Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A fully automated instrument for multiple simultaneous peptide synthesis was constructed to provide large numbers of peptides for immunological research. The synthesis is performed in a flow-through mode with the conventional solid supports contained in 48 individual reaction columns. The instrument is based on a commercial autosampler equipped with a motor-driven syringe for accurate delivery of reagents and a robot arm carrying a dispenser needle. Dedicated software was developed to compile overlapping peptides from a given protein sequence and to control all functions of the robot. In situ activation by BOP was chosen as the optimized chemistry protocol. The peptides are cleaved from the resin in the reactors used for synthesis, thus minimizing handling. Performance of the instrument was demonstrated by synthesis of overlapping 14-mer peptides derived from the sequence of HIV reversed transcriptase. A second mode of operation allows the synthesis to be carried out on the surface of polyethylene pins. Peptides derived from the sequence of human TNF were synthesized using this method and used to characterize antibodies raised against the intact protein.
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PMID:Automated multiple peptide synthesis. 128 42

To extent our knowledge on the cytokines possibly involved in the pathophysiology of B-cell chronic lymphocytic leukemia (B-CLL), the mRNA expression of a panel of 10 cytokines was investigated on purified B-CLL cells using a reverse-transcriptase polymerase chain reaction method. Whereas negative RT-PCR signals were recorded for interleukin-1 alpha (IL-1 alpha), IL-2, IL-3, IL-4, IL-5, IL-7, tumor necrosis factor beta (TNF beta), and granulocyte-macrophage colony-stimulating factor, we detected the expression of IL-1 beta, IL-6 and TNF alpha. Furthermore, the constitutive expression of IL-8 mRNA was observed in all 17 B-CLL samples analyzed. mRNA expression was associated with the capacity of the leukemic cells to release IL-8 both constitutively (4.6 +/- 8.1 SD ng/mL) and, to a further extent, after stimulation (14.5 +/- 19.4 ng/mL). The circulating levels of IL-8 were also evaluated in 12 untreated B-CLL sera samples and the overall mean level was significantly higher (P < .01) than in normal sera. In addition, supernatants of purified B-CLL cells cultured in the presence of 12-O-tetradecanoylphorbol-13-acetate showed chemotactic activity towards neutrophils; this activity was neutralized in the presence of an anti-IL-8 antiserum. The mRNA for IL-8 was absent in five B-cell preparations from hairy cell leukemia cases and in four B-cell lines. Normal tonsil CD5+ B cells showed a low expression of IL-8 mRNA only in two of the nine preparations tested and the overall quantity of IL-8 released by these cells after 3 days' incubation was significantly lower compared with that released by B-CLL cells (0.4 +/- 0.3 and 1.6 +/- 0.9 ng/mL under basal and stimulated conditions, respectively). These findings point to an involvement of a member of the proinflammatory chemokine supergene family in human CD5+ B lymphocytes. The different IL-8 behavior observed between B-CLL cells and their normal counterpart is likely to reflect an activation state of the leukemic population.
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PMID:Cytokine gene expression in B-cell chronic lymphocytic leukemia: evidence of constitutive interleukin-8 (IL-8) mRNA expression and secretion of biologically active IL-8 protein. 751 9

This study describes the distinct roles of B7 and LFA-3 in the regulation of T cell responses. Activation of CD4+ T cells with Chinese hamster ovary (CHO)-DR4/B7 and CHO-DR4/LFA-3 cells that present the superantigen staphylococcal enterotoxin A resulted in significant T cell proliferation and substantial production of TNF and IFN-gamma. Strong IL-2 production was recorded in B7-costimulated, but not LFA-3-costimulated, cultures. The presence of B7 induced a more vigorous and prolonged proliferative T cell response compared with LFA-3 costimulation. In contrast, LFA-3 was more efficient than B7 in mediating cell adhesion of CD4+ T cells. Costimulation with the CHO-DR4/B7/LFA-3 triple transfectant resulted in enhanced cell adhesion, proliferation, and cytokine production compared with either DR4/B7 or DR4/LFA-3 alone. Optimal production of IL-2 by naive and memory CD4+ T cells was seen only when cells were costimulated with B7, whereas IFN-gamma production was induced in memory cells by both LFA-3 and B7. The Jurkat T cell line responded to CHO-DR4/B7/LFA-3 in a manner similar to peripheral blood CD4+ T cells. Reverse transcriptase-PCR analysis of Jurkat cells stimulated with staphylococcal enterotoxin E and the different CHO transfectants revealed that the cooperative effect of B7 and LFA-3 on IL-2 production was also seen at the mRNA level. The large amounts of IL-2 produced by B7 costimulation indicate a paracrine function of the B7/CD28 pathway, whereas the LFA-3/CD2 pathway provides strong adhesion and may facilitate autocrine T cell expansion. Combined expression of the B7 and LFA-3 molecules seems to provide an optimal Ag-presenting function that ensures strong adhesion and optimal signal transduction.
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PMID:Costimulation of human CD4+ T lymphocytes with B7 and lymphocyte function-associated antigen-3 results in distinct cell activation profiles. 752 63

