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

Tissue factor activity was detected in human mononuclear cells cultured with E. coli endotoxin in vitro. The effecient concentration of endotoxin inducing significant tissue factor development was in excess of 10(-3) microgram/ml, and a dose response type relationship was seen between them. The activity developed after culturing for 2 hr and increased up to 6 hr, and thereafter no significant increase was observed. Although the activity was detected both in cell extract and on cell surface, the main activity seemed to exist on the cell surface. No correlation was observed between the synthetic rate of nucleic acid and the rate of development of the activity. Although the activity was detected also in a granulocyte preparation, it was significantly less than that in mononuclear cells. The development of activity was observed when lymphocytes were cultured in RPMI 1640 medium, MEM, and in autologous serum. However, the activity was not observed when cultured in Hanks solution and PBS, in which the main difference from RPMI was the absence of amino acids, and in autologous serum containing sodium citrate which was added for elimination of Ca++ from serum. Moreover, actinomycin D suppressed the development of activity, and the same was noted at low culture temperature. These results suggest that some metabolic change of the cell membrane triggered by endotoxin may induce the development of tissue factor in cells.
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PMID:Development of tissue factor activity in mononuclear cells cultured in vitro. 35 95

An improved rapid cell enzyme-linked immunosorbent assay (CELISA) is described which is suitable for the large scale screening of monoclonal antibodies to islet cell surface antigens. 5 x 10(4) insulin-producing rat insulinoma (RIN) cells were seeded per well in a 96-well flat-bottomed polystyrene plate coated one day before a 0.01% poly-D-lysine solution in PBS. After culture for 4 days in 200 microliters/well RPMI 1640 supplemented with 7.5% heat-inactivated fetal calf serum, the cell number per well was up to 2.1 x 10(5). These monolayer RIN cell cultures were used as a target for the detection of islet cell surface antibodies (ICSA) in the supernatants of hybridomas. The cells were used without fixation to avoid modification of sensitive surface antigens. Poly-D-lysine did not cause non-specific binding of immunoglobulins to the plastic wells as tested with irrelevant monoclonals. The specificity and sensitivity of the method is comparable to indirect immunofluorescence. All mc-ICSA primary screened by indirect immunofluorescence using viable RIN cell suspensions were positive in this CELISA. There was a correlation (r = 0.7; n = 44) between the antibody binding measured by CELISA and the indirect immunofluorescence technique. The advantage of this CELISA is that cell surface structures are well preserved in a viable cell monolayer used as target without chemical fixation. This assay procedure should be generally suitable for the initial screening of monoclonal antibodies to cell surface antigens of cells growing under culture conditions.
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PMID:CELISA for rapid screening of monoclonal islet cell surface antibodies using living rat insulinoma cells as target. 181 97

Flies of two colonies of Phlebotomus perniciosus established in Italy and Spain respectively, were infected with a Leishmania infantum isolate from Spain. Only P. perniciosus from Spain was completely found susceptible to the infection. Experimental infections of P. perniciosus from Spain with the same L. infantum isolate were carried out using RPMI or PBS as infective feeding mediums. The results demonstrate that using PBS it is possible to re-feed a great quantity of infected flies.
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PMID:Experimental infections of a Phlebotomus perniciosus colony using different procedures. 184 Dec 39

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

Essentially pure (97%) alveolar macrophages were isolated by bronchoalveolar lavage of rats with warm (37 degrees C) PBS solution. These cells were allowed to adhere to the inside walls of open-ended glass cylinders which were closed off at each end by three-way stopcocks. The adhering cells were perifused with RPMI-1640 medium supplemented with 5% fetal bovine serum for 18 hr at the rate of 1 mL/hr, and the effluent medium was collected automatically in 2-mL aliquots. Cell recoveries and viabilities did not differ from those found for Petri cultures treated similarly, indicating that the perifusion method under study offered an adequate milieu for short-term primary cultures. The alveolar macrophages in culture were subjected to the presence of particulate (chrysotile asbestos) and soluble (phorbol myristate) toxicants, and their response was monitored in the effluent medium by measuring the release of prostaglandins (PGE) by radioimmunoassay. A significant increase in the sequential release of PGE was observed in the presence of asbestos (100 micrograms/mL) or phorbol myristate (200 ng/mL). Treatment of the cells with indomethacin (20 microM) completely abolished the release of PGE stimulated with phorbol myristate. A cumulative response to the toxicants was also observed when cells were harvested manually from the chambers: asbestos caused a 2-fold increase in cell mortality relative to control, while phorbol myristate brought about a 3-fold increase in the number of dead cells. This effect was not prevented by the presence of indomethacin. Cell aggregation was also observed when cells were perifused in the presence of phorbol myristate, whether indomethacin was present or absent. Our results indicate that the cell perifusion system combines the advantages of conventional adherent cell cultures (viability, aggregation) with those of perifusion techniques (sequential metabolism studies).
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PMID:An adherent cell perifusion technique to study the overall and sequential response of rat alveolar macrophages to toxic substances. 664 51

