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)

By screening an expression library of the yeast form of Candida albicans with a serum directed against whole fungal cells, a cDNA (2,325 bp) encoding a stress protein of C. albicans was cloned and sequenced. The cloned sequence (CaRLV130) identified a single open reading frame with a length of 1,968 bp coding for a protein containing 656 amino acid residues (70 kDa). The deduced amino acid sequence was 84% similar to the sequence of the Saccharomyces cerevisiae SSA1 gene, which encodes one member of the 70-kDa heat shock protein (Hsp70) family. The relevant gene (C. albicans HSP70 gene [CaHSP70]) was localized on the highest-M(r) (R1; approximately 3.8 Mb) chromosome of C. albicans as determined by pulse-field electrophoresis. CaHSP70 was expressed after heat shock, as demonstrated by Northern (RNA) blotting and reverse transcriptase-PCR with specific pairs of oligonucleotide sequences and gene probes. A recombinant protein was obtained in Escherichia coli after cloning of the full coding sequence into the BamHI site of the pDS56/RBSII6xhisE- plasmid and purification by nickel chelate affinity chromatography. The recombinant protein (6xhis-CaHsp70) was efficiently recognized in immunoblots by a monoclonal antibody directed against a common epitope of eukaryotic Hsp70 proteins, as well as by sera from normal human subjects. Moreover, immune mouse sera against the purified recombinant protein recognized native, heat-inducible constituents with sizes of around 70 kDa in whole-cell protein extracts of C. albicans. Overall, our data demonstrate that CaHSP70 encodes one member of a family of proteins (Hsp70) which usually represent highly conserved immunodominant antigens of infectious agents.
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PMID:Molecular cloning and expression of a 70-kilodalton heat shock protein of Candida albicans. 755 17

The presence of Cryptosporidium in drinking water supplies is a significant problem faced by the water industry. Although a variety of methods exist for the detection of waterborne oocysts, water utilities currently have no way of assessing the infectivity of detected oocysts and consequently are unable to accurately determine the risks posed to public health by waterborne Cryptosporidium. In this paper, the development of an infectivity assay for waterborne Cryptosporidium parvum is described. Oocysts were inoculated onto monolayers of Caco-2 cells and grown on microscope slides, and infections were detected by C. parvum specific reverse transcriptase PCR of extracted mRNA, targeting the heat shock protein 70 (hsp70) gene. A single infectious oocyst was detected by this experimental procedure. The use of concentrated samples obtained from 250 liters of finished water had no observable effect on the integrity of cell monolayers or on the infectivity of oocysts seeded into the concentrate. Intracellular developmental stages of the parasite were also detected by using fluorescently labeled antibodies. One pair of PCR primers targeting the hsp70 gene was specific for C. parvum, while a second pair recognized all species of Cryptosporidium tested. The C. parvum-specific primers amplified DNA from 1 to 10 oocysts used to seed 65 to 100 liters of concentrated environmental water samples and were compatible with multiplex PCR for the simultaneous detection of C. parvum and Giardia lambia. This paper confirms the utility of PCR for the detection of waterborne C. parvum and, most importantly, demonstrates the potential of an in vitro infectivity assay.
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PMID:An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum. 914 32

Animal experiments have shown that members of the heat shock protein (HSP) family have cytoprotective properties against ischaemia. In experimentally induced cardiac ischaemia, the induction of HSP70s correlates with reduced infarct size and enhanced myocardial function and endothelial recovery. Direct evidence that increased myocardial HSP70 expression result in cytoprotection during ischaemia has also been obtained using transgenic mice overexpressing either rat or human HSP72. This study examined the induction and expression of myocardial HSP70s after an obligatory period of ischaemia in patients during cardiac surgery. The level of HSP72/HSC73 protein in Tru-cut biopsies of the myocardium, taken before and after an acute ischaemic insult, was examined using a polyclonal antibody. The amount of HSP72 mRNA in the biopsies was also determined by reverse transcriptase polymerase chain reaction (RT-PCR) and correlated HSP72/HSC73 protein expression. In four patients subjected to brief alternating periods of normothermic ischaemia and reperfusion, the amount of myocardial HSP72/HSC73 protein was increased several fold after ischaemic insult. This was accompanied by increased expression of HSP72 mRNA. In contrast, the amounts of myocardial HSP72/HSC73 protein and HSP72 mRNA were unchanged in a patient subjected to a single prolonged period of hypothermic ischaemia. Given the proven myocardial protective properties of HSP72 in experimental models, it is postulated that the observed induction of HSP72 may have a similar function in man.
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PMID:Induction of myocardial heat shock protein 70 during cardiac surgery. 934 41

