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)

Study of early and transient response gene expression is important for understanding the mechanisms of response to growth stimuli and exogenous agents such as microbes, stress, and radiation. Many of the cytokines, proto-oncogenes, and other transiently expressed gene products are encoded by mRNAs that contain AU-rich elements (AREs) in their 3' untranslated regions (UTRs). In this article, we describe an approach to selectively synthesize ARE-containing cDNA (ARE-cDNA) using an innovative combination of culture treatment, thermostabilization of reverse transcriptase (RT) by the disaccharide trehalose, and use of optimized ARE-specific oligomers. The monocytic cell line, THP-1, was treated with cycloheximide and endotoxin to enrich for ARE-mediated gene expression followed by the RT procedure. Selection of ARE-cDNA with simultaneous suppression of abundant cDNA was made possible using the procedure as monitored by the preferential expression of IL-8, an ARE-cDNA molecule, over the abundant housekeeping cDNA, beta-actin. The use of trehalose dramatically reversed cDNA abundance, resulting in almost complete suppression of housekeeping cDNA. Finally, construction of specialized ARE-cDNA libraries confirmed the selectivity of ARE-cDNAs and the presence of rare genes. The ability to reverse the abundance of housekeeping and other highly expressed genes toward ARE genes facilitates the discovery and study of rare early response and transiently expressed genes.
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PMID:Selection of AU-rich transiently expressed sequences: reversal of cDNA abundance. 1503 83

Induction of Mycobacterium avium proteins labelled with [35S]methionine and mRNAs upon infection of the human macrophage cell line THP-1 was investigated by two-dimensional gel electrophoresis-mass spectrometry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. M. avium overexpressed proteins within the macrophages that are involved in fatty acids metabolism (FadE2, FixA), cell wall synthesis (KasA), and protein synthesis (EF-tu). The correlation of differential protein and mRNA expression varied between good and no correlation. Overall, these four proteins may be involved in the adaptation and survival of M. avium within human macrophages.
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PMID:Induction of Mycobacterium avium proteins upon infection of human macrophages. 1537 97

Genes induced or suppressed by oxidized low-density lipoproteins (oxLDL) in human monocytic THP-1 cells were searched using the differential display reverse transcriptase polymerase chain reaction. One of the differentially expressed (up-regulated) cDNA fragments was found to contain sequences corresponding to monocyte chemotactic protein-3 (MCP-3). The stimulatory effect of the oxLDL on the expression of MCP-3 mRNA was both time- and dose-dependent. Treatment with GF109203X and genistein, inhibitors of protein kinase C and tyrosine kinase, respectively, had no effect on the induction of MCP-3 mRNA by oxLDL, while treatment with cycloheximide inhibited the induction. The induction was reproduced by the lipid components in oxLDL such as 9-HODE and 13-HODE, which are known to activate the peroxisome proliferator-activated receptor gamma (PPARgamma). Introduction of an endogenous PPARgamma ligand, 15d-PGJ2, in the culture of THP-1 cells resulted in the induction of MCP-3 gene expression. Furthermore, analyses of human atherosclerotic plaques revealed that the expressional pattern of MCP-3 in the regions of neointimal and necrotic core overlapped with that of PPARgamma. These results suggest that oxLDL delivers its signal for MCP-3 expression via PPARgamma, which may be further related to the atherogenesis.
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PMID:Oxidized low-density lipoproteins may induce expression of monocyte chemotactic protein-3 in atherosclerotic plaques. 1538 Oct 85

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NAT1 (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NAT1 is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5' non-coding region of NAT1. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NAT1 expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforms were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NAT1 transcripts may contribute to the variation in NAT1 activity in vivo.
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PMID:Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities. 1548 85

Low-density lipoprotein (LDL) in patients with diabetes is subject to modification by both oxidation and glycation. In contrast to oxidized LDL, the biological effects of glycoxidized LDL have not been well characterised. In this study, the effects of oxidized, glycated, glycoxidized and oxidized LDL on scavenger receptor gene expressions, and the induction of oxidized LDL uptake and cholesteryl ester accumulation in THP-1 macrophages were compared. Modified LDL was incubated with THP-1 macrophages. Gene expression of scavenger receptor class A (SR-A), CD36 and scavenger receptor class B type I (SR-BI) was determined by quantitative reverse transcriptase PCR (RT-PCR). Glycoxidized LDL was able to significantly induce SR-A and CD36 expression by 3- and 4.5-fold, respectively, in macrophages whereas SR-BI expression was suppressed by glycoxidized LDL, glycated LDL and oxidized LDL. Incubation with glycoxidized LDL enhanced the uptake of DiI-labeled oxidized LDL by macrophages to a greater extent than that of glycated LDL or oxidized LDL. Glycoxidized LDL also induced a significant degree of intracellular cholesteryl ester accumulation. Taken together, our results would suggest that glycoxidized LDL might be an important candidate in the initiation of foam cell formation and might play a significant role in the pathogenesis of atherosclerosis in diabetes mellitus.
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PMID:Glycoxidized low-density lipoprotein regulates the expression of scavenger receptors in THP-1 macrophages. 1553 Sep 5

The CpG motif of bacterial DNA (CpG-DNA) is a potent immunostimulating agent whose mechanism of action is not yet clear. Here, we used both DNA microarray and proteomic approaches to investigate the effects of oligodeoxynucleotides containing the CpG motif (CpG-ODN) on gene transcription and protein expression profiles of CpG-ODN responsive THP-1 cells. Microarray analysis revealed that 2 h stimulation with CpG-ODN up-regulated 50 genes and down-regulated five genes. These genes were identified as being associated with inflammation, antimicrobial defense, transcriptional regulation, signal transduction, tumor progression, cell differentiation, proteolysis and metabolism. Longer stimulation (8 h) with CpG-ODN enhanced transcriptional expression of 58 genes. Among these 58 genes, none except one, namely WNTI inducible signaling pathway protein 2, was the same as those induced after 2 h stimulation. Proteomic analysis by two-dimensional gel electrophoresis, followed by mass spectrometry identified several proteins up-regulated by CpG-ODN. These proteins included heat shock proteins, modulators of inflammation, metabolic proteins and energy pathway proteins. Comparison of microarray and proteomic expression profiles showed poor correlation. Use of more reliable and sensitive analyses, such as reverse transcriptase polymerase chain reaction, Western blotting and functional assays, on several genes and proteins, nonetheless, confirmed that there is indeed good correlation between mRNA and protein expression after CpG-ODN treatment. This study also revealed that several anti-apoptotic and neuroprotective related proteins, not previously reported, are activated by CpG-DNA. These findings have extended our knowledge on the activation of cells by CpG-DNA and may contribute to further understanding of mechanisms that link innate immunity with acquired immune response(s).
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PMID:A transcriptomic and proteomic analysis of the effect of CpG-ODN on human THP-1 monocytic leukemia cells. 1569 60

Tolerance to bacterial cell-wall components may represent an essential regulatory mechanism during bacterial infection. We have demonstrated previously that the inhibition of nuclear factor (NF)-kappaB and mitogen-activated protein kinase activation was present in bacterial lipoprotein (BLP) self-tolerance and its cross-tolerance to lipopolysaccharide (LPS). In this study, the effect of BLP-induced tolerance on the myeloid differentiation factor 88 (MyD88)-dependent upstream signaling pathway for NF-kappaB activation in vitro was examined further. When compared with nontolerant human monocytic THP-1 cells, BLP-tolerant cells had a significant reduction in tumor necrosis factor alpha (TNF-alpha) production in response to a high-dose BLP (86+/-12 vs. 6042+/-245 ng/ml, P < 0.01) or LPS (341+/-36 vs. 7882+/-318 ng/ml, P < 0.01) stimulation. The expression of Toll-like receptor 2 (TLR2) protein was down-regulated in BLP-tolerant cells, whereas no significant differences in TLR4, MyD88, interleukin-1 receptor-associated kinase 4 (IRAK-4), and TNF receptor-associated factor 6 expression were observed between nontolerant and BLP-tolerant cells, as confirmed by Western blot analysis. The IRAK-1 protein was reduced markedly in BLP-tolerant cells, although IRAK-1 mRNA expression remained unchanged as revealed by real-time reverse transcriptase-polymerase chain reaction analysis. Furthermore, decreased MyD88-IRAK immunocomplex formation, as demonstrated by immunoprecipitation, was observed in BLP-tolerant cells following a second BLP or LPS stimulation. BLP pretreatment also resulted in a marked inhibition in total and phosphorylated inhibitor of kappaB-alpha (IkappaB-alpha) expression, which was not up-regulated by subsequent BLP or LPS stimulation. These results demonstrate that in addition to the down-regulation of TLR2 expression, BLP tolerance is associated with a reduction in IRAK-1 expression, MyD88-IRAK association, and IkappaB-alpha phosphorylation. These findings further elucidate the molecular mechanisms underlying bacterial peptide tolerance.
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PMID:Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation. 1646 41

Chlamydia trachomatis is a human pathogen that causes multiple diseases worldwide. Despite appropriate therapy with existing antichlamydial antibiotics, chronic exacerbated diseases often occur and lead to serious sequelae. Since C. trachomatis has been found to enter a persistent state after exposure to deleterious conditions, the role of persistence in the failure of chlamydial antibiotherapy is questioned. HeLa, THP-1 and U-937 cells were infected with 10(4)C. trachomatis serovar L2 infectious particles. Three days later the infected cells were treated with minimal bactericidal concentrations of doxycycline (DOX), erythromycin (ERY) or tetracycline (TET) for 24 days or 30 days. Antibiotic efficacy was assessed by measuring chlamydial inclusions and infectious particles, by investigating the resumption of chlamydial growth after antibiotic removal and by testing Chlamydia viability using reverse transcriptase polymerase chain reaction targeting unprocessed 16S rRNA, processed 16S rRNA and Omp-1 mRNA. Treatment of infected HeLa cells with the usual antichlamydial antibiotics suppressed chlamydial active growth. The infection remained unapparent. However, 24 days post treatment the bacterium was found to be viable, as proved by continued expression of unprocessed and processed 16S rRNA and Omp-1 mRNA. This inactive unapparent chlamydial state is not infectious, suggesting Chlamydia persistence. Chlamydia trachomatis also developed persistence both in permissive THP-1 and non-permissive U-937 cells. Unlike in HeLa cells, persistent chlamydial infection in THP-1 and U-937 cells was resolved after 30 days of DOX treatment. Of interest, we noticed that only THP-1 and U-937 cells that were persistently infected following their interaction with infected HeLa cells remained capable of transmitting active infection to HeLa cells. These findings suggest that DOX, TET and ERY, usually administered to combat chlamydial diseases, fail to resolve persistent infection occurring during treatment in non-immune HeLa cells. However, in immune THP-1 and U-937 cells, the persistent infection is resolved by therapy with DOX. Epithelial cells could be the reservoir of persistent chlamydial particles.
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PMID:Effects of sustained antibiotic bactericidal treatment on Chlamydia trachomatis-infected epithelial-like cells (HeLa) and monocyte-like cells (THP-1 and U-937). 1652 61

We assessed the effect of voriconazole (VRC) on the expression and release of selected cytokines and chemokines in the THP-1 human monocytic cell line in response to Aspergillus fumigatus hyphal fragments (HF) by cDNA microarray analysis, reverse transcriptase (RT) PCR, and enzyme-linked immunosorbent assay. Stimulation of THP-1 cells by HF alone caused a significant up-regulation of CCL4 (MIP1B) and CCL16, while CCL2 (MCP1) was down-regulated. By comparison, in the presence of VRC, a large number of genes such as CCL3 (MIP1A), CCL4 (MIP1B), CCL5 (RANTES), CCL7 (MCP3), CCL11 (EOTAXIN), CCL15 (MIP1Delta), CXCL6, and CXCL13 were strongly up-regulated in THP-1 cells challenged by HF, whereas CCL20 (MIP3A) and CCL21 (MIP2) were down-regulated. Among five genes differentially expressed in THP-1 cells, IL12A, IL12B, and IL-16 were down-regulated whereas IL-11 and TGFB1 were significantly up-regulated in the presence of VRC. The inflammation-related genes IFNgamma, IL1R1, and TNFA were also up-regulated in THP-1 cells exposed to HF only in the presence of VRC. RT-PCR of four selected genes validated the results of microarrays. The release of interleukin 1beta (IL-1beta) and IL-12 was significantly increased from monocytes stimulated either by HF alone (P < 0.05) or in the presence of VRC (P < 0.01 and P < 0.05, respectively). In contrast, tumor necrosis factor alpha release from monocytes was enhanced only in the presence of VRC (P < 0.01). The chemokines monocyte chemoattractant protein 1 and macrophage inflammatory protein 1beta were decreased under both conditions (P < 0.01). These results demonstrate that in the presence of VRC, HF induces a more pronounced profile of gene expression in THP-1 cells than HF alone, potentially leading to more-efficient host resistance to A. fumigatus.
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PMID:Expression of immunomodulatory genes in human monocytes induced by voriconazole in the presence of Aspergillus fumigatus. 1717 97

Immune response is greater in females than in males and lymphocytes/monocytes from female subjects (or tested in vitro with estrogens) show higher immune/inflammatory reactivity. In order to test in vitro the interactions between 17beta-estradiol (E2--10(-9) M), testosterone (T--10(-8) M) and the antiproliferative/immune suppressive drug Leflunomide metabolite A77 1726 (LEF-M--30 microM) employed in rheumatoid arthritis (RA), their combined effects were evaluated on inflammatory cytokine (CK) expression/production in cultures of differentiated macrophages (M) (from activated THP-1 monocytes) and primary cultures of RA synovial macrophages (SM). TNFalpha, IL-6 and TGFbeta were detected by immunocytochemistry (ICC), Western blot analysis (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR). The ICC, WB and RT-PCR showed a significant down-regulation induced by LEF-M on CK expression by cultured M when compared to untreated cells (IL-6 p < 0.01, TNFalpha p < 0.001, TGFbeta p < 0.01). At ICC analysis E2 increased CK expression, whereas T decreased the expression, confirmed by WB and RT-PCR (range between p < 0.05 and p < 0.001). LEF-M treatment significantly downregulated the CK expression in E2/T treated M: the effect was more significant in LEF-M plus T-treated cells versus controls (range between p < 0.01 and p < 0.001). Concerning the RA SM, the results were replicated (range between p < 0.05 and p < 0.001). E2 seems to contrast, but T seems to synergize the LEF-M activity. Results might support a stronger therapeutical efficacy, at least for LEF, in male RA patients, as already reported by clinical evidences.
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PMID:Sex hormones modulate the effects of Leflunomide on cytokine production by cultures of differentiated monocyte/macrophages and synovial macrophages from rheumatoid arthritis patients. 1932 22


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