The steady-state transmembrane potentials of P815 mastocytoma cells were recorded when the cells were bathed in salines of different compositions. In the normal growth medium (RPMI 1640 with added fetal calf serum) the mean membrane potential was -8.7 mV (SEM +/- 0.4, n = 22). A family of Tris-buffered salines (TBS), modeled from Dulbecco's modified PBS (289 mosmol, 169 milliionic strength units, pH 7.5), having different K+ and different C1- concentrations, were designed and used to bathe the tumor cells. All of the TBS solutions had constant, but reduced levels of ionized Ca2+. In the absence of external C1-, an increase of external K+ from 2 to 20 mM results in a 5.7 mV depolarization. In the presence of external C1- the same increase in external K+ results in a 2.1 mV depolarization. The presence of 145 mM C1- resulted in a steady-state depolarization (for either level of K) of about 50%. One explanation for these results would be the presence of an inward-going active C1- transport.
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PMID:Effects of potassium and chloride on the membrane potentials of P815 mastocytoma tumor cells. 681 35

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

Electroejaculates from eight snow leopards were used to determine how the motility of spermatozoa was influenced by (i) type of media (Ham's F10, PBS, human tubal fluid or RPMI-1640); (ii) holding temperature (23 degrees C versus 37 degrees C); (iii) washing of spermatozoa and (iv) a sperm metabolic enhancer, pentoxifylline. The duration of sperm motility was assessed by evaluating samples in each treatment every hour for 6 h and a sperm motility index (a value combining percentage sperm motility and rate of forward progression) calculated. Spermatozoa from the Ham's F10, PBS and PBS plus pentoxifylline treatments were also co-incubated with zona-intact, domestic cat eggs that were fixed and evaluated for spermatozoa bound to the zona pellucida, penetrating the outer and inner layers of the zona pellucida and within the perivitelline space. During the 6 h co-incubation, the sperm motility index in PBS with pentoxifylline was greater (P < 0.05) than in PBS alone which, in turn, was greater (P < 0.05) than in the other three test media. Washing the spermatozoa enhanced (P < 0.05) motility in both PBS and PBS plus pentoxifylline relative to unwashed samples, but there was no effect (P > 0.05) of holding temperature. Pentoxifylline supplementation enhanced (P < 0.05) the proportion of cat eggs with bound, but not penetrated, snow leopard spermatozoa in the inner layer of the zona pellucida, and there were no spermatozoa in the perivitelline space.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Function and culture requirements of snow leopard (Panthera uncia) spermatozoa in vitro. 796 9

Oxidation of lipids and lipoproteins by macrophages is an important event during atherogenesis. Activation of monocytic cells by zymosan and other agonists results in the release of multiple oxidant species and consequent oxidation of LDL. We now show evidence that ceruloplasmin, a copper-containing acute phase reactant, is secreted by zymosan-activated U937 monocytic cells, and that the protein has an important role in LDL oxidation by these cells. In one approach, ceruloplasmin has been shown to exhibit oxidant activity under the appropriate conditions. Exogenous addition of purified human ceruloplasmin stimulates U937 cell oxidation of LDL to nearly the same extent as activation by zymosan. In contrast to previous cell-free experiments (Ehrenwald, E., G.M. Chisom, and P.L. Fox. 1994. Intact human ceruloplasmin oxidatively modifies low density lipoprotein. J. Clin. Invest. 93:1493-1501.) in which ceruloplasmin by itself (in PBS) oxidizes LDL, under the conditions of the current experiments (in RPMI 1640 medium) ceruloplasmin only oxidizes LDL in the presence of cells; the mechanism by which cells overcome the inhibition by medium components has not been ascertained. As further evidence for a role of ceruloplasmin, activation of U937 cells with zymosan induces ceruloplasmin mRNA and ceruloplasmin protein synthesis after a 5-6 h lag that is consistent with that preceding LDL oxidation. Finally, neutralization by a highly specific polyclonal antibody to human ceruloplasmin inhibits LDL oxidation by at least 65%. Moreover, multiple antisense oligodeoxynucleotides targeted to different regions of the ceruloplasmin mRNA block LDL oxidation by up to 95%. The specific action of the antisense oligonucleotides has been verified by showing inhibition of ceruloplasmin synthesis and by the ability of exogenous ceruloplasmin to overcome the inhibition. In summary, these results are consistent with a mechanism in which cell-derived ceruloplasmin participates in oxidation of LDL by U937 monocytic cells. The data also show that cellular factors in addition to ceruloplasmin, possibly active oxygen species and/or lipoxygenases, are essential and act synergistically with ceruloplasmin to oxidize LDL.
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PMID:Role of endogenous ceruloplasmin in low density lipoprotein oxidation by human U937 monocytic cells. 860 49

Intracellular pH has been measured by laser microspectrofluorimetry, using the pH-sensitive dyes SNARF-1, SNARF-calcein and SNARF-1-dextran. By this technique it was possible to accurately determine pH in volumes as small as 0.5 x 0.5 x 1 microns 3. The probes were loaded into the cells either by diffusion of their acetoxymethylester derivatives (SNARF-1-AM, SNARF-calcein-AM) or by microinjection (SNARF-1-dextran). When the five types of cells were studied in RPMI medium, the nuclear pH was consistently found to be 0.3 to 0.5 units above that of the cytosol. Although the presence of pores in the nuclear membrane has been taken as evidence that free diffusion of ions and small molecules can occur in and out the nucleus, we conclude that the nuclear membrane of these cells presents a permeability barrier to H+. The pH gradient was not observed in cells suspended in PBS.
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PMID:Nuclear pH gradient in mammalian cells revealed by laser microspectrofluorimetry. 883 10


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