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)

Using differential mRNA display to uncover potential mediators associated with chronic rejection, we identified a cDNA fragment induced in Lewis to F344 rat cardiac allografts with arteriosclerosis but not Lewis syngrafts. The full-length cDNA (1.4 kb) isolated from a rat cardiac allograft cDNA library was 99% identical to galactose/N-acetylgalactosamine (Gal/GalNAc) macrophage lectin, a cell-surface receptor. This cDNA hybridized in Northern analysis with total RNA from eight cardiac allografts but not with host hearts, syngrafts, or other organs. There was a significant allograft-specific increase in transcript levels measured by reverse transcriptase PCR at days 7, 14, 28, and 75 in comparison with paired F344 host hearts (subject to same circulation but histologically normal), day-0 hearts, and syngrafts (P < 0.008, n = 4 at each time). Transcript levels in cardiac allografts were higher than those in paired host spleens (a major source of inflammatory cells) (P < 0.0001), indicating the localized nature of Gal/GalNAc lectin induction. By in situ hybridization and immunostaining, Gal/GalNAc lectin expression localized to a subset of inflammatory cells in cardiac allografts. These findings link Gal/GalNAc macrophage lectin to the chronic rejection process, as a possible mediator of macrophage infiltration.
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PMID:Identification and upregulation of galactose/N-acetylgalactosamine macrophage lectin in rat cardiac allografts with arteriosclerosis. 804 Mar 27

We evaluated the ability of a replication-deficient, recombinant adenoviral vector to transfer the bifunctional gene GAL-TEK, which expresses a marking/therapeutic gene product, to naturally occurring cat fibrosarcomas in situ. GAL-TEK contains an in-frame fusion of the bacterial LacZ gene for histochemical marking of tumors with beta-galactosidase (beta-Gal) and the HSV tk gene for enzyme-prodrug activation of the prodrug ganciclovir (GCV) to induce selective tumor cell killing. GAL-TEK bifunctional marking and cell killing activities were tested in vitro after adenoviral vector infection of HT1080 human fibrosarcoma cells. The tk activity of GAL-TEK is shown to be almost as potent as HSV tk to catalyze conversion of GCV to GCV nucleotides and promote selective cell killing. Using 8 cats with recurring 2.5-cm2 fibrosarcomas that either arose spontaneously or were induced by vaccine, we determined experimentally the administration routes and times required for optimum GAL-TEK gene transfer by beta-Gal histological staining and reverse transcriptase polymerase chain reaction to the multiple compartments of the growing fibrosarcomas consonant with minimizing collateral infection of neighboring tissues and other unwanted side effects.
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PMID:In vivo marking of spontaneous or vaccine-induced fibrosarcomas in the domestic house cat, using an adenoviral vector containing a bifunctional fusion protein, GAL-TEK. 852 80

In the pathogenesis of allergic reactions, T cells and cytokines play a major role. However, characterizations of food allergen-specific T cells are very limited. In this study, we screened the peripheral blood mononuclear cells (PBMC) of 14 patients for reactivity to ovomucoid (Gal d I), the major hen's egg allergen, and ovalbumin (Gal d II). Cell lines and clones specific to ovomucoid were generated from PBMC of four egg-allergic subjects, in order to study antigen domain specificity and cell cytokine production profiles. The results demonstrated, firstly, that egg-allergic patients respond to ovomucoid rather than to ovalbumin, and, secondly, that antigen specificity is predominantly directed toward the second and third domains of ovomucoid. The T-cell cytokine message was characterized by reverse transcriptase polymerase chain reaction (RT-PCR). Cell lines and clones from all four patients consistently expressed interleukin (IL)-5. IL-4, IL-13, and interferon-gamma were found to be expressed only by certain lines or clones. This observation suggests a central pathogenic role for IL-5 in food allergy-related symptoms.
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PMID:Characterization of ovomucoid-specific T-cell lines and clones from egg-allergic subjects. 879 79

Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in unwanted events, such as the formation of recombinant viruses and the mobilization of therapeutic vectors. Using sensitive reverse transcriptase PCR assays, we investigated human and murine gene therapy packaging cell lines for incorporation of endogenous retrovirus transcripts into murine leukemia virus (MLV) vector particles and, conversely, whether vector genomes are incorporated into human endogenous retrovirus (HERV) particles. VL30 endogenous retrovirus sequences were efficiently packaged in particles produced by the murine AM12 packaging system. For every seven MLV-derived beta-galactosidase (beta-Gal) vector genomes present in the particles, one copy of VL30 was also packaged. Although human FLY packaging cells expressed several classes of HERV transcripts (HERV-K, HuRT, type C, and RTVL-H), none was detectable in the MLV vector particles released from the cells. Nonspecific packaging of the MLV Gag-Pol expression vector transcripts was detected in the FLY virions at a low level (1 in 17,000 sequences). These findings indicate that human packaging cells produce retrovirus particles far less contaminated by endogenous viral sequences than murine packaging cells. Human teratocarcinoma cells (GH cells), which produce HERV-K particles, were transduced with an MLV-derived beta-Gal vector. Although both HERV-K and RTVL-H sequences were found in association with the particles, beta-Gal transcripts were not detected, indicating that HERV Gag proteins do not efficiently package MLV-based vectors.
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PMID:Packaging of endogenous retroviral sequences in retroviral vectors produced by murine and human packaging cells. 952 84

Expression of inducible nitric oxide (NO) synthase (NOS-2) occurs during inflammation in the central nervous system (CNS) and has been linked to demyelination accompanying certain CNS inflammatory diseases. Although astrocytes and microglia are thought to be the major sources of NOS-2 expression in the CNS in vivo, recent evidence suggested that the myelin-producing oligodendrocytes (OLs) themselves can express NOS-2 in culture. Given the potentially important pathological implications of this finding, the purpose of this study was to examine further the expression of NOS-2 by OLs in vitro. After exposure to lipopolysaccharide (LPS) and interferon-gamma (IFNgamma), primary cultures enriched for mature OLs released NO in a time-dependent manner, although the amount varied considerably between different culture preparations. Increased NO production was accompanied by expression of NOS-2 mRNA and protein, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Immunofluorescence analysis revealed that the cell-type expressing NOS-2 in these cultures was galactocerebroside (Gal C)-negative but CD11b-positive. Further, NO production could be attenuated in cultures treated with the microglial/macrophage toxin, leucine methyl ester, prior to LPS/IFNgamma stimulation. Thus, microglia were the source of NOS-2 catalytic activity in these cultures. The present results indicate that LPS and IFNgamma are not effective stimuli for induction of NOS-2 in OLs in primary cell culture.
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PMID:Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia. 1049 7

The cysteine-rich region of the 170-kDa subunit galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica is a subunit vaccine candidate and a protective antigen in the gerbil model of amebiasis. Macrophage-mediated immunity is important for protection against E. histolytica and is activated by Th1 cytokines. As Th1 differentiation is promoted by IL-12, we investigated what portion of the Gal-lectin could stimulate IL-12 in human THP-1 macrophages. Native Gal-lactin stimulated IL-12 p40 / p35 mRNA expression in a dose- and time-dependent manner as measured by reverse transcriptase-PCR. Human immune serum and Gal-lectin mAb inhibition studies identified amino acids (aa) 596 - 998 as immunogenic and containing the IL-12 inducing domain. IFN-gamma priming augmented Gal-lectin-induced IL-12 mRNA expression independent of TNF-alpha and IL-1beta, and was required for IL-12 p70 protein production from macrophages and human peripheral blood mononuclear cells. Gal-lectin plus IFN-gamma stimulated IL-12 p40 and p35 gene transcription with stable mRNA transcripts and a differential requirement for protein synthesis. These results suggest that aa 596 - 998 of the Gal-lectin can confer Th1-mediated protection against amebiasis through IL-12 induction.
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PMID:A subunit vaccine candidate region of the Entamoeba histolytica galactose-adherence lectin promotes interleukin-12 gene transcription and protein production in human macrophages. 1067 Nov 97

Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR-PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.
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PMID:Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent. 1091 34

The impact of a pathogen-induced inflammatory response on dendritic cells (DCs) and on their expression of galectin-3 (Gal-3) was studied on splenic DCs (sDCs) from Trypanosoma cruzi-infected mice. We determined the lectin expression and also presentation of ligands using the labeled galectin as probe. By reverse transcriptase polymerase chain reaction, western blot analysis, quantitative glycocytochemistry, and computer-assisted quantitative microscopy, we demonstrate that, in sDCs from infected mice, expression of Gal-3 and Gal-3-specific ligands were markedly up-regulated and adhesiveness was increased with Gal-3-coated substratum. Gal-3 expression was also enhanced in T. cruzi-infected D2SC-1 cells. To assess influence on migration, we had to work exclusively with D2SC-1 cells because sDCs rapidly lost their capacity to adhere to substratum. Migration of infected- and TCM-treated D2SC-1 cells were reduced when substratum was coated with Gal-3. Expression of Gal-3 by D2SC-1 was reduced when they were incubated with anti-Gal-3 antisense oligonucleotide without effect on cell invasion by the parasite. By using seven neoglycoconjugates, we probed the cellular capacity to specifically bind carbohydrate ligands. Similar to Gal-3, an up-regulation was noted with respect to sites specific for Man and alpha-GalNAc, respectively, revealing that infection-dependent changes are not confined to Gal-3-dependent parameters. Considered together, these data document for the first time that a parasitic infection can modulate both in vivo and in vitro the expression of Gal-3 and of ligands for this lectin in DCs with functional consequences on their capacities of adhesion and migration. These results suggest a new immunomodulatory property of T. cruzi.
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PMID:Up-regulation of galectin-3 and its ligands by Trypanosoma cruzi infection with modulation of adhesion and migration of murine dendritic cells. 1504 84

