Calf thymus histone H2A. Purification and tryptic peptides.
(41/8961)
One of the five main histone molecular species, H2A, of calf thymus was fractionated and purified on a large scale for chemical and physical studies. This was achieved by three methods, using different combinations of our CM-cellulose chromatographic technique with other chromatographic systems reported. Method I consists of chromatography first on CM-cellulose and then on Sephadex G-100, Method II first on Amberlite CG-50 and then on CM-cellulose and then on Bio-Gel P-10. Method I was successful when the starting material obtained by Johns' fractionation methods was contaminated by a small amount of H3 histone. Method II did not suffer from such a limitation but gave a low recovery of H2A on the first chromatography. Method III provided the purest preparations of H2A, together with highly purified H3, H4, and others, and is superior to methods previously reported for the large-scale preparation of H2A and other species from whole histone as regards the simplicity of the procedures and the purity and yield of the products. The preparation obtained by Method I was digested with trypsin [EC 3.4.21.4]. The resulting soluble and insoluble fractions of the digest were fractionated by column chromatography to give 20 small peptides and 2 large peptides, respectively, with high recoveries. The sequences of almost all the soluble peptides were determined; these, taking into account the recoveries of these peptides and the compositions of the insoluble peptides (19 and 29 residues), accounted for all the 129 amino acid residues of this histone. (+info)
Murine macrophage-lymphocyte interactions: scanning electron microscopic study.
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Light and scanning electron microscopic observations revealed murine macrophage-lymphocyte interactions involving the initial contact of peritoneal, spleen, or thymus lymphocytes with peritoneal macrophage processes or microprocesses followed by clustering of lymphocytes over the central nuclear area of the macrophages. Lymphocyte-lymphocyte clustering was not observed in the absence of macrophages. Attachment and subsequent clustering appeared not to require the presence of serum or antigen; the attachment of allogeneic or xenogeneic lymphocytes was comparable to that seen in the syngeneic system, but central clustering of these lymphocytes failed to occur. No attachment or clustering was observed when thymic lymphocytes were cultured with thymus derived fibroblasts rather than with peritoneal macrophages. Lymphocyte attachment to immune, antigen-activated, syngeneic macrophages occurred more rapidly than that to normal unstimulated syngeneic macrophages; however, lymphocytes attached to the "activated" macrophages appeared to be killed by a nonphagocytic mechanism. A similar increase in the rate of lymphocyte attachment to macrophages occurred in the presence of migration inhibitory factor. Subsequent lymphocyte clustering on macrophages was observed in the migration inhibitory factor-stimulated cultures. In addition, lymphocyte-macrophage interactions similar to those in vitro were observed to occur in vivo on intraperitoneally implanted cover slips. (+info)
Inhibition of poly(ADP-ribose) synthetase (PARS) and protection against peroxynitrite-induced cytotoxicity by zinc chelation.
(43/8961)
Peroxynitrite, a potent oxidant formed by the reaction of nitric oxide and superoxide causes thymocyte necrosis, in part, via activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS). The cytotoxic PARS pathway initiated by DNA strand breaks and excessive PARS activation has been shown to deplete cellular energy pools, leading to cell necrosis. Here we have investigated the effect of tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) a heavy metal chelator on peroxynitrite-induced cytotoxicity. TPEN (10 microM) abolished cell death induced by authentic peroxynitrite (25 microM) and the peroxynitrite generating agent 3-morpholinosidnonimine (SIN-1, 250 microM). Preincubation of TPEN with equimolar Zn2+ but not Ca2+ or Mg2+ blocked the cytoprotective effect of the chelator. TPEN (10 microM) markedly reduced the peroxynitrite-induced decrease of mitochondrial transmembrane potential, secondary superoxide production and mitochondrial membrane damage, indicating that it acts proximal to mitochondrial alterations. Although TPEN (1 - 300 microM) did not scavenge peroxynitrite, it inhibited PARS activation in a dose-dependent manner. The cytoprotective effect of TPEN is only partly mediated via PARS inhibition, as the chelator also protected PARS-deficient thymocytes from peroxynitrite-induced death. While being cytoprotective against peroxynitrite-induced necrotic death, TPEN (10 microM), similar to other agents that inhibit PARS, enhanced apoptosis (at 5-6 h after exposure), as characterized by phosphatydilserine exposure, caspase activation and DNA fragmentation. In conclusion, the current data demonstrate that TPEN, most likely by zinc chelation, exerts protective effects against peroxynitrite-induced necrosis. Its effects are, in part, mediated by inhibition of PARS. (+info)
ZAP-70 protein promotes tyrosine phosphorylation of T cell receptor signaling motifs (ITAMs) in immature CD4(+)8(+) thymocytes with limiting p56(lck).
