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)

We have isolated and characterized cDNA and genomic clones that encode a 70 kDa heat shock protein (Hsp70) from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. The gene encodes a 649-amino-acid protein showing high identity with other members of the hsp70 gene family. The hsp70 gene is induced during both heat shock of yeast cells at 42 degrees C and the mycelial to yeast transition. A differential expression of this gene can be observed between mycelial and yeast forms, with a much higher level of expression in the yeast. We found two introns of 178 and 72 nucleotides in the P. brasiliensis hsp70 gene. Splicing of these introns is regulated during the heat shock process and possibly during infection. In order to analyse the differential accumulation of unspliced mRNA following cellular differentiation and/or heat shock, reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were carried out. The temperature-induced mycelial to yeast transition results in the transient accumulation of unspliced hsp70 mRNA transcripts. Yeast cells, after adaptation at 36 degrees C, seem to be more proficient at splicing, at least with respect to hsp70 mRNA because, during a severe heat shock (42 degrees C), the unspliced form of this mRNA does not accumulate. The mycelial to yeast differentiation will have the adaptational effect of increasing the resistance of the organism to environmental stress, which may be necessary for parasite survival in the mammalian host.
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PMID:Differential expression of an hsp70 gene during transition from the mycelial to the infective yeast form of the human pathogenic fungus Paracoccidioides brasiliensis. 1009 73

Activated microglia regulate immune and inflammatory responses in the CNS under a variety of stresses due to infection, injury and disease. In this study, we show that a stress-inducible small heat shock protein, alpha-crystallin, induces in vitro activation of microglia cultured from newborn rat brain. Exposure of microglia to alpha-crystallin resulted in an increased production of nitric oxide (NO) and the expression of the inducible NO synthase (iNOS) as determined by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. Alpha-crystallin also stimulated the synthesis of the pro-inflammatory cytokine, TNF alpha. The results presented showing microglial induction of the two key immune regulatory and inflammatory molecules, i.e., NO and TNF alpha, in response to a stress-inducible protein, suggest a link between environmental stress and the CNS immune response.
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PMID:Microglial activation by the small heat shock protein, alpha-crystallin. 1051 55

Although our understanding of effects of space flight on human physiology has advanced significantly over the past four decades, the potential contribution of stress at the cellular and gene regulation level is not characterized. The objective of this ground-based study was to evaluate stress gene regulation in cells exposed to altered gravity and environmentally suboptimal conditions. We designed primers to detect message for both the constitutive and inducible forms of the heat shock protein, HSP-70. Applying the reverse transcriptase-polymerase chain reaction (RT-PCR), we probed for HSP-70 message in human acute T-cell leukemia cells, Jurkat, subjected to three types of environmental stressors: (1) altered gravity achieved by centrifugation (hypergravity) and randomization of the gravity vector in rotating bioreactors, (2) serum starvation by culture in medium containing 0.05% serum, and (3) temperature elevation (42 degrees C). Temperature elevation, as the positive control, significantly increased HSP-70 message, while centrifugation and culture in rotating bioreactors did not upregulate heat shock gene expression. We found a fourfold increase in heat shock message in serum-starved cells. Message for the housekeeping genes, actin and cyclophilin, were constant and comparable to unstressed controls for all treatments. We conclude that gravitational perturbations incurred by centrifugal forces, exceeding those characteristic of a Space Shuttle launch (3g), and culture in rotating bioreactors do not upregulate HSP-70 gene expression. In addition, we found RT-PCR useful for evaluating stress in cultured cells.
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PMID:Regulation of heat shock protein message in Jurkat cells cultured under serum-starved and gravity-altered conditions. 1067 23

Prolonged exposure of rodent beta-cells to combinations of cytokines induces the inducible form of nitric oxide synthase (iNOS) expression and Fas expression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifaceted actions. Recent studies have shown that NO, in addition to having cytotoxic actions, may regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determined by nuclear dyes, of pancreatic islets or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) plus gamma-interferon (1,000 U/ml) plus tumor necrosis factor-alpha (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a >3-fold increase in Fas and MnSOD mRNA expression in wild-type (WT) and iNOS-/- islets. On the other hand, hsp 70 was induced in WT but not in iNOS-/- islets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in WT (39 +/- 3%) and iNOS4-/- (33 +/- 4%) beta-cells. On the other hand, cytokines increased necrosis in WT (20 +/- 4%) but not in iNOS-/- (7 +/- 3%) beta-cells. From these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines induce both apoptosis and necrosis in mouse beta-cells, and 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation.
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PMID:Cytokines induce apoptosis in beta-cells isolated from mice lacking the inducible isoform of nitric oxide synthase (iNOS-/-). 1090 67

By use of reverse transcriptase-polymerase chain reaction, abundant expression of the mRNA of 27 kDa heat shock protein (Hsp27) was revealed in the sympathetic and parasympathetic ganglia as well as in the sensory ganglia of unstressed adult rats. In situ hybridization and immunohistochemistry further localized Hsp27 mRNA and protein to both neurons and satellite cells in all types of ganglia examined. Schwann cells in the ganglia and peripheral nerve fibers were devoid of Hsp27 signal. These results suggested that Hsp27 is constitutively expressed in neurons and satellite cells in the entire peripheral nervous system of the rat.
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PMID:Constitutive expression of the 27-kDa heat-shock protein in neurons and satellite cells in the peripheral nervous system of the rat. 1116 16

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (epsilon) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by epsilon and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of epsilon-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.
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PMID:Reconstitution of a functional duck hepatitis B virus replication initiation complex from separate reverse transcriptase domains expressed in Escherichia coli. 1146 13

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-epsilon interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in epsilon binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.
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PMID:In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins. 1173 92

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

By adapting a semi-quantitative reverse transcriptase-PCR (RT-PCR) method, we investigated kinetics of gene expression at different developmental stages of Trichinella spiralis and T. pseudospiralis. The analyzed genes included four kinds of excretory and secretory (ES) proteins, a heat shock protein (HSP) and a DNA binding protein and showed that T. spiralis and T. pseudospiralis expressed ES proteins in a stage-specific manner. The gene encoding a 43 kDa ES protein was expressed by muscle larvae, either pre-cyst or post-cyst larvae. The genes encoding: the 53 kDa ES protein of T. spiralis; 53 kDa ES protein of T. pseudospiralis; and 19.6 kDa ES protein of T. spiralis were expressed by post-cyst larvae and adult worms, but not expressed by pre-cyst larvae or newborn larvae. The results showed that pre-cyst larvae and post-cyst larvae are similar but different in the expression of 53 and 19.6 kDa ES proteins. On the other hand, genes of housekeeping proteins, such as HSP and the DNA binding protein, were expressed at all stages although there were some differences in the expression level.
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PMID:Expression of excretory and secretory protein genes of Trichinella at muscle stage differs before and after cyst formation. 1211 53

A new RTE-like, non-long terminal repeat retrotransposon, termed SjR2, from the human blood fluