UNIPROT:P30536 - FACTA Search

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Query: UNIPROT:P30536 (PBS)
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Studies were undertaken to identify intracellular mediators of prolactin inhibition of glucocorticoid-induced apoptosis in Nb2 lymphoma cells. A short-term assay was implemented that quantitates fragmented DNA released from the genome by reaction with diphenylamine. Induction and inhibition of internucleosomal DNA cleavage (indicative of apoptosis) was verified by agarose gel electrophoresis of extracted cellular DNA. Synchronized Nb2 cells (G0/G1) exhibited increased DNA fragmentation after 4-hr incubation with dexamethasone (DEX) (25-100 nM) which was inhibited by ovine prolactin (oPRL) (0.1-1 ng/ml), the glucocorticoid receptor antagonist, RU486 (500 nM), and the nuclease inhibitor, aurintricarboxylic acid (100 microM). Signals previously implicated in prolactin induction of mitogenesis in Nb2 cells were investigated for their role in prolactin inhibition of apoptosis including: protein kinase C activation, arachidonic acid metabolism, polyamine production, tyrosine phosphorylation, and extracellular calcium. Protein kinase C agonists, phorbol-12-myristate-13-acetate, and 1,2-dioctanoyl-sn-glycerol, +/- the calcium ionophore, A23187 (200 nM), did not mimic oPRL inhibition of DEX-induced DNA fragmentation. Protein kinase C inhibitors, gossypol and quercetin, did not block prolactin action. Arachidonic acid did not mimic prolactin protection against DEX-induced DNA fragmentation. Inhibitors of arachidonic acid metabolism, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and indomethacin did not block prolactin action. The polyamine, spermine, inhibited DEX-induced DNA fragmentation at 1.5 to 2.5 mM. However, inhibition of polyamine synthesis with alpha-difluoromethyl ornithine or methylglyoxal bis(guanylhydrazone) did not inhibit prolactin action. Prolactin action was not blocked by inhibitors of tyrosine kinase activation, genistein and tyrphostin-47. On the other hand, pervanadate, a potent tyrosine phosphatase inhibitor, consistently inhibited DEX-induced DNA fragmentation. Prolactin action and DEX-induced apoptosis both occurred in calcium-free PBS. In summary, protein kinase C activation and eicosanoid production do not appear to mediate this prolactin action. Although spermine could block DNA fragmentation, blockade of the polyamine cascade did not inhibit prolactin action, suggesting that polyamines do not mediate this prolactin effect. While inhibitors of tyrosine kinase activation did not block prolactin action, tyrosine phosphatase inhibition in the presence of basal tyrosine kinase activity mimicked prolactin action, suggesting tyrosine phosphorylation participation in the anti-apoptotic effect. Extra-cellular calcium was not required for prolactin or DEX action.
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PMID:Investigation of intracellular signals mediating the anti-apoptotic action of prolactin in Nb2 lymphoma cells. 777 88

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examined the effect of Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO4 or CuSO4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn2+ by EDTA indicated that Zn2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn2+. When the spirochetes were washed after incubation in the presence of ZnSO4 for 2 cycles and incubated in fresh PBS-GR without Zn2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO4 was due to a direct action of ZnSO4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO4, the inhibitory effect of Zn2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO4 revealed clumping of ribosomes and clearing of cell cytoplasm.
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PMID:Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper. 780 26

The effect of varying external concentrations of Mg2+ has been studied on NMDA induced toxicity, increases in the intracellular calcium concentration, [Ca2+]i, and cGMP production in cultured neocortical neurons. The neurotoxic potency of NMDA during a 5 h exposure period in 7-day-old cultures was examined in three different exposure media: phosphate buffered saline, PBS (137 mM NaCl, 2.7 mM KCl, 7.3 mM Na2HPO4, 1.5 mM KH2PO4, 0.9 mM CaCl2, 0.6 mM MgCl2; pH 7.4); PBS without addition of Mg2+; and Neuronal Dulbecco's Minimal Essential Medium (NDMEM = DMEM with a final concentration of KCl of 25.5 mM KCl; cf. Experimental Procedures). In the presence of Mg2+, no toxicity of NMDA was observed in PBS (ED50 > 1000 microM) whereas omission of Mg2+ in PBS resulted in an ED50 value for NMDA of 9 +/- 3 microM. Using NDMEM as the exposure medium, an intermediate neurotoxic potency of NMDA (ED50 = 40 +/- 5 microM) was observed. This intermediate value is probably due to partial attenuation of the Mg2+ block of the NMDA associated cation channel by the depolarizing conditions in NDMEM (with 25.5 mM KCl). Diminishing the external Mg2+ concentration also potentiated the increase in [Ca2+]i after stimulation with NMDA. This potentiation was maximally 3-fold (obtained at 300 microM NMDA), and the IC50 was 15 +/- 2 microM. Surprisingly, the NMDA induced production of cGMP was not sensitive to variations in the external Mg2+ concentration. These findings of distinct regulatory mechanisms of different NMDA receptor coupled responses may indicate the existence of several types of NMDA receptors.
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PMID:Effect of magnesium on NMDA mediated toxicity and increases in [Ca2+]i and cGMP in cultured neocortical neurons: evidence for distinct regulation of different responses. 782 63

Treatment of human lymphocytes with hydrogen peroxide (10 microM, 30 min, 37 degrees C in PBS) or with 1 cGy X-rays evoked about a 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). In addition to a lower micronuclei frequency, we also found an increase in the sedimentation distance of the nucleoids, when measured 90 min (duration of the isolation procedure carried out at 4 degrees C) after the adaptive dose (hydrogen peroxide or X-rays) and preceding the challenge dose. To test whether Ca2+ is involved in the induction of the adaptive response pathway, we treated cells with the calcium chelator, EGTA. When EGTA was given at the same time as the adaptive dose, it prevented the development of the adaptive response. In addition, the calcium antagonist, TMB-8, also prevented the development of the adaptive response as it prevented the reduction of both micronuclei and increased nucleoid sedimentation. Cellular treatment with TMB-8 increased the free [Ca2+] by 40%, when given together with hydrogen peroxide. The faster sedimenting nucleoids from adapted cells were also examined by ethidium bromide titration; there was no indication of any change in supercoil density or loop size. Psi-tectorigenin, an inhibitor of phosphatidylinositol turnover, did not modify the adaptive response, indicating that inositol (1,4,5)-trisphosphate is not involved in the induction of the adaptive response, but free Ca2+ ions are.
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PMID:Calcium antagonist, TMB-8, prevents the induction of adaptive response by hydrogen peroxide or X-rays in human lymphocytes. 802 16

In order to study the mechanism of cancer metastasis, AH100B cells, an ascitic hepatoma cell line, were transplanted into the small intestine of male Donryu rats. Each metastatic nodule in the liver was collected with the respective intestinal lesion. Each sample thus obtained was injected into the peritoneal cavity of male Donryu rats to make free cancer cells. Then, the cancer cells, having an intact cell surface, of the metastatic and primary intestinal lesion were collected respectively. After washing in Dolbecco's PBS (Ca2+ and Mg(2+)-free, pH 7.2), the definite numbers of cancer cells of the metastatic and primary intestinal lesion were incubated in the PBS containing [1-14C]-AA at 25 degrees C for 30 min, respectively. AA metabolites formed during the incubation period were extracted and subjected to TLC, followed by autoradiography. Each radioactive part was scraped off the plate and measured for its radioactivity. The pattern of the ability to synthesize PGs was different between the cancer cells which metastasized to the liver and those of the primary lesion, that is, percentage values of PGE2 and PGF2 alpha were higher (p < 0.01) in the cancer cells which metastasized to liver as compared with those of the primary intestinal lesion. These results suggest that PGs produced by hepatic metastatic cancer cells might play an important role in cancer metastasis.
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PMID:Some features in prostaglandin synthesis of the cancer cells which metastasized into liver from intestinal cancer lesions. 826 26

The purpose of these experiments was to determine the effect of electroporation of IP3 into the cytosol of murine secondary oocytes and evaluate any alterations in [Ca2+]i resulting from Ca2+ release from intracellular stores. In addition, we evaluated the effect of ethanol (ETOH) on the release of Ca2+ from intracellular stores. Oocytes were loaded with the Ca2+ indicator fluo-3 by incubation in 100 microliters drops of medium containing 2 microM fluo-3/AM for 60 min at 37 degrees C. Changes in fluorescence were monitored by use of an inverted microscope which had been connected to a spectrofluorometer. Fluorescent intensity measurements were acquired for a minimum of 416 sec time span or up to 1,248 sec, with integration readings of 1 sec duration obtained every 2 sec throughout the measurement period. The experimental design consisted of comparing the rise in [Ca2+]i of fluo-3 loaded secondary oocytes subjected to electroporation in PBS and Ca(2+)-free PBS, each containing 25 microM IP3, to that elicited by PBS and Ca(2+)-free PBS containing a final concentration of 7% ETOH. Non-pulsed control secondary oocytes were placed in PBS + 25 microM IP3 during monitoring of [Ca2+]i fluorescence. Pulsed control secondary oocytes were placed in Ca(2+)-free PBS, subjected to electroporation pulse, and monitored for [Ca2+]i fluorescence. Electroporation of IP3 was accomplished by placing fluo-3 loaded secondary oocytes between the electrodes of a glass slide fusion chamber which had been overlaid with 300 microliters of PBS + 25 microM IP3 or Ca(2+)-free PBS + 25 microM IP3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Electroporation of inositol 1,4,5-triphosphate induces repetitive calcium oscillations in murine oocytes. 842 41

