Role of class B scavenger receptor type I in phagocytosis of apoptotic rat spermatogenic cells by Sertoli cells.
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Rat Sertoli cells phagocytose apoptotic spermatogenic cells, which consist mostly of spermatocytes, in primary culture by recognizing phosphatidylserine (PS) exposed on the surface of degenerating spermatogenic cells. We compared the mode of phagocytosis using spermatogenic cells at different stages of spermatogenesis. Spermatogenic cells were separated into several groups based on their ploidy, with purities of 60-90%. When the fractionated spermatogenic cell populations were subjected to a phagocytosis assay, cells with ploidies of 1n, 2n, and 4n were almost equally phagocytosed by Sertoli cells. All the cell populations exposed PS on the cell surface, and phagocytosis of all cell populations was similarly inhibited by the addition of PS-containing liposomes. Class B scavenger receptor type I (SR-BI), a candidate for the PS receptor, was detected in Sertoli cells. Overexpression of the rat SR-BI cDNA increased the PS-mediated phagocytic activity of Sertoli cell-derived cell lines. Moreover, phagocytosis of spermatogenic cells by Sertoli cells was inhibited in the presence of an anti-SR-BI antibody. Finally, the addition of high density lipoprotein, a ligand specific for SR-BI, decreased both phagocytosis of spermatogenic cells and incorporation of PS-containing liposomes by Sertoli cells. In conclusion, SR-BI functions at least partly as a PS receptor, enabling Sertoli cells to recognize and phagocytose apoptotic spermatogenic cells at all stages of differentiation. (+info)
Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin.
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ZO-2, a member of the MAGUK family, was thought to be specific for tight junctions (TJs) in contrast to ZO-1, another MAGUK family member, which is localized at TJs and adherens junctions (AJs) in epithelial and nonepithelial cells, respectively. Mouse ZO-2 cDNA was isolated, and a specific polyclonal antibody was generated using corresponding synthetic peptides as antigens. Immunofluorescence microscopy with this polyclonal antibody revealed that, similarly to ZO-1, in addition to TJs in epithelial cells, ZO-2 was also concentrated at AJs in nonepithelial cells such as fibroblasts and cardiac muscle cells lacking TJs. When NH2-terminal dlg-like and COOH-terminal non-dlg-like domains of ZO-2 (N-ZO-2 and C-ZO-2, respectively) were separately introduced into cultured cells, N-ZO-2 was colocalized with endogenous ZO-1/ZO-2, i.e. at TJs in epithelial cells and at AJs in non-epithelial cells, whereas C-ZO-2 was distributed along actin filaments. Consistently, occludin as well as alpha catenin directly bound to N-ZO-2 as well as the NH2-terminal dlg-like portion of ZO-1 (N-ZO-1) in vitro. Furthermore, immunoprecipitation experiments revealed that the second PDZ domain of ZO-2 was directly associated with N-ZO-1. These findings indicated that ZO-2 forms a complex with ZO-1/occludin or ZO-1/alpha catenin to establish TJ or AJ domains, respectively. (+info)
Skeletal muscle type ryanodine receptor is involved in calcium signaling in human B lymphocytes.
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The regulation of intracellular free Ca2+ concentration ([Ca2+]i) in B cells remains poorly understood and is presently explained almost solely by inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ release, followed by activation of a store-operated channel mechanism. In fact, there are reports indicating that IP3 production does not always correlate with the magnitude of Ca2+ release. We demonstrate here that human B cells express a ryanodine receptor (RYR) that functions as a Ca2+ release channel during the B cell antigen receptor (BCR)-stimulated Ca2+ signaling process. Immunoblotting studies showed that both human primary CD19(+) B and DAKIKI cells express a 565-kDa immunoreactive protein that is indistinguishable in molecular size and immunoreactivity from the RYR. Selective reverse transcription-polymerase chain reaction, restriction fragment length polymorphism, and sequencing of cloned cDNA indicated that the major isoform of the RYR expressed in primary CD19(+) B and DAKIKI cells is identical to the skeletal muscle type (RYR1). Saturation analysis of [3H]ryanodine binding yielded Bmax = 150 fmol/mg of protein and Kd = 110 nM in DAKIKI cells. In fluo-3-loaded CD19(+) B and DAKIKI cells, 4-chloro-m-cresol, a potent activator of Ca2+ release mediated by the ryanodine-sensitive Ca2+ release channel, induced Ca2+ release in a dose-dependent and ryanodine-sensitive fashion. Furthermore, BCR-mediated Ca2+ release in CD19(+) B cells was significantly altered by 4-chloro-m-cresol and ryanodine. These results indicate that RYR1 functions as a Ca2+ release channel during BCR-stimulated Ca2+ signaling and suggest that complex Ca2+ signals that control the cellular activities of B cells may be generated by cooperation of the IP3 receptor and RYR1. (+info)
Bovine endometrial epithelial cells as a model system to study oxytocin receptor regulation.
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Endometrial epithelial cell cultures were established from bovine uterine tissue collected during the oestrous cycle from commercially slaughtered animals. These cells were shown to express moderately high levels of oxytocin receptors (OTR) (up to 30000 per cell) after about one week in culture. These receptors have been characterized at the molecular, pharmacological and functional level and shown to be identical to those expressed in the bovine endometrium in vivo. Preliminary experiments to investigate the regulation of the OTR and its gene using this system, have shown that expression is to a large degree constitutive, the receptors being spontaneously upregulated during culture. Sex steroids at concentrations close to or above the serum limits observed in vivo appeared to have no effect, although the cells were shown to express mRNA for the specific steroid receptors throughout culture. Only the blastocyst product, interferon-tau, showed a significant effect, downregulating both OTR and their gene transcripts in the cultured endometrial epithelial cells. Although more extensive studies are necessary, these results support the view that the OTR gene is controlled in part at least by a combination of constitutive and inhibitory elements. (+info)
Regulation of the human oxytocin receptor in the uterus: a molecular approach.
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The oxytocin-oxytocin receptor (OTR) system plays important roles in the human uterus, and the effectiveness of oxytocin is greatly influenced by the pattern of receptor expression in vivo. To investigate OTR expression at the molecular level, we have established a bioassay system for the specific transcripts using Xenopus laevis oocytes, cloned the OTR cDNA, raised anti-OTR antibodies, and characterized OTR genomic clones and systems to study the transcriptional regulation of the gene. Using these molecular tools, we have examined OTR expression in the endometrium and myometrium of non-pregnant and pregnant women. OTR expression appears mainly to be regulated at the transcription level. Analysis of the 5' flanking region of this gene indicates constitutionally active promoter activity when transfected into cultured HeLa and SKN cells. We are currently developing these techniques to analyse OTR regulation in the uterus. (+info)
Pharmacology of LY315920/S-5920, [[3-(aminooxoacetyl)-2-ethyl-1- (phenylmethyl)-1H-indol-4-yl]oxy] acetate, a potent and selective secretory phospholipase A2 inhibitor: A new class of anti-inflammatory drugs, SPI.
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LY315920 is a potent, selective inhibitor of recombinant human, group IIA, nonpancreatic secretory PLA2 (sPLA2). In a chromogenic isolated enzyme assay, LY315920 inhibited sPLA2 activity with an IC50 of 9 +/- 1 nM or 7.3 x 10(-6) mole fraction, which approached the stiochiometric limit of this assay. The true potency of LY315920 was defined using a deoxycholate/phosphatidylcholine assay with a mole fraction of 1.5 x 10(-6). LY315920 was 40-fold less active against human, group IB, pancreatic sPLA2 and was inactive against cytosolic PLA2 and the constitutive and inducible forms of cyclooxygenase. Human sPLA2-induced release of thromboxane A2 (TXA2) from isolated guinea pig lung bronchoalveolar lavage cells was inhibited by LY315920 with an IC50 of 0.79 microM. The release of TXA2 from these cells by N-formyl-methionyl-leucyl-phenylalanine or arachidonic acid was not inhibited. The i.v. administration of LY315920, 5 min before harvesting the bronchoalveolar lavage cells, resulted in the inhibition of sPLA2-induced production of TXA2 with an ED50 of 16.1 mg/kg. Challenge of guinea pig lung pleural strips with sPLA2 produced contractile responses that were suppressed in a concentration-dependent manner by LY315920 with an apparent KB of 83 +/- 14 nM. Contractile responses induced by arachidonic acid were not altered. Intravenous or oral administration of LY315920 to transgenic mice expressing the human sPLA2 protein inhibited serum sPLA2 activity in a dose-related manner over a 4-h time course. LY315920 is a potent and selective sPLA2 inhibitor and represents a new class of anti-inflammatory agent designated SPI. This agent is currently undergoing clinical evaluation and should help to define the role of sPLA2 in various inflammatory disease states. (+info)
Identification of a clinically relevant immunodominant region of collagen IV in Goodpasture disease.
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BACKGROUND: The characteristic feature of Goodpasture disease is the occurrence of an autoantibody response to the noncollagenous domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1] in the alveolar and glomerular basement membrane. These antibodies are associated with the development of a rapidly progressive glomerulonephritis, with or without lung hemorrhage, whereas autoantibodies specific for the other alpha chains of the heterotrimeric type IV collagen probably do not cause disease. In this study, we have investigated whether differences in fine specificity of autoimmune recognition of the alpha3(IV)NC1 correlate with clinical outcome. METHODS: For mapping of antibody binding to type IV collagen, chimeric collagen constructs were generated in which parts of the alpha3(IV)NC1 domain were replaced by the corresponding sequences of homologous nonreactive alpha1(IV). The different recombinant collagen chimeras allowed the analysis of antibody specificities in 77 sera from well-documented patients. RESULTS: One construct that harbors the aminoterminal third of the alpha3(IV)NC1 was recognized by all sera, indicating that it represents the dominant target of the B-cell response in Goodpasture disease. Seventy percent of the samples recognized other parts of the molecule as well. However, only reactivity to the N-terminus of the alpha3(IV)NC1 correlated with prognosis, that is, kidney survival after six months of follow-up. CONCLUSION: The results indicate the crucial importance of antibody recognition of this particular domain for the pathogenesis of Goodpasture disease, thereby opening new avenues for the development of better diagnostic and therapeutic procedures. (+info)
Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium.
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Lindane-degrading activity under aerobic conditions has been observed in two bacterial strains: UT26, phenotypically identified as Sphingomonas paucimobilis, and a new single unidentified isolate named RP5557T. The rrs (16S rDNA) sequences for both strains and the phenotypic characteristics for the unidentified isolate RP5557T were determined. RP5557T does not have high identity (less than 90% in all cases) with any sequence in the GenBank or RDP databases. A phylogenetic analysis based on rrs sequences indicated that RP5557T belongs to the gamma-Proteobacteria in a coherent phylum that includes the genera Xanthomonas and Xylella (100% bootstrap), whereas UT26 is clearly separate from the Xanthomonas cluster. Based on the phylogenetic analyses and on the phenotypic characteristics, a new genus, Rhodanobacter, containing a single species, Rhodanobacter lindaniclasticus, is proposed for strain RP5557T (= LMG 18385T), which becomes the type strain. (+info)