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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.
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PMID:Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. 215 62

The aim of this study was to investigate mechanisms of anti-tumour activity and necrosis induced by combinations of tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma). In a breast cancer xenograft model, locally injected recombinant human TNF-alpha arrested growth of established tumours in the absence of overt necrosis. Macroscopic necrosis occurred when rat IFN-gamma, which had no anti-tumour activity as a single agent, was given systemically. Treatment with TNF-alpha and IFN-gamma caused focal engorgement of tumour capillaries with erythrocytes, intravascular recruitment of polymorphonuclear cells and platelet adherence to the tumour vascular endothelium 4 h after the combined treatment. This was followed by destruction of tumour vascular endothelium and both necrosis and apoptosis of tumour cells. Concomitant with these changes, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the increase of stromal (murine) mRNA levels for TNF-alpha, TNF receptor 55 kDa, TNF receptor 75 kDa, intracellular adhesion molecule 1, vascular cell adhesion molecule 1, P-selectin and interleukin 6 (IL-6). Thus, the effect of the combined TNF-alpha and IFN-gamma therapy involved the selective destruction of the tumour vasculature, death of tumour cells and increased expression of a series of stromal cytokines, cytokine receptors and adhesion molecules, which could be implicated in the observed events.
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PMID:Changes in endogenous cytokines, adhesion molecules and platelets during cytokine-induced tumour necrosis. 757 63

Continuous ambulatory peritoneal dialysis is known to interfere with the normal inflammatory responses of macrophages in the peritoneal cavity. Commercial peritoneal dialysis solution (CDS) has been shown to inhibit tumor necrosis factor alpha (TNF alpha) release from LPS stimulated peritoneal macrophages. To further dissect the mechanism of this inhibition, we used human blood-derived macrophages or the murine macrophage cell line, P388D1, that were stimulated with LPS after pretreatment with CDS, and tested TNF alpha mRNA levels by Northern hybridization or reverse transcriptase polymerase chain reaction. Time course studies demonstrated that CDS lowered TNF alpha mRNA levels within 15 minutes of pretreatment of cells. In addition, the CDS inhibited DNA binding activity of NF-kappa B that is probably involved in regulation of LPS-mediated transcriptional activation of the TNF alpha gene. Inhibition was dependent on both the low pH and the lactate in the CDS, but was independent of the osmolarity or glucose concentration. The rate of catabolism of TNF alpha mRNA was not affected by CDS as demonstrated by actinomycin D chase experiments. Thus, impairment of LPS-stimulated macrophage function by CDS is associated with low TNF alpha mRNA which may be the result of the low activity of NF-kappa B. Since NF-kappa B is involved in transcription regulation of a large number of "early activation" genes, CDS may interfere with the production of additional immunomodulatory proteins that are encoded by genes possessing NF-kappa B site(s) in their promoter region.
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PMID:Commercial dialysate inhibits TNF alpha mRNA expression and NF-kappa B DNA-binding activity in LPS-stimulated macrophages. 764 22

Administration of interleukin-2 (IL-2) leads to pulmonary vascular leak. This form of pulmonary edema has previously been postulated to be due to the in vivo induction of tumor necrosis factor-alpha (TNF-alpha). To determine whether TNF-alpha plays a role in IL-2-induced pulmonary vascular leak, we performed in situ hybridization of lung sections and reverse transcriptase-polymerase chain reaction of bronchoalveolar lavage macrophages from IL-2-challenged mice. The results confirm an in situ upregulation of TNF-alpha mRNA expression in the lungs associated with vascular leak. In addition, a significant increase in TNF-alpha protein production was found in the lung following IL-2 administration, as measured by TNF-alpha-specific ELISA of lung supernatants (P = 0.028). Intravenous administration of a soluble TNF receptor significantly diminished IL-2-induced pulmonary vascular leak (P = 0.006). These findings confirm a central role for TNF-alpha in mediating the pulmonary vascular leak associated with IL-2 toxicity.
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PMID:Tumor necrosis factor-alpha plays a central role in interleukin-2-induced pulmonary vascular leak and lymphocyte accumulation. 803 44

In previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR) and that its expression the important for TNF alpha-mediated upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes. Because factors that either reduce or enhance TNFR expression are likely to have a major impact on the biological effects of TNF alpha on keratinocytes, these studies were conducted to determine the factors that regulate its expression on keratinocytes. Using reverse transcriptase polymerase chain reaction, human keratinocytes were shown to lack 75-kD TNFR expression, indicating that TNF responsiveness of human keratinocytes critically depended on regulation of 55-kD TNFR expression. Human keratinocyte 55-kD TNFR surface and mRNA expression was found to be regulated in vitro by recombinant human (rh) TNF alpha. Stimulation of keratinocytes with rhTNF alpha initially decreased, but later increased, 55-kD TNFR surface expression. This biphasic modulation of 55-kD TNFR surface expression was associated with concomitant changes in 55-kD TNFR mRNA expression. Ultraviolet B (UVB) radiation, a well-known inducer of synthesis and secretion of TNF alpha by human keratinocytes, was found to mimic TNF alpha-induced modulation of 55-kD TNFR surface and mRNA expression via a TNF alpha-mediated autocrine regulatory mechanism. Production of soluble 55-kD TNFR by human keratinocytes remained unaffected by TNF alpha stimulation or UVB irradiation. These studies provide clear evidence that membrane expression of the human 55-kD TNFR may be regulated in human keratinocytes by the ligand itself: TNF alpha. Since in previous studies UVB irradiation transiently inhibited TNF alpha-induced human keratinocyte ICAM-1 expression, it is proposed that UVB radiation-induced biphasic modulation of human keratinocyte 55-kD TNFR expression may affect the capacity of these cells to respond to TNF alpha.
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PMID:The 55-kD tumor necrosis factor receptor on human keratinocytes is regulated by tumor necrosis factor-alpha and by ultraviolet B radiation. 839 91

Although tumor necrosis factor-alpha (TNF) is constitutively expressed in human and mouse thymus, the effects of TNF on thymocyte proliferation, differentiation and survival suggest that its influence in the thymus is complex. To determine if this complexity results from changes in the expression of the two TNF receptors during thymocyte differentiation, we examined the expression of the 55 kDa TNF receptor (TNF-R1) and the 75 kDa TNF receptor (TNF-R2) on postnatal human thymocytes. Both TNF-R1 and TNF-R2 mRNA were found in resting human thymocytes by reverse transcriptase-polymerase chain reaction (RT-PCR). Using mAb which specifically react with the respective TNF receptors and a highly sensitive, three-step method of immunofluorescence, cell surface TNF-R1 was detected on the vast majority of thymocytes. In contrast, detectable cell surface TNF-R2 was present on a mean of only 12.9% of thymocytes. TNF conjugated to phycoerythrin (TNF-PE) also reacted with a small population of thymocytes and was found to specifically block binding of the TNF-R2 mAb and not the TNF-R1 mAb, implicating preferential binding of TNF-PE to TNF-R2. Using dual-color immunofluorescence with TNF-PE we found that the population of cells which express TNF-R2 also express high levels of the TCR alpha, beta-CD3 complex, CD4 or CD8, and IL-2 receptor alpha chain. Thus, immature (TCRneg/low) thymocytes express TNF-R1 while mature (TCRhigh) thymocytes can also express TNF-R2. This differential expression of TNF receptors provides a mechanism for distinct effects of TNF on immature vs. mature thymocytes.
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PMID:Characterization of TNF receptors on human thymocytes. 852 4

The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) is readily detected after human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages in vitro and is present in plasma and tissues of patients with AIDS. Previous studies have shown that human recombinant TNFalpha (hrTNFalpha) enhances HIV replication in both chronically infected promonocytic and T-lymphoid cell lines in vitro. We report here that in contrast to untreated tissue culture-differentiated macrophages (TCDM), in which the proviral long terminal repeat (LTR) could be detected as soon as 8 h postinfection by a PCR assay, TCDM pretreatment for 3 days by hrTNFalpha markedly delayed its appearance until 72 h after infection with the HIV-1 Ada monocytotropic strain. Moreover the inhibition of formation of the proviral LTR in HIV-1-infected TCDM was directly proportional to the concentration of hrTNFalpha used. To determine if the inhibition of LTR formation results from blockade of viral entry, we performed a reverse transcription PCR assay to detect intracellular genomic viral RNA as early as 2 h after infection. Pretreatment of primary TCDM by hrTNFalpha for 3 days and even for only 2 h inhibits 75% of the viral entry into the cells. The inhibition of viral entry by hrTNFalpha was totally abolished by the use of anti-human TNFalpha monoclonal antibody. By using TNFalpha mutants specific for each human TNFalpha receptor, we showed that the inhibition of HIV-1 entry into TCDM was mediated not through the 55-kDa TNF receptor but through the 75-kDa TNF receptor. Although prolonged (1 to 5 days) TNFalpha treatment can downregulate CD4 expression in primary human TCDM, surface CD4 levels were not reduced by 2 h of treatment and was therefore not a limiting step for HIV-1 entry. In contrast to the inhibition of viral entry into primary TCDM, pretreatment with hrTNFalpha did not modify HIV-1 entry into phytohemagglutinin A-activated peripheral blood lymphocytes. TNFalpha-pretreatment inhibited HIV-1 replication in primary TCDM but not in phytohemagglutinin A-activated peripheral blood lymphocytes as assessed by decreased reverse transcriptase activity in culture supernatants. These results demonstrate that TNFalpha is able to enhance host cellular resistance to HIV-1 infection and that selective inhibition of HIV-1 entry into primary TCDM by TNFalpha involves the 75-kDa TNF receptor but not the 55-kDa TNF receptor.
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PMID:Tumor necrosis factor alpha inhibits entry of human immunodeficiency virus type 1 into primary human macrophages: a selective role for the 75-kilodalton receptor. 889 57

We report in this study that repeated tumor necrosis factor alpha (TNF-alpha) pretreatment, starting before and continued after infection by human immunodeficiency virus type 1 (HIV-1), inhibits replication of the monocytotropic Ada strain in primary tissue culture-differentiated macrophages (TCDM), as assessed by sixfold lower levels of reverse transcriptase (RT) activity than that in untreated cells and absence of syncytium formation in TCDM cultures. In order to determine the pathways involved in inhibition of HIV-1 replication in primary TCDM pretreated with TNF-alpha, we tested TNF-alpha mutants T55 and T75, which recognize either the 55-kDa (TNF-R1) or the 75-kDa (TNF-R2) TNF receptor, respectively. Pretreatment of TCDM with the T75 mutant decreased the RT activity compared with that in untreated infected control cells fivefold and almost totally inhibited syncytium formation. In contrast, when TCDM were pretreated with the T55 mutant alone, syncytia were observed and RT activity was decreased about one-half. These results suggest that the inhibition of HIV-1 replication in TCDM pretreated with TNF-alpha might be mediated mainly through the 75-kDa TNF receptor (TNF-R2) rather than through the 55-kDa receptor (TNF-R1). Inhibition of HIV-1 replication in TCDM was observed with both T75 mutant pretreatment and posttreatment, starting at 1 h or 3 days after infection, whereas posttreatment with the T55 mutant, but not pretreatment, stimulated HIV-1 growth in primary TCDM. Both pre- and posttreatment with TNF-alpha inhibited HIV-1 replication in primary TCDM. The stimulation of HIV-1 replication by TNF-alpha in a chronically infected promonocytic cell line, U1, which contains two copies of integrated provirus, was mediated through the 55-kDa TNF-R1 alone and not through the 75-kDa TNF-R2. These results demonstrate that the 55-kDa TNF-R1 is involved in postintegration stimulation of HIV-1 while the 75-kDa TNF-R2 is involved in the inhibition of an early step of the viral life cycle in primary human TCDM.
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PMID:55- and 75-kilodalton tumor necrosis factor receptors mediate distinct actions in regard to human immunodeficiency virus type 1 replication in primary human macrophages. 909 99

Experimental and clinical studies have yet to determine the extent to which methotrexate (MTX) or cyclosporine A (CSA) treatment alone affects the expression in vivo of tumour necrosis factor-alpha (TNF alpha), a cytokine produced primarily by macrophages and believed to be directly involved in the pathogenesis of cardiac allograft rejection. In light of previously published findings from this laboratory examining the effects of combination CSA/MTX treatment, these studies were designed to examine the individual effects of CSA and MTX upon TNF alpha gene expression post-transplant (post-tx) using an accessory cervical heart transplant model in the rat. These studies have focused on a highly sensitive method with which to detect changes in gene expression, reverse transcriptase-polymerase chain reaction (RT-PCR) methodology and enzyme-linked immunosorbent assay (ELISA) assessment of transplant TNF alpha protein levels. Both techniques consistently demonstrated biphasic TNF alpha expression in cardiac transplant tissue obtained from untreated allograft recipients during the first week post-tx in contrast to isograft recipients. Previously demonstrated in combination to prolong cardiac allograft survival, low-dose MTX and low-dose CSA were each evaluated alone in the course of these studies to determine their impact on TNF alpha gene expression. While TNF alpha levels were up-regulated during untreated allograft rejection, both TNF alpha RNA and protein were significantly diminished with low-dose combination CSA/MTX treatment, with CSA alone, but not significantly with MTX treatment alone. In conclusion, TNF alpha gene expression in untreated allografts is consistent with the hypothesis that TNF alpha may play a role in events leading to allograft rejection. Results of these studies indicate that TNF alpha levels are significantly regulated in vivo by CSA but not by MTX treatment. These studies further implicate a role for low-dose MTX in mediating statistically significant immunosuppressive effects in conjunction with low-dose CSA.
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PMID:Effects of cyclosporine A and methotrexate on induction of tumour necrosis factor-alpha in rat cardiac allografts. 910 31

The present study was conducted to examine the expression of tumor necrosis factor-alpha (TNF-alpha) and its receptors (types I and II, designated TNFR-I and TNFR-II, respectively) in human oocytes and cumulus cells at the mRNA and protein levels. mRNA expression was investigated using a reverse transcriptase-polymerase chain reaction (PCR)/Southern hybridization procedure. DNA-free RNA was isolated from the oocytes/cumulus cells, reverse-transcribed, and PCR-amplified using specific oligonucleotide primers based upon genomic/cDNA sequences. The expected bands of 303 bp and 513 bp were observed in oocytes and cumulus cells using primers based on genomic/cDNA sequences of TNF-alpha and TNFR-II, respectively, that hybridized with specific cDNA probes in Southern blot hybridization procedure. The expected band of 368 bp was not observed in oocytes and cumulus cells using primers based on the TNFR-I cDNA sequence. Similar results were observed for expression at the protein level, as seen by the immunoreactivity of the specific antibodies with the paraformaldehyde-fixed oocytes and cumulus cells in the indirect immunofluorescence technique (IFT). These results indicate that human oocytes and cumulus cells express TNF-alpha and its receptor type II (TNFR-II), and not type I (TNFR-I), both at the mRNA and protein levels. These findings provide further evidence and substantiate the proposed physiologic role of TNF-alpha in ovarian function, and may lead to clinical applications in in vitro fertilization programs and in diagnosis and treatment of infertility in women, especially in cases attributed to ovarian dysfunction.
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PMID:Expression of tumor necrosis factor-alpha and its receptors type I and type II in human oocytes. 913 12


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