The cadherins are a family of calcium-dependent cell adhesion molecules that are regulated both spatially and temporally during development. Epithelial cadherin (E-cadherin) is present in epithelial cells in both the embryo and yolk sac during organogenesis. The consequences of disrupting the expression of E-cadherin at this stage of development are poorly understood. We report here our studies on the effects of antisense oligonucleotides on E-cadherin in the rat whole embryo culture system. Four 18-base single strand phosphorothioate oligodeoxynucleotides (AS-oligos), complementary to various regions of the mouse E-cadherin cDNA sequence, were dissolved in saline and injected into the amniotic cavities of 5-7 somite rat embryos; a sense (S-oligo) to oligo-1, an 18-base random sequence oligo (C-oligo), and PBS were used as controls. Embryos were cultured for up to 45 h; embryo morphology and the relative concentrations of E-cadherin protein were examined. All six oligonucleotides (AS-oligos and control oligos) induced malformations when amounts ranging from 25 to 50 pmol of oligonucleotide were injected per embryo. The malformations induced by all the oligos included craniofacial hypoplasia, an enlarged pericardium, twisted spinal cord, swelling of the rhombencephalon, and underdeveloped forelimb. Injection of AS-oligo-1, a sequence starting at the tenth base downstream from the translation initiation codon (ATG), resulted in malformed embryos with a high incidence of cranial neural tube malformations. The effects of AS-oligo-1 on the relative abundance of E- and neural (N)-cadherin proteins were examined by Western blot analysis. In the AS-oligo-1-exposed malformed embryos, the relative abundance of E- and N-cadherin proteins was not altered up to 24 h after injection; E- and N-cadherin concentrations in the embryo were decreased at 45 h postinjection. In contrast, the relative abundance of the E-cadherin protein in the yolk sac was reduced at 1-2 h after injection of AS oligo-1 and returned to control levels by 4 h. S-oligo-1 did not induce any change in the relative abundance of E- or N-cadherins. Thus, there was a tissue-specific and temporary "knockdown" of E-cadherin expression in the yolk sac of embryos exposed to antisense (AS-oligo-1); the down-regulation of yolk sac E-cadherin appears to lead to the induction of neural tube defects in the embryo. The exposure of whole embryos in culture to antisense oligonucleotides provides a model system in which the roles of developmentally important molecules and their spatial and temporal contributions to embryogenesis can be elucidated.
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PMID:Antisense oligonucleotide down-regulation of E-cadherin in the yolk sac and cranial neural tube malformations. 852 29

The lethal effect of near ultraviolet (NUV) with low intensity on cultured RPE cells has been investigated. RPE cultures with various cell densities were exposed to NUV (peaking at 365 nm) with or without ambient oxygen in phenol-red-free Dulbecco's PBS containing Ca2+, Mg2+ and glucose (PBS+). The cell viability was determined by dye exclusion and was expressed as cell death ratio (CDR, dead cells/total cells). When RPE cells at 5 x 10(3) cells/cm2, a non-contact low density, were irradiated either at a fixed irradiance (900 microW/cm2) with different exposure times (4 to 8h) or vice versa (8 h with irradiance from 430 to 900 microW/cm2), the change of CDR represented a similar linear function. The replotted data from both the time- and the irradiance-dependent curves indicated that the killing of RPE cells is dependent on the total energy dose of NUV. When a single NUV energy (19.44 J/cm2) was used, CDR was RPE cell density dependent. At confluence, NUV at the highest dosage tested (26 J/cm2) did not show any lethality. An oxygen-free condition abolished the NUV lethality on RPE cells even though the RPE cells were at a non-contact state. The presence of an antioxidant enzyme, catalase, in oxygen-saturated PBS+ protected RPE cells against NUV killing, but superoxide dismutase did not protect the RPE cells against NUV killing. These findings demonstrate that NUV possesses a lethal effect on RPE cells in vitro. Two key factors determine the magnitude and nature of this lethal effect: first, total NUV energy dose determines the nature of NUV's lethal effect; second, RPE growth conditions suggest the importance of cell-cell interaction in protecting these cells from NUV injury. Because an oxygen-free condition abolishes NUV lethality, it suggests that the oxidative stress is directly related to NUV lethal action. The selective inhibition by catalase of NUV killing of RPE cells suggests that the killing is oxidative species specific. NUV radiation might be highly risky to RPE viability in vivo, especially when the integrity of the RPE layer has been lost.
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PMID:Characterization of lethal action of near-ultraviolet on retinal pigment epithelial cells in vitro. 897 37