The 70 kDa heat shock protein (HSP70) is induced in cells exposed to chemical or physical stress. HSP70 facilitates cell survival by preventing protein denaturation and incorrect assembly of polypeptides. Induction of HSP70 messenger RNA (mRNA) synthesis also inhibits transcription of genes coding for pro-inflammatory cytokines. We analyzed whether HSP70 mRNA was expressed in a cultured human cervical cell line (HeLa cells) following exposure to human semen, or in cells obtained from the endocervices of sexually active women. HeLa cells were co-cultured with a 1:50 dilution of semen from four men, with purified spermatozoa, or with cell-free seminal fluid. Endocervical swabs were obtained at mid-cycle from 53 women. HSP70 mRNA was detected in HeLa cells by a reverse transcriptase-polymerase chain reaction (RT-PCR) and analysis on agarose gels. HSP70 mRNA in cervical cells was measured by RT-PCR followed by hybridization with an HSP70-specific internal probe and detection by ELISA. Cervical IgA antibodies to HSP70 were measured by ELISA. HeLa cell-semen co-culture led in each case to induction of HSP70 mRNA. Cell-free seminal fluid and isolated motile spermatozoa also induced HSP70 mRNA when incubated individually with HeLa cells. HSP70 mRNA was detected in 28 (52.8%) of 53 endocervical cell samples obtained from women at varying times after sexual intercourse. The percentage of samples expressing HSP70 mRNA was 37.5% at <10 h, 64.3% at 10 h, 70.0% at 11 h and between 36 and 50% at later times after semen exposure. Cervical IgA antibodies to HSP70 were also detected in some women and their occurrence was highly correlated with HSP70 gene transcription (P < 0.0001). The data demonstrate that exposure to semen induces HSP70 mRNA in endocervical cells.
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PMID:Induction of messenger RNA for the 70 kDa heat shock protein in HeLa cells and the human endocervix following exposure to semen: implications for antisperm antibody production and susceptibility to sexually transmitted infections. 936 6

In experimental models, the synthesis of heat shock protein 70 (HSP 70) has been recognized as an intracellular response to ischemia and reperfusion, insults inherent to transplantation. In this study, the HSP response in early stages of human liver transplantation was investigated. HSP 70 mRNA expression was detected by means of reverse transcriptase (RT)-PCR in liver biopsies (n = 28) and in cells obtained from the organ perfusate (n = 14) following cold preservation. The expression of HSP 70 differed substantially between individuals. Retrospective analysis revealed a close correlation of the amount of HSP 70 mRNA in perfusate cells and biopsies with the onset of organ dysfunction due to early graft rejection. Patients with early graft rejection had a significantly lower amount of HSP 70 mRNA than patients without rejection. These results suggest a protective role of HSP 70 expression. Low levels of HSP 70 may, therefore, represent a prognostic marker for early graft rejection.
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PMID:Expression of HSP 70 as a potential prognostic marker for acute rejection in human liver transplantation. 956 74

Human hepatoma cells (HepG2) were exposed to several heavy metal salts and the induction of heat shock protein 70 (hsp70) mRNA was analysed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Metals were added to the cell medium at concentrations ranging from 0.1 to 100 microM and incubation was continued for 4 h. In addition we analysed the time dependence of hsp70 induction by adding each metal at a certain concentration followed by an incubation for 0.5 to 24 h. CdCl2, NaAsO2, AgNO3 could be classified as very strong inducers (20-, 13- and 10-fold above control level) and they reached their maximum level of induction at 1-10 microM after 2 h. CuCl2, MnCl2, Pb(NO3)2, TlNO3, CoCl2 and NiCl2 were also strong inducing agents, giving a 4-6 fold induction at 10-100 microM after 4-8 h. ZnSO4, Hg(NO3)2 and AlCl3 were only weak inducers (1.5-2 fold at 50-100 microM after 4-8 h) of hsp70 mRNA. Cytotoxic effects (measured by release of lactate dehydrogenase) could only be detected for 100 microM Hg2+ after 4 h and when the cells were incubated with 5 microM Cd2+ for more than 8 h. We also tested a few combinations of these heavy metal salts for their hsp70-inducing ability. Zn2+ and Mn2+ were able to diminish Cd2+ induced hsp70 mRNA levels by 65%. Ag+ mediated induction was reduced by 40% when combined with Cu2+, whereas Hg2+ increased induction by Ag+ about 3-fold and led to a dramatic decrease in cell viability. In our study we were able to demonstrate that the analysis of hsp70 mRNA levels in chemically stressed HepG2 cells by RT-PCR can be a valuable tool for studying mechanisms of toxicity associated with elevated expression of hsp70.
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PMID:Analysis of hsp70 mRNA levels in HepG2 cells exposed to various metals differing in toxicity. 982 Jun 63