Tumor necrosis factor alpha (TNF alpha) has previously been immunolocalized within mouse oocytes. Our first objective was to examine TNF alpha immunolocalization in ovaries of adult, fetal, and neonatal rats. Our second objective was to examine TNF alpha mRNA in ovaries by Northern blot analysis and in oocytes by reverse-transcriptase polymerase chain reaction (RT-PCR). Our final objective was to determine whether oocytes contained bioactive TNF alpha. Ovaries and oviducts were collected throughout the estrous cycle in adult rats, fetal ovaries were obtained 1 day before expected delivery, and neonatal ovaries were collected 2 days after birth. TNF alpha was localized in tissues by a biotin-avidin immunocytochemical procedure. Immunocytochemistry in adult ovaries showed that the ooplasm of the oocyte was the primary site of TNF alpha localization within the follicle. Immunostaining was present in all oocytes in the adult, including ovulated oocytes within the oviduct. Oocytic TNF alpha immunostaining was also present within oocytes in the neonate; however, fetal oocytes did not contain immunoreactive TNF alpha. Northern blots showed that ovaries in the adult, neonate, and fetus all contained TNF alpha mRNA. RT-PCR analysis of oocytes collected from preovulatory follicles generated a cDNA band of 500 bp, corresponding to the predicted size for amplified TNF alpha cDNA. Subsequent Southern blot analysis showed that the 500-bp band hybridized to the TNF alpha probe, indicating that preovulatory oocytes contain TNF alpha mRNA. Preovulatory oocytes were used in TNF alpha cytotoxicity assays with L929 cells. Oocytes contained TNF alpha bioactivity that was similar to that of recombinant murine TNF alpha in the bioassay. Our results provide evidence for the identification of immunoreactive and bioactive TNF alpha within oocytes in the rat, which is further supported by the presence of TNF alpha mRNA within the oocyte. These studies also indicate that TNF alpha may appear in the oocyte around the time of birth.
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PMID:Oocytic tumor necrosis factor alpha: localization in the neonatal ovary and throughout follicular development in the adult rat. 752 75

Increasing evidence suggests that regulation of transcription at the level of elongation or processivity may be an important mechanism governing expression of eukaryotic genes. In this study we compared LPS- and IFN-gamma-induced transcription of the TNF-alpha gene in two murine macrophage cell lines, ANA1 and Pu5-1.8. Our data from nuclear run-on analysis indicate that in ANA-1 cells TNF-alpha expression is regulated at the transcriptional level, as previously found in primary macrophages. In contrast, in Pu5-1.8 cells the TNF gene is constitutively transcribed. Using several short probes spanning the TNF gene we find that in ANA-1 cells transcription can be initiated before activation, but such transcripts have low processivity and are prematurely terminated or arrested within the gene. Induction with LPS alone or with LPS plus IFN-gamma results both in increased transcription initiation, and in the increased processivity of these transcripts. In Pu 5-1.8 cells neither type of transcriptional regulation of the TNF gene is observed. Our results indicate that the TNF gene is preactivated in ANA-1 cells, and RNA polymerase is allowed to initiate transcription, but due to the low processivity of the transcripts very little mRNA is formed. After LPS stimulation the TNF gene is maximally activated both by increased initiation and by higher processivity of the transcript, and each of these components of activation do not require a new protein synthesis. Our findings are consistent with a recently proposed model that the same transcriptional activators contribute to both initiation and processivity of transcription. In the case of LPS and LPS+IFN-gamma stimulation of macrophages, inducible members of NF-kappa B/Rel family are likely candidate transcriptional activators.
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PMID:Lipopolysaccharide-induced expression of TNF-alpha gene in the macrophage cell line ANA-1 is regulated at the level of transcription processivity. 760 44

Graft-versus-host disease (GVHD) is one of the major complications which should be resolved to improve the survival rates in allogeneic bone marrow transplantation (BMT). Recently, several cytokines have been identified, suggesting that they form a cytokine network and play an important role in immune system and hematopoiesis. Among several cytokines, it has been reported that tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) are mainly involved in GVHD. In the present report, we analyzed the role of cytokines in GVHD. When we measured serum cytokine levels, IL-6, interferon gamma (IFN gamma), and TNF alpha levels were increased prior to the onset of acute GVHD. For chronic GVHD, a similar pattern of cytokine increment was observed. Interestingly, these cytokines appeared to interact synergistically to induce clinical GVHD, suggesting that none of those cytokines does not function solely. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that increased IL-1 beta mRNA expression was also observed in acute GVHD in addition to increased IL-6 and TNF alpha mRNA expressions. Unexpectedly, no increased IL-2 levels were observed in both assays. In hyperacute GVHD, only IL-6 level was increased. However, in vivo administration of IL-6 into allogeneic bone marrow chimeras did not induce severe GVHD. Therefore, some other factors also appeared to be involved in inducing hyperacute GVHD. Furthermore, it is important to consider the role of inhibitory cytokines such as transforming growth factor beta (TGF beta) or IL-10.
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PMID:Cytokines involved in graft-versus-host disease. 770 47

In this study, we showed that systemic administration of SSG, a highly branched soluble (1-->3)-beta-D-glucan obtained from Sclerotinia sclerotiorum, induced immunological changes in the alveolar space of mice in vivo, assessed by analysing some immune mediators in bronchoalveolar lavage (BAL) fluid. A single i.v. administration of SSG (250 micrograms/mouse) induced a rapid but transient leakage of the serum components, IgG and fibronectin, into the alveolar space. This was apparent 12 h post-administration and reached a peak on day 2. Similar kinetic changes were found for lysosomal enzyme activities and interferon gamma (IFN gamma) concentrations in BAL which are markers of activated alveolar macrophages (AMs) or pulmonary T cells. BAL prepared from SSG-treated mice stimulated lysosomal enzyme release from AMs in vitro. However, SSG did not provoke the chronic accumulation of serum proteins in alveoli and did not induce the release of detectable amounts of nitric oxide and the inflammatory cytokines, IL-1, IL-6 and TNF alpha, into BAL. However, their mRNAs were detected in lung tissue using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique. Similar results were observed for multiple i.v. administration (250 micrograms, once a day for 10 consecutive days), and there were a little differences between single and multiple administration. In summary, systemic administration of SSG induces immune responses, including activation of AMs and lymphocytes, but does not provoke chronic inflammation in the alveolar space when administered either as single or multiple doses. This finding is very important for the clinical application of SSG in immunocompromised hosts as a biological response modifier (BRM) without toxic-side effects on lung tissue.
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PMID:Changes in immune mediators in mouse lung produced by administration of soluble (1-->3)-beta-D-glucan. 792 Apr 19

Rapid and severe rejection remains a major obstacle to successful clinical intestinal transplantation (IT). The aggressive nature of rejection in IT has been attributed to the increased massive immune stimulus provided by large numbers of resident lymphocytes, antigen presentation capacity of enterocytes, and graft damage mediated by luminal microflora. Early small bowel expression of proinflammatory cytokines, MHC class II, and adhesion molecules may also promote IT rejection, but the lack of a mouse model has hampered extensive studies of gene expression in IT. Using a recently developed surgical model, we examined the temporal pattern of gene expression in CB6F1 (H-2b/d) vascularized, heterotopic intestinal allografts transplanted into BALB/c (H-2d) mice. Although histological evidence of rejection was not present until day 7 in allografts, Northern blot analysis demonstrated increases in TNF alpha gene transcripts as early as day 3, followed by the expression of IL-1 beta, intercellular adhesion molecule-1, and MHC class II by day 5. Using reverse-transcriptase polymerase chain reaction, IFN-gamma was detected in allografts by day 3 and persisted to day 10. In contrast, IL-2, IL-4, IL-5, and IL-6 mRNA transcripts peaked by day 5 and then decreased, suggesting that both Th1 and Th2 subsets are involved in the rejection of unmodified small bowel allografts. The early and progressive expression of TNF alpha and IL-1 beta as well as IFN-gamma, intercellular adhesion molecule-1, and MHC class II in IT rejection may contribute to the difficulty in controlling IT rejection with present immunosuppression.
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PMID:Altered gene expression of cytokine, ICAM-1, and class II molecules precedes mouse intestinal allograft rejection. 794 Jul 16

Human peritoneal mesothelial cells (HPMC) respond to tumor necrosis factor alpha (TNF alpha) by releasing various cytokines that may activate the endothelium and induce recruitment of leukocytes during peristonitis. We characterized the receptors for TNF on HPMC to elucidate their functions in peritonitis. Scatchard analysis determined the presence of 70 x 10(3) TNF receptors/cell with a kDa of 0.44 nM. TNF receptor 1 (TNF-R1, p55) and TNF-R2 (p75) mRNA were demonstrated by reverse-transcriptase-PCR (RT-PCR). TNF-R1 protein was solely detected by flow cytometry (FCM). Interleukin-1 alpha (IL-1 alpha) induced down-regulation of TNF-R1. This was concomitant with accumulation of soluble TNF-R1 (sTNF-R1) detected by specific ELISA. LPS had a lower TNF-R1-shedding activity while TNF alpha did not induce shedding. The IL-1-induced-sTNF-R1-shedding was suppressed by the protein-kinase-A (PKA) inhibitor, H-8, or by H-7, the inhibitor of both PKC and PKA, but not by the specific PKC inhibitor GF. These experiments suggest a role for PKA in the IL-1-shedding signal. No change in TNF-R1 mRNA levels was observed after IL-1 alpha or TNF alpha stimulation while TNF-R2 (p75) mRNA basal levels transiently increased three to fivefold, reaching a peak after four hours followed by an accumulation of sTNF-R2 in the supernatant. Our data suggest that the main receptor expressed on HPMC is TNF-R1. Down-regulation and shedding of TNF-R1 induced by IL-1, and the transient expression of TNF-R2 induced by IL-1 and TNF, may regulate the responses to TNF by HPMC. These results may be important in understanding the inflammatory process of peritonitis were TNF plays a major role.
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PMID:TNF-receptors on human peritoneal mesothelial cells: regulation of receptor levels and shedding by IL-1 alpha and TNF alpha. 880 91

Mononuclear phagocytes are important regulators of normal immune, inflammatory, and fibrotic responses. These functions are mediated through the production of several cytokines, including tumor necrosis factor-alpha (TNF-alpha), which regulate the activity of inflammatory and tissue structural cells such as fibroblasts. It is increasingly evident that fibroblasts are also capable of releasing a number of cytokines and soluble factors that can, in turn, interact with monocytes and thereby modulate the inflammatory process. In this study we provide evidence that human lung fibroblasts, through the release of soluble factors such as prostaglandin E2 (PGE2), inhibit both TNF messenger ribonucleic acid (mRNA) accumulation and TNF-alpha protein release by lipopolysaccharide (LPS)-activated human peripheral blood monocytes (PBM). Reverse transcriptase-polymerase chain reaction (RT-PCR) results showed that fibroblast-conditioned medium (FCM) caused a 50% reduction of the TNF-alpha transcript accumulation in LPS-stimulated monocytes. Furthermore, FCM induced a significant decrease in the release of TNF-alpha by LPS-activated PBM. This effect was dependent on the concentration of the FCM and the number of fibroblasts producing it. The maximal effect was seen with monocytes cultured in 100% FCM produced by 2 x 10(6) fibroblasts. This indicated that one or possibly more soluble factors released by fibroblasts were responsible for the effect. Considering that exogenous PGE2 can inhibit TNF-alpha production by PBM, and that fibroblasts are a good source of PGE2, we determined the content of PGE2 in the FCM used in our experiments. We found a good correlation (r = 0.949) between the amount of PGE2 produced by fibroblasts and the degree of TNF-alpha inhibition exerted. In addition, the inhibitory effect of FCM was mimicked by the addition to PBM cultures of exogenous PGE2 in amounts similar to those spontaneously released by fibroblasts in FCM. All of these data suggest a molecular and cellular interaction between PBM and fibroblasts that could contribute to those modulatory mechanisms involved in the self-limitation of the fibrotic process.
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PMID:Human lung fibroblasts inhibit tumor necrosis factor-alpha production by LPS-activated monocytes. 887 79


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