream.
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PMID:Serine protease-induced enhancement of blood-borne metastasis of rat ascites tumour cells and its prevention with deoxyribonuclease. 212 Dec 20

The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.
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PMID:A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase. 234 31

Optimization of microcarrier processes is dependent upon efficient, serial subcultivation routines. Established methods have been modified for a high degree of cell detachment from microcarriers, and transfer of a maximum number of viable cells from one culture to the next during the scale up process. Cultures of MRC-5 and Vero cells were studied, and cell inocula were obtained from different growth phases (i.e. exponential versus stationary) to investigate growth in subsequent cultures. Microcarrier cultures containing confluent cells were washed with EDTA-PBS and then exposed to trypsin (185 U/ml) in PBS (pH 8.0, 37 degrees C). 95-100% of the cells detached from the microcarriers with a viability greater than 95% following a 10 minute exposure to the trypsin. The presence of residual trypsin in the inoculum was investigated with respect to subsequent growth, and no significant effect was found. The methods developed at the laboratory scale (0.25 to 1.5 l cultures) were successfully applied to pilot scale (1 to 100 l cultures), and resulted in split ratios of up to 1:10 for MRC-5 cells and 1:100 for Vero cells. These results show that the modified subpassaging method and an optimal cell inoculum are vital in establishing efficient, industrial scale microcarrier processes.
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PMID:Subpassaging cells on microcarriers: the importance for scaling up to production. 243 73

C1q was isolated from human serum by dialysis in 0.24 M EDTA, followed by affinity chromatography on immobilized IgG and removal of IgG traces in a column with anti-IgG antibodies. Microplates were coated with C1q in PBS at 10-20 mg/l, nonspecific binding sites were saturated with human serum albumin. The sera were diluted 16-fold in 0.05 M PBS, 0.01 M EDTA, 0.05% Tween. After incubation with diluted samples the plates were treated with horseradish peroxidase--anti-human IgG conjugates. Enzymic activity was measured by adding p-phenylenediamine (0.2 g/l) in acetate buffer, pH 5.9, containing 0.05% H2O2. The sensitivity of the assay ranged between 2.5 and 300 mg/l.
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PMID:[A method of isolating C1q from human serum and its use in the solid-phase immunoenzyme determination of immune complexes]. 246 33

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

Centrifugal elutriation was used further to isolate human peripheral blood monocytes (HPBM) from mononuclear-enriched cells harvested as a secondary component following platelet concentration collection samples. HPBM were recovered in either one or two populations consisting of either total HPBM or small (SM) and large monocytes (LM). The elutriation was carried out at 3,500 +/- 5 rpm for the separation of lymphocytes and HPBM in Ca++- and Mg++-free PBS without EDTA. An average of 5.05 +/- 1.50 X 10(8) HPBM were recovered in the total HPBM with a purity of 95% +/- 3%. The SM and LM were obtained by splitting the total HPBM into two equal populations with an HPBM purity of 92% +/- 3% and 93% +/- 3, respectively, by nonspecific esterase staining. The elutriation media were shown to have no effect on viability by trypan blue exclusion. All three HPBM populations were shown to be histochemically (lack of reactivity to leu-1 and leu-7) and functionally (depletion of NK cell activity) purified from the lymphocyte population. The HPBM populations were enriched in HLA-Dr, OKM-1, OKM-5, MY-8, and leu M-3 monoclonal antibody marker staining. There were no differences in percent positive cells between SM and LM populations for any of the monocyte-specific monoclonal antibodies. All three monocyte populations mediated antibody-dependent cell-mediated cytotoxicity to human red blood cells, with LM mediating more lysis (27.0% +/- 5%) than SM (7% +/- 3%).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Centrifugal elutriation as a method for isolation of large numbers of functionally intact human peripheral blood monocytes. 308 57


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