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)

Pulmonary tissue injury and repair processes involve complex and coordinated cellular events such as necrosis, inflammation, cell growth/differentiation, apoptosis, and remodeling of extracellular matrix. These processes are regulated by expression of multiple mediator genes. Commercially available microarray blots and slides allow screening of hundreds to thousands of genes in a given tissue or cell preparation. However, often these blots do not contain cDNAs of one's interest and are difficult to interpret. In order to analyze the tissue expression profile of a large number of genes involved in pulmonary injury and pathology, we developed a rat gene array filter using array technology. This array consisted of 27 genes representing inflammatory and anti-inflammatory cytokines, growth factors, adhesion molecules, stress proteins, transcription factors and antioxidant enzymes; 3 negative controls, and 2 blank spots. Using rat gene-specific polymerase chain reaction (PCR) primer pairs, cDNAs for these genes were amplified and cloned into a TA vector. Plasmids with recombinant cDNA inserts were purified and blotted onto a nylon membrane. Lung total RNA was isolated at 3 or 24 h following intratracheal (IT) exposure of male Sprague Dawley rats to either saline (control), residual oil fly ash (ROFA; 3.3 mg/kg) or metals found in one instillate of ROFA: nickel (NiSO(4); 1. 3 micromol/kg) or vanadium (VSO(4); 2.2 micromol/kg). (32)P-Labeled cDNA was generated from RNA samples in a reverse transcriptase reaction and subsequently hybridized to array blots. Densitometric scans of array blots revealed a twofold induction of interleukin (IL)-6 and TIMP-1 at 24 h post ROFA or Ni exposure. The pulmonary expressions of cellular fibronectin (cFn-EIIIA), ICAM-1, IL-1beta, and iNOS genes were also increased 24 h post ROFA-, V-, or Ni-exposure. Consistent hybridization of beta-actin in all array blots and absence of hybridization signals in negative controls indicated gene specific hybridization. ROFA or metal-induced increase in the expression of IL-6 observed in array blot was validated by Northern blot hybridization. Developing a pulmonary rat gene array may provide a tool for screening the expression profile of tissue specific markers following exposure to toxic air contaminants.
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PMID:A pulmonary rat gene array for screening altered expression profiles in air pollutant-induced lung injury. 1111 90

T-type calcium channels may be involved in the maintenance of myogenic tone. We tested their role in isolated rat cremaster arterioles obtained after CO(2) anesthesia and decapitation. Total RNA was analyzed by RT-PCR and Southern blotting for calcium channel expression. We observed expression of voltage-operated calcium (Ca(V)) channels Ca(V)3.1 (T-type), Ca(V)3.2 (T-type), and Ca(V)1.2 (L-type) in cremaster arterioles (n = 3 rats). Amplification products were observed only in the presence of reverse transcriptase and cDNA. Concentration-response curves of the relatively specific L-type blocker verapamil and the relatively specific T-type blockers mibefradil and nickel were made on cannulated vessels with either myogenic tone (75 mmHg) or a similar level of constriction induced by 30 mM K(+) at 35 mmHg. Mibefradil and nickel were, respectively, 162-fold and 300-fold more potent in inhibiting myogenic tone compared with K(+)-induced constriction [log(IC(50), M): mibefradil, basal -7.3 +/- 0.2 (n = 9) and K(+) -5.1 +/- 0.1 (n = 5); nickel, basal -4.1 +/- 0.2 (n = 5) and K(+) -1.6 +/- 0.5 (n = 5); means +/- SE]. Verapamil had a 17-fold more potent effect [log(IC(50), M): basal -6.6 +/- 0.1 (n = 5); K(+) -5.4 +/- 0.3 (n = 4); all log(IC(50)) P < 0.05, basal vs. K(+)]. These data suggest that T-type calcium channels are expressed and involved in maintenance of myogenic tone in rat cremaster muscle arterioles.
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PMID:Role of T-type calcium channels in myogenic tone of skeletal muscle resistance arteries. 1238 44

The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-beta-cyclodextrin (mbetacd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ETA receptor) was reduced by mbetacd and increased by cholesterol. Neither ligand binding nor colocalization of ETA and caveolin-1 was affected by mbetacd. Ca2+ inflow via store-operated channels after depletion of intracellular Ca2+ stores was reduced in mbetacd-treated caudal arteries, as shown by Mn2+ quench rate and intracellular [Ca2+] response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase-polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by mbetacd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni2+ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1-induced contractions were insensitive to the TRPC1 blocking antibody and to mbetacd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to mbetacd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1.
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PMID:Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TRPC1. 1455 Dec 43