(44/8961)
As a result of interaction with epithelial cells in the thymic cortex, immature CD4(+)8(+) (double positive, DP) thymocytes express relatively few T cell receptors (TCRs) and contain diminished numbers of coreceptor-associated p56(lck) (lck) PTK molecules. As a result, TCR signal transduction in DP thymocytes is significantly impaired, despite its importance for repertoire selection. We report here that, in DP thymocytes, tyrosine phosphorylation of TCR signaling motifs (ITAMs) by lck, an early event in TCR signal transduction, is dependent upon ZAP-70 protein independent of ZAP-70's kinase activity. Furthermore, the dependence on ZAP-70 protein for ITAM phosphorylation diminishes as available lck increases. Importantly, ZAP-70's role in ITAM phosphorylation in DP thymocytes is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this study indicates that ZAP-70 protein augments ITAM phosphorylation in DP thymocytes and so compensates in part for the relative deficiency of coreceptor-associated lck. (+info)
Functional Fas expression in human thymic epithelial cells.
(45/8961)
Fas, a cell surface receptor, can induce apoptosis after cross-linking with its ligand. We report that Fas antigen is constitutively expressed in medullary epithelial cells of the human thymus. Expression is decreased in cultured thymic epithelial cells (TEC), similarly to HLA-DR antigen. TEC are resistant to anti-Fas-induced apoptosis after 4 days of primary culture, and this resistance is reversed by concomitant addition of cycloheximide. Cycloheximide also downregulated the expression of Fas-associated phosphatase-1, which has been found to inhibit Fas-induced apoptosis. This phosphatase could be involved in the resistance to Fas-induced apoptosis observed on day 4 of TEC culture. When TEC were subcultured after 10 to 13 days of primary culture, exposure to interleukin-1-beta, tumor necrosis factor-alpha, and interferon-gamma, alone or together, reinduced Fas mRNA and protein expression. In coculture with activated thymocytes, TEC also upregulated Fas protein expression. Cytokine-activated TEC became sensitive to apoptosis induced by an agonistic anti-Fas antibody. This apoptosis was inhibited by Z-VAD-fmk but not by Z-DEVD-fmk and DEVDase activity was slightly increased in Fas-stimulated TEC, suggesting that DEVDase activity is not sufficient to induce TEC apoptosis. Taken together, these data show that the Fas receptor is expressed in medullary epithelial cells of the human thymus and is able to induce apoptosis. (+info)
Somatostatin receptor subtype expression in cells of the rat immune system during adjuvant arthritis.