In this study, we have found that IGF-binding protein-3 (IGFBP-3) in calf serum added to tissue culture medium is degraded by cultured FRTL-5 cells and a major 31 kDa fragment of IGFBP-3 is produced. When FRTL-5 rat thyroid cells were cultured in 6H medium (modified F-12M medium containing TSH, insulin, hydrocortisone, somatostatin, transferrin, and glycyl-histidyl-lysine) containing 5% calf serum, both 44-46 and 31 kDa IGFBPs were found in conditioned medium by ligand blot analysis using 125I-labelled IGF-II. However, predominantly the 44-46 kDa IGFBP was detected in unconditioned 6H medium containing 5% calf serum. When calf serum in the media was replaced by human serum similar results were obtained, and the 44-46 kDa and 31 kDa IGFBPs were recognized using a human IGFBP-3 antibody following Western blot analysis. FRTL-5 cells secreted only small amounts of an endogenous 29 kDa IGFBP, thought to be IGFBP-5. To separate the 31 kDa fragment of IGFBP-3 from the endogenous IGFBP-5, culture media were fractionated by concanavalin-A-Sepharose chromatography and aliquots of both flow-through and eluate from the column were analyzed by ligand blotting. A 31 kDa IGFBP was found in the eluate fractions from concanavalin-A-Sepharose chromatography following the separation of conditioned 6H medium supplemented with calf serum, suggesting that this species was an N-linked glycoprotein and could be derived from the degradation of serum IGFBP-3 by FRTL-5 cells. Using a modified zymographic assay, we examined whether the degradation of IGFBP-3 could depend on the cell membrane. Confluent FRTL-5 cells were washed with PBS and overlaid with liquid agarose solution. After the agarose had solidified, unconditioned 6H medium containing 5% calf serum was incubated with the cells at 37 degrees C for 16 h. Both 44-46 and 31 kDa IGFBP species were found in the overlying, conditioned medium by ligand blot. However, the 31 kDa IGFBP was not found in medium in the absence of FRTL-5 cells, and no IGFBP could be found in serum-free conditioned medium from agarose-covered FRTL-5 cells. This suggests that the 44-46 kDa IGFBP-3 in serum was degraded to yield a 31 kDa fragment, while any endogenous IGFBP-5 could not pass out of the agarose. The degradation of 44-46 kDa IGFBP-3 in the modified zymographic assay was inhibited by phenylmethylsulfonyl fluoride, EDTA, and aprotinin, but not by leupeptin. In summary, these results indicated that IGFBP-3 in calf serum added to culture medium could be degraded by FRTL-5 cells and that this may involve calcium-dependent serine proteases.
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PMID:Degradation of IGF-binding protein-3 by proteases in cultured FRTL-5 rat thyroid cells. 907 84

We have recently demonstrated the presence of phospholipase A2 (PLA2) activity in cells from bovine retinal pigment epithelium (RPE) [Jacob et al. (1996) J. Biol. Chem. 271, 19209-19218]. We report here our results on the characterization of this RPE-PLA2 activity. We show that RPE probably contains two types of PLA2 enzyme, as indicated by the results obtained with different PLA2-active fractions eluted from cation-exchange columns and treated with Ca2+/EGTA, dithiothreitol, p-bromophenacyl bromide or heat. These results, in addition to those from PLA2 assays using different substrates, also suggest that RPE-PLA2 enzymes are different from the well-known secretory, cytoplasmic and Ca2+-independent forms. Sequential extraction of RPE with (1) isotonic, (2) hypertonic and (3) detergent-containing PBS argues for the presence of weakly membrane-associated enzymes. Control experiments using 'back and forth' TLC allowed us to discriminate between PLA2 and phospholipase C/diacylglycerol lipase activity and confirmed that, in our assay conditions, the release of fatty acids was indeed due to PLA2 enzymes. These results, together with those obtained by treating RPE homogenates with H2SO4, guanosine 5'-[gamma-thio]triphosphate, ATP and different protease inhibitors, permitted us to make the first characterization of these RPE-PLA2 enzymes. We conclude that RPE contains novel types of PLA2 that are different from the secretory, cytoplasmic and Ca2+-independent forms.
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PMID:Bovine retinal pigment epithelium contains novel types of phospholipase A2. 935 16

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