This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-gamma via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2beta. The levels of the CRF receptor type 2beta were not altered in cells treated with activators of protein kinase C, Ca2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-beta3, -gamma1, or -gamma2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-gamma1 and -gamma2 were phosphorylated. The increase in phospholipase C-gamma phosphorylation was concentration-dependent with an EC50 of 4.2+/-0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2beta that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2iminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated the phospholipase C-gamma phosphorylation. In summary, CRF induces phospholipase C-gamma phosphorylation at tyrosine residues, which depends on Ca2+ and is mediated by activation of protein tyrosine kinases via the CRF receptor type 2beta.
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PMID:Corticotropin-releasing factor induces phosphorylation of phospholipase C-gamma at tyrosine residues via its receptor 2beta in human epidermoid A-431 cells. 988 91

The influence of deep hypothermia (4 degrees C) during a substrate-free, hypoxia-reoxygenation treatment was investigated on cardiomyocytes (CM) prepared from newborn rat heart in culture in an in vitro, substrate-free model of ischemia-reperfusion. The transmembranous potentials were recorded with standard microelectrodes. The contractions were monitored photometrically. The RNA messenger (mRNA) and protein expression for protein (HSP70) were analysed by RT-PCR (reverse transcriptase-polymerase chain reaction) and Western blotting, respectively. Simulated ischemia (SI) caused a gradual decrease and then a cessation of the spontaneous electromechanical activity. During the reoxygenation, the CM recovered normal function, provided that SI did not exceed 2.5 h. When SI duration was increased up to 4 h, reoxygenation failed to restore the spontaneous electromechanical activity. Conversely, the exposure of the CM to SI together with deep hypothermia decreased the functional alterations observed, and provided a complete electromechanical recovery after 2.5 h as well as after 4 h of SI. Deep hypothermia alone failed to induce HSP70 mRNA and protein production. On the contrary, HSP70 mRNA production increased after 2.5 and 4 h of deep hypothermia followed by 1 h of rewarming, proportionally to the duration of the cooling period. This augmentation in mRNA was associated with a rise in HSP70 protein content. In summary, it appeared that deep hypothermia exerts a strong cytoprotective action during SI only, whereas cooling CM before SI has no beneficial effect on subsequent SI. Moreover, these results suggested the persistence of a signaling system and/or transduction in deeply cooled, functionally depressed cells. Finally, CM in culture appeared to be a model of interest for studying heart graft protection against ischemia-reperfusion and contributed to clarifying the molecular and cellular mechanisms of deep hypothermia on myocardium.
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PMID:Influence of deep hypothermia on the tolerance of the isolated cardiomyocyte to ischemia-reperfusion. 1173 54

We examined whether heat shock response is affected by experimental hyperlipidemia in rat hearts. Therefore, isolated hearts of male Wistar rats fed a 2% cholesterol-enriched diet or standard diet for 12 weeks were subjected to either 20 min heat stress at 42 degrees C or global normothermic ischemia followed by 120 min normothermic, normoxic perfusion. Both heat stress and ischemia resulted in a significant increase in cardiac mRNA and protein levels of the inducible member of the 70-kDa heat shock protein family (HSP70) when compared to time-matched controls as assessed by reverse transcriptase polymerase chain reaction and Western blotting in hearts of normal rats. However, in hyperlipidemic groups, increase in cardiac hsp70 mRNA and HSP70 protein in response to heat stress and ischemia was markedly attenuated. We further observed that the basal level of hsp70 mRNA was significantly higher in the hyperlipidemic group when compared to normal controls; however, the HSP70 protein level was not different. This is the first demonstration that hyperlipidemia inhibits cardiac heat shock response. We further conclude that basal HSP70 expression might be downregulated at a posttranscriptional level in hyperlipidemia.
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PMID:Hyperlipidemia induced by high cholesterol diet inhibits heat shock response in rat hearts. 1182 Jul 96

Among the parasitic helminths schistosomes are of high medical and economic importance. Despite of the world-wide relevance of this parasite, very little is known about the cellular mechanisms controlling its development and concerning the host-parasite interaction. Within the last decade a great effort has been made in this blood fluke to identify genes which play important roles during these processes. However, molecular analysis was limited by the fact, that neither function nor regulation of candidate genes could be investigated in this organism due to the lack of transformation protocols. Here, we present the strategy of ballistic gene transfer to introduce and characterize transgenes in different schistosome life stages. As a transformation vector, the heat shock protein 70 (hsp70) gene promoter and terminator from Schistosoma mansoni were cloned and fused to the green fluorescent protein (GFP) reporter gene. In a first attempt, the hsp70--GFP vector was successfully tested in a eukaryotic cell line. Thereafter, adult male schistosomes and sporocysts were transformed with this vector, and GFP expression was demonstrated using molecular and microscopical methods. PCR, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses confirmed the presence, transcription and translation of the transgene in adults. Confocal laser scanning microscopy revealed GFP-activity at various sites along the surface of the worms after hs induction and within sporocysts. These results suggest diverse roles for hsp70 during the development of schistosomes. Furthermore, the results demonstrate the feasibility of this method and open the perspective to analyze a variety of molecular functions in schistosomes.
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PMID:HSP70-controlled GFP expression in transiently transformed schistosomes. 1184 13


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