(46/8961)
Somatostatin is a neuropeptide that is widely distributed throughout the body. It acts as a neurohormone and a neurotransmitter and may also have an immunomodulatory role. The genes for five subtypes of somatostatin receptors (sst) have been cloned, suggesting that the diverse effects of the peptide might be mediated by different receptors. We are interested in studying the role of sst ininflammation, using an animal model. Because of the up-regulation of sst expression in inflamed joints in human rheumatoid arthritis, we chose rat adjuvant arthritis as an experimental model. In order to determine which of the sst subtypes might be important in immune modulation, subtype expression in leukocytes isolated from different lymphoid tissues of the rat was studied. Also, the expression levels of the most abundantly expressed sst mRNAs in leukocytes from spleen and blood were compared in rats with adjuvantarthritis and controls, using a semi-quantitative approach. Furthermore, the effect of systemic administration of a long-acting somatostatin analogue, octreotide, which binds selectively to sst subtypes 2 and 5 (sst2 and sst5), on the incidence and the severity of rat adjuvant arthritis, was studied. The main sst expressed in cells of the rat immune system, both resting and activated, were found to be sst3 and sst4. This contrasts with the human and murine situations, in which sst2 appears to be the main subtype expressed in the immune system. No quantitative differences in sst subtype mRNA levels in leukocytes from spleen and blood were found between rats with adjuvant arthritis and controls. Finally, no effect of systemic administration of octreotide on either the incidence or severity of adjuvant arthritis in Lewis rats was found. As octreotide binds selectively to sst2 and sst5, the absence of an immunomodulatory effect of this analogue in rat adjuvant arthritis corroborates our finding that these sst subtypes are not expressed in cells of the rat immune system. In conclusion, cells of the rat immune system appear to express a spectrum of sst (sst3 and sst4) different from that found in human granulomatous and autoimmune disease (mainly sst2). Therefore, the rat adjuvant arthritis model appears to be suitable only for studying the immunomodulatory effects of somatostatin analogues which have a high affinity for sst3 and sst4, but not for studying the immunomodulatory effects of octreotide, which has a high affinity only for sst2 and sst5. (+info)
Gastrointestinal epithelium is an early extrathymic site for increased prevalence of CD34(+) progenitor cells in contrast to the thymus during primary simian immunodeficiency virus infection.
(47/8961)
The objective of this study was to determine the effects of primary simian immunodeficiency virus (SIV) infection on the prevalence and phenotype of progenitor cells present in the gastrointestinal epithelia of SIV-infected rhesus macaques, a primate model for human immunodeficiency virus pathogenesis. The gastrointestinal epithelium was residence to progenitor cells expressing CD34 antigen, a subset of which also coexpressed Thy-1 and c-kit receptors, suggesting that the CD34(+) population in the intestine comprised a subpopulation of primitive precursors. Following experimental SIVmac251 infection, an early increase in the proportions of CD34(+) Thy-1(+) and CD34(+) c-kit+ progenitor cells was observed in the gastrointestinal epithelium. In contrast, the proportion of CD34(+) cells in the thymus declined during primary SIV infection, which was characterized by a decrease in the frequency of CD34(+) Thy-1(+) progenitor cells. A severe depletion in the frequency of CD4-committed CD34(+) progenitors was observed in the gastrointestinal epithelium 2 weeks after SIV infection which persisted even 4 weeks after infection. A coincident increase in the frequency of CD8- committed CD34(+) progenitor cells was observed during primary SIV infection. These results indicate that in contrast to the primary lymphoid organs such as the thymus, the gastrointestinal epithelium may be an early extrathymic site for the increased prevalence of both primitive and committed CD34(+) progenitor cells. The gastrointestinal epithelium may potentially play an important role in maintaining T-cell homeostasis in the intestinal mucosa during primary SIV infection. (+info)
Expression of the lymphotoxin beta receptor on follicular stromal cells in human lymphoid tissues.
(48/8961)
The lymphotoxin beta receptor (LTbetaR), and its ligand, LTalpha1beta2, have been proposed to play a key role in the development and organization of lymphoid tissues. The LTbetaR is expressed on a variety of human primary and transformed cells, but strikingly absent on T or B lymphocytes and primary monocytes or peripheral dendritic cells, although LTbetaR is detected on some myeloid leukemic lines. In the developing thymus LTbetaR is prominent along the trabeculae and into the medulla upto corticomedullary junction. In the spleen, LTbetaR is prominently expressed by cells in the red pulp and along the borders of red and white pulp which colocalizes with reticular stromal cells. The LTbetaR is expressed on a human follicular dendritic cell line, FDC-1, and signals expression of CD54 when ligated with the LTalpha1beta2 complex. These results support the concept that directional interactions between LTalpha1beta2 bearing lymphocytes and LTbetaR bearing stromal cells are involved in the organization of lymphoid tissue. (+info)