The standard procedure for diagnosing allergic contact dermatitis is to perform a patch test. Because this has several disadvantages, the development of a new in vitro test system would be of immense value. Gene transcripts that distinguish allergics from non-allergics may have the potential to serve as the molecular basis for such a diagnostic tool. In this study, we use the microarray technology in the identification of differentially expressed genes in allergen-stimulated peripheral blood mononuclear cells (PBMCs) from 3 chromium-allergic patients versus 3 healthy controls. Using an Affymetrix GeneChip, the gene expression was analysed in PBMC cultures grown with 100 microg/ml CrCl3 or in media alone for 24 hr. A total of 26 genes were differentially expressed by more than twofold (P < 0.01) in allergen-activated PBMCs from patients compared with controls. 18 of these were upregulated, whereas 8 were downregulated. The expression of 1 downregulated gene, CASP8, was also found specifically and significantly reduced in an expanded population including 4 additional chromium allergic patients and 1 additional control subject by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The expression of 2 upregulated genes, ETS2 and CISH, correlated with a high-proliferative response following CrCl3 exposure. Additionally, real-time RT-PCR analysis indicated that the same gene expression changes are valid for nickel allergics, potentially making the expression profile more widely available. The 26 differentially expressed genes identified in this study may potentially function as diagnostic markers for contact sensitivity.
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PMID:Gene transcripts as potential diagnostic markers for allergic contact dermatitis. 1603 4

Partial hoxH genes of 2 cyanobacterial genera, including 5 strains of Arthrospira and 2 strains of Spirulina, were cloned and characterized. This gene encodes the large subunit of nickel-iron hydrogenase. The results showed that these genes comprised 1349 nucleotides in Arthrospira and 1343 nucleotides in Spirulina, respectively. The GC contents of hoxH were 45.7% to 47.3% in Arthrospira and up to 50.4% to 50.9% in Spirulina. The hoxH gene was demonstrated to be single copy by Southern analysis, and its transcription was verified by reverse transcriptase polymerase chain reaction in Arthrospira platensis FACHB341. The similarities of nucleotide sequences among the 5 strains of Arthrospira ranged from 95.7% to 99.8%, which are higher than those between Arthrospira and Spirulina (72.9-77.0%). However, similarity between the 2 Spirulina strains was only 72.5%, slightly lower than that between the 2 genera. A phylogenetic tree based on hoxH was constructed. All 5 strains of Arthrospira formed a monophyletic clade, which was highly supported by bootstrap value, while the 2 strains of Spirulina were separated into 2 different clades.
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PMID:Cloning and characterization of hoxH genes from Arthrospira and Spirulina and application in phylogenetic study. 1604 66

We utilized a diaminobenzidine reaction enhanced with nickel to compare dorsal vagal complex (DVC) and myenteric neuronal Fos-Like immunoreactivity (Fos-LI), in response to sulfated cholecystokinin-8 (CCK-8) (5, 10, 20, 40 microg/kg), among Sprague-Dawley (SD), Standard Long-Evans (SLE), Otsuka Long-Evans Tokushima Fatty (OLETF), and Long-Evans Tokushima Otsuka (LETO) rats. All rat strains but OLETF expressed Fos-LI in response to CCK-8. In addition, SD rats expressed more Fos-LI in the area postrema and myenteric neurons than SLE and LETO rats. To investigate the basis for these differences, we utilized cuprolinic blue staining, which stains neuronal cell bodies, to quantify the number of myenteric neurons, and a reverse transcriptase chain polymerase reaction to measure the gene expression of CCK(1) receptor in the gut. We found that SD rats have significantly more duodenal myenteric neurons than the other strains. In addition, this strain expressed significantly higher levels of the CCK(1) gene in both the duodenum and jejunum than the other strains. In conclusion, SD rats may express more myenteric Fos-LI in response to CCK due to increased numbers of myenteric neurons or more intestinal CCK(1) receptors than the other strains of rats.
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PMID:Strain differences in myenteric neuron number and CCK1 receptor mRNA expression may account for differences in CCK induced c-Fos activation. 1616 31

Amiodarone (AM) is a potent vasodilator and exhibits diverse cardiovascular protective effects in vivo, but their underlying mechanisms remain unsettled. We investigated the effects of AM and N-desethylamiodarone (DEA), the major metabolite of AM, on endothelial nitric oxide (NO) production using cultured human umbilical vein endothelial cells (HUVECs). The release of NO was evaluated as measured by nitrite, a stable metabolite of NO, using the Griess reaction and also measured directly by a NO-selective electrode. The expression of each nitric oxide synthase (NOS) mRNA was examined by reverse transcriptase-polymerase chain reaction (RT-PCR), and the effects of AM on eNOS mRNA expression were studied by quantitative real-time RT-PCR. AM and DEA (1-30 microM) enhanced NO production in a concentration-dependent manner. DEA was capable of producing more NO than AM. L-NAME, a nonselective NOS inhibitor, EGTA, a Ca(2+)-chelating agent, and nickel, a nonspecific Ca(2+) blocker, all inhibited AM-induced NO production. However, LY294002, an Akt pathway inhibitor and SB202190, a MAP kinase inhibitor, did not significantly suppress the production. In RT-PCR analysis, only eNOS mRNA was detected. Treatment with AM for 4 hours did not show a significant increase in the expression of eNOS mRNA. AM lower than 30 microM did not induce apoptosis, net cell loss, or LDH release from cells. The present study provides the first evidence that therapeutic concentrations of AM and DEA enhance eNOS-mediated NO production without any toxic or apoptotic effects. This mechanism may underlie the cardiovascular protective effects of AM and its metabolite observed in a clinical setting.
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PMID:Amiodarone and N-desethylamiodarone enhance endothelial nitric oxide production in human endothelial cells. 1647 44

