Tyrosines 60, 64, and 101 of Epstein-Barr virus LMP2A are not essential for blocking B cell signal transduction.
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Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed on the membrane of B-lymphocytes and blocks B cell receptor (BCR) signaling in EBV-transformed B-lymphocytes in vitro. The LMP2A amino-terminal domain, which is essential for the LMP2A-mediated block of B cell signal transduction, contains eight tyrosine residues. Three of these tyrosine residues (Y74, Y85, and Y112) have been demonstrated to be essential for the LMP2A-mediated block on protein tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression after BCR activation. To investigate the importance of tyrosines at positions 60, 64, and 101 on B cell signaling, EBV recombinants were constructed containing a tyrosine-to-phenylalanine point mutation at amino acid 60, 64, or 101 within LMP2A. Tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression were not affected by any of the tyrosine point mutations after BCR activation. In addition, constitutive phosphorylation of LMP2A was unaffected by any of the tyrosine point mutations. These data indicate that tyrosines 60, 64, and 101 are not essential for the LMP2A-mediated block of B cell signal transduction in transformed cell lines. (+info)
Luteal regression in the primate: different forms of cell death during naturaland gonadotropin-releasing hormone antagonist or prostaglandin analogue-induced luteolysis.
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Morphological changes in the corpus luteum following natural and induced luteolysis in the marmoset were investigated by light and electron microscopy. Functional corpora lutea were studied in the mid and late luteal phase, naturally regressed corpora lutea in the early and late follicular phase, and corpora lutea induced to regress by administration of GnRH antagonist or prostaglandin F(2alpha) analogue in the midluteal phase. Natural luteolysis was associated with lutein cell atrophy, condensation of cytoplasmic inclusions and organelles, and accumulation of lipid. GnRH antagonist treatment resulted in aggregations of smooth membranes and myelin-like bodies in the cytoplasm of the lutein cells together with complex aggregations of degenerative cells. After prostaglandin treatment, the lutein cells contained numerous small and large vesicles; as the degenerative changes advanced, these vesicles coalesced into alveolar-type vacuoles, and nuclei involuted. These results show that in the marmoset, natural luteolysis and the two luteolytic treatments reveal different forms of luteal degeneration and cell death, none of which fit the ultrastructural criteria for apoptosis. More emphasis needs to be placed on understanding these predominant nonapoptotic forms of cell death in order to elucidate the process of luteolysis in the primate. (+info)
Primary CA1 and conditionally immortal MHP36 cell grafts restore conditional discrimination learning and recall in marmosets after excitotoxic lesions of the hippocampal CA1 field.
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Common marmosets (Callithrix jacchus, n = 18) were trained to discriminate between rewarded and non-rewarded objects (simple discriminations, SDs) and to make conditional discriminations (CDs) when presented sequentially with two different pairs of identical objects signifying reward either in the right or left food well of the Wisconsin General Test Apparatus. After bilateral N-methyl-D-aspartate (0.12 M) lesions through the cornu ammonis-1 (CA1) field (7 microl in five sites), marmosets showed profound impairment in recall of CDs but not SDs, and were assigned to lesion only, lesion plus CA1 grafts and lesion plus Maudsley hippocampal cell line, clone 36 (MHP36) grafts groups matched for lesion-induced impairment. Cell suspension grafts (4 microl, 15-25 000 cells/microl) of cells dissected from the CA1 region of foetal brain at embryonic day 94-96, or of conditionally immortalized MHP36 cells, derived from the H-2Kb-tsA58 transgenic mouse neuroepithelium and labelled with [3H]thymidine, were infused at the lesion sites. The lesion plus MHP36 grafts group was injected five times per week with cyclosporin A (10 mg/kg) throughout testing. Lesion, grafted and intact control marmosets (n = 4-5/group) were tested on recall of SDs and CDs learned before lesioning and on acquisition of four new CDs over a 6-month period. Lesioned animals were highly impaired in recall and acquisition of CD tasks, but recall of SDs was not significantly disrupted. Both grafted groups of marmosets showed improvement to control level in recall of CDs. They were significantly slower in learning the first new CD task, but mastered the remaining tasks as efficiently as controls and were substantially superior to the lesion-only group. Visualized by Nissl staining, foetal grafts formed clumps of pyramidal-like cells within the denervated CA1 field, or jutted into the lateral ventricles. MHP36 cells, identified by beta-galactosidase staining and autoradiography, showed neuronal and astrocytic morphology, and were distributed evenly throughout the CA1 region. The results indicate that MHP36 cell grafts are as functionally effective as foetal grafts and appear to integrate into the host brain in a structurally appropriate manner, showing the capacity to differentiate into both mature neurons and glia, and to develop morphologies appropriate to the site of migration. These findings, which parallel the facilitative effects of foetal and MHP36 grafts in rats with ischaemic CA1 damage, offer encouragement for the development of conditionally immortal neuroepithelial stem cell lines for grafting in conditions of severe amnesia and hippocampal damage following recovery from cardiac arrest or other global ischaemic episodes. (+info)
Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe.
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In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events. (+info)
Effects of beta-blockers and nicardipine on oxotremorine-induced tremor in common marmosets.
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Effects of beta-blockers (propranolol, arotinolol and nipradilol) and a Ca2+ channel blocker (nicardipine) on oxotremorine-induced tremor were studied in common marmosets. Generalized tremor was elicited by an intraperitoneal administration of 0.25 mg/kg oxotremorine. Intensity of the tremor was classified into 7 degrees, and it was evaluated every 10 min. The total intensity of oxotremorine-induced tremor for each drug was expressed as "points", which were the sum of tremor intensity scores evaluated every 10 min up to 190 min following the administration of oxotremorine. Beta-blockers significantly suppressed the tremor. On the other hand, the Ca2+ channel blocker exacerbated the tremor. (+info)
Centromere repositioning.
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Primate pericentromeric regions recently have been shown to exhibit extraordinary evolutionary plasticity. In this paper we report an additional peculiar feature of these regions that we discovered while analyzing, by FISH, the evolutionary conservation of primate phylogenetic chromosome IX. If the position of the centromere is not taken into account, a relatively small number of rearrangements must be invoked to account for interspecific differences. Conversely, if the centromere is included, a paradox emerges: The position of the centromere seems to have undergone, in some species, an evolutionary history independent from the surrounding markers. A significant number of additional rearrangements must be proposed to reconcile the order of the markers with centromere position. Alternatively, the evolutionary emergence of neocentromeres can be postulated. (+info)
Expression of growth factor ligand and receptor genes in preimplantation stage water buffalo (Bubalus bubalis) embryos and oviduct epithelial cells.
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The temporal pattern of expression of genes for several growth factor ligands and receptors was examined in preimplantation water buffalo embryos and oviduct epithelial cells using RT-PCR. The identity of the resulting PCR products was confirmed by their expected size, restriction analysis, Southern blot hybridization and nucleotide sequence analysis. Preimplantation stage embryos from the one-cell to the blastocyst stage were derived after maturation, fertilization and culture of oocytes in vitro. Expression of members of the insulin-like growth factor (IGF) family was observed predominantly in preimplantation stage embryos and oviduct epithelial cells. Similarly, transcripts encoding insulin and IGF-I receptors were detected at each stage of embryonic development. The mRNA transcript of the IGF-I receptor was not detected in oviduct epithelial cells, but a prominent band corresponding to the insulin receptor was observed. Insulin and IGF-II mRNA were expressed as maternal transcripts that were not detected at the two- to four-cell stage but were present as zygotic transcripts at the eight-cell stage. Transcripts encoding IGF-I were detected in oviduct epithelial cells, but were not observed in any of the preimplantation stage embryos. Transforming growth factor (TGF) alpha and beta and epidermal growth factor mRNA transcripts were not detected in any of the preimplantation stage embryos. These results indicate that IGF-I acts via a paracrine mechanism to promote growth and development of preimplantation water buffalo embryos. Similarly, IGF-II appears to act through a heterologous autocrine mechanism via the IGF-I or the insulin receptor. Furthermore, the presence of TGF-alpha in oviduct epithelial cells indicates that it may have a critical role during development. (+info)
Marmoset monkey trophoblastic tissue growth and matrix metalloproteinase secretion in culture.
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Marmoset monkey blastocysts maintained in culture produced trophoblastic vesicles up to 4 mm in diameter that were subdivided into fragments and subcultured to produce new vesicles. These tissues are composed of an outer layer of trophoblast-like cells and an inner layer of endoderm-like cells, and resemble a blastocyst wall. When such vesicles were cultured in serum-free medium for 14 days, they increased in size but there was no significant difference in their protein content at the end of culture. The proliferation index, measured by BrdU incorporation, varied considerably within and between vesicles. The purpose of this investigation was to determine which matrix metalloproteinases are secreted by marmoset monkey trophoblastic tissue in vitro, and the effect of extracellular laminin on this secretion. It was determined by zymography that the vesicles secreted matrix metalloproteinase 2, but not matrix metalloproteinase 9, and that matrix metalloproteinase 2 was secreted as the proenzyme (72 kDa). Matrix metalloproteinases 1, 3 and 7 were not detectable in the culture medium. The addition of laminin (5-20 micrograms ml-1), either as a substrate or in solution in the medium, did not have a consistent effect on matrix metalloproteinase 2 secretion during the culture period. The vesicles were found to express both matrix metalloproteinases 2 and 9 in both types of cell when examined by immunohistochemistry. The expression of matrix metalloproteinase 9 in the vesicles, but the absence of its secretion, indicates that specific factors, possibly of endometrial origin, may be required for inducing secretion. (+info)