A genome-wide transcriptional analysis of Entamoeba histolytica was performed on trophozoites isolated from the colon of six infected mice and from in vitro culture. An Affymetrix platform gene expression array was designed for this analysis that included probe sets for 9435 open reading frames (ORFs) and 9066 5' and 3' flanking regions. Transcripts were detected for > 80% of all ORFs. A total of 523 transcripts (5.2% of all E. histolytica genes) were significantly changed in amebae isolated from the intestine on Days 1 and 29 after infection: 326 and 109 solely on Days 1 and 29, and 88 on both days. Quantitative real-time reverse transcriptase PCR confirmed these changes in 11/12 genes tested using mRNA isolated from an additional six mice. Adaptation to the intestinal environment was accompanied by increases in a subset of cell signaling genes including transmembrane kinases, ras and rho family GTPases, and calcium binding proteins. Significant decreases in mRNA abundance for genes involved in glycolysis and concomitant increases in lipases were consistent with a change in energy metabolism. Defense against bacteria present in the intestine (but lacking from in vitro culture) was suggested by alterations in mRNA levels of genes similar to the AIG1 plant antibacterial proteins. Decreases in oxygen detoxification pathways were observed as expected in the anaerobic colonic lumen. Of the known virulence factors the most remarkable changes were a 20-35-fold increase in a cysteine proteinase four-like gene, and a 2-3-fold decrease in two members of the Gal/GalNAc lectin light subunit family. Control of the observed changes in mRNA abundance in the intestine might potentially rest with four related proteins with DNA binding domains that were down-regulated 6-16-fold in the intestinal environment. In conclusion, the first genome-wide analysis of the transcriptome of E. histolytica demonstrated that the vast majority of genes are transcribed in trophozoites, and that in the host intestine trophozoites altered the expression of mRNAs for genes implicated in metabolism, oxygen defense, cell signaling, virulence, antibacterial activity, and DNA binding.
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PMID:Impact of intestinal colonization and invasion on the Entamoeba histolytica transcriptome. 1656 49

Chondroitin sulfate/dermatan sulfate (CS/DS) chains regulate the development of the central nervous system in vertebrates. Previously, we demonstrated that CS/DS hybrid chains from embryonic pig brain exhibit neuritogenic and growth factor binding activities, which depended on their IdoUA content defining the DS-like structure. To elucidate the distribution of such functional sugar chains during the development of the brain, in situ hybridization was performed to examine expression of three CS/DS GalNAc 4-O-sulfotransferases, D4ST-1, C4ST-1, and C4ST-2, and a single uronyl 2-O-sulfotransferase (UST) involved in the biosynthesis of DS in addition to CS intermediates. C4ST-1 and C4ST-2 were ubiquitously expressed in the postnatal mouse brain, whereas the expression of D4ST-1 and UST was restricted in the developing cerebellum and culminated at postnatal day 14 as shown by reverse transcriptase-PCR analysis. In situ analysis of the disaccharides of CS/DS in brain sections revealed that the concentration of CS/DS increases 2-fold during development (postnatal day 7 to 7 weeks). The proportions of DS-specific, principal disaccharides, IdoUA-Gal-NAc(4-O-sulfate) (iA) and IdoUA(2-O-sulfate)-GalNAc(4-O-sulfate) (iB), produced by the sequential actions of D4ST-1 and UST, were higher in the CS/DS chains from cerebellum than those from whole brain sections. A dramatic increase (10-fold) in the proportion of iB during development was noteworthy. In contrast, GlcUA/IdoUA(2-O-sulfate)-GalNAc(6-O-sulfate) (D/iD) and GlcUA/IdoUA-GalNAc(4, 6-O-disulfate) (E/iE) decreased to 50 and 30%, respectively, in the developing cerebellum. These results suggest that the IdoUA-containing iA and iB units along with D/iD and E/iE units in the CS/DS hybrid play important roles in the formation of the cerebellar neural network during postnatal brain development.
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PMID:Chondroitin sulfate/dermatan sulfate hybrid chains in the development of cerebellum. Spatiotemporal regulation of the expression of critical disulfated disaccharides by specific sulfotransferases. 1670 20


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