The rabbit is frequently employed in small animal models of in-vivo coagulation and fibrinolysis, but the degree to which its plasma proteins resemble their human counterparts is incompletely known. Our aims were: to determine the nucleotide sequence of a full-length rabbit liver alpha(2)-antiplasmin (alpha(2)AP) cDNA; compare it with its human, murine, and bovine counterparts; and express it in functional form. Partial cDNAs encoding 5' and 3' portions of the alpha(2)AP mRNA were obtained by reverse transcriptase (RT)-polymerase chain reaction (PCR), using rabbit liver RNA and 'guessmer' oligonucleotides based on known sequences. This information was employed to design additional oligonucleotides for RT-PCR and rapid amplification of cDNA ends (RACE) procedures yielding overlapping clones corresponding to the entire rabbit alpha(2)AP mRNA sequence. Mature alpha(2)AP was expressed as a hexahistidine-tagged recombinant protein in Escherichia coli, purified by nickel-chelate affinity and ion exchange chromatography, and its reaction with plasmin and soluble fibrin assessed electrophoretically and compared with an analogous recombinant human alpha(2)AP. The consensus rabbit alpha(2)AP cDNA is 2,157 bp long, and encodes a 464-residue mature sequence and a 27-amino-acid presequence. The mature protein is 82% identical to its human counterpart, and its recombinant form produced denaturation-resistant complexes with both plasmin and fibrin. The region of greatest sequence divergence lies within the C-terminal extension of alpha(2)AP, unique in the serpin family of proteins to which alpha(2)AP belongs. The highly conserved nature of rabbit alpha(2)AP reinforces its role as a vital antifibrinolytic protein and supports fibrinolytic investigations in models employing this small animal.
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PMID:Molecular cloning and functional expression of rabbit alpha2-antiplasmin. 1665 71

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family. It is required for B cell development. Deregulation of BLyS was involved in the pathogenesis of B cell-related autoimmune diseases and multiple myeloma. To prepare monoclonal antibodies (MAbs) against BLyS, cDNA encoding soluble BLyS (sBLyS) was first amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers, and then inserted into a prokaryotic expression vector pET-30a. Right recombinant plasmid was expressed in Escherichia coli strain BL21(DE3), purified by nickel affinity chromatography. Isolated sBLys was used as an antigen to immunize mice. Splenocytes of one immunized mouse were fused with NS- 1. Hybridomas secreting antibodies against sBLyS were identified by ELISA. One positive clone was selected to produce antibody by injecting the hybridoma into the peritoneal cavity of mice. After collecting ascites, the antibody was purified by protein A affinity chromatography. Western blot and immunoflourescence demonstrated that the antibody could bind recombinant sBLyS and genuine membrane-bound BLyS (mBLyS) on U937. This MAb can be used as a detecting reagent to analyze the serum level of BLyS in patients with autoimmune diseases and the expression profile of mBLyS on multiple myeloma cells.
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PMID:Preparation and characterization of a monoclonal antibody against human B lymphocyte stimulator. 1670 8

The aim of the present study was to investigate the effects of environmentally important heavy metals and organochlorines on the transcriptional profiles of genes coding for heat shock cognate 70 (hsc70) and inducible heat shock protein 70 (hsp70) in a black sea bream fibroblast cell line. Using the nucleotide sequence information, from the cloned genes, specific reverse transcriptase-polymerase chain reaction (RT-PCR) methods were devised to test the effects of heavy metals (Cd2+, Cu2+ and Ni2+) and organochlorines (aroclor 1254, hexachlorobenzene and 2-4-dichloroaniline) on the cell stress response. Hsp70 was induced in fibroblasts upon heavy metal exposure concentrations as low as 0.01 microM whereas hsc70 expression was induced upon organochlorine exposure concentrations as low as 0.001 microM. Overall, our findings demonstrate that gene members of the HSP70 family are responsive to environmentally important chemicals.
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PMID:Impact of heavy metals and organochlorines on hsp70 and hsc70 gene expression in black sea bream fibroblasts. 1678 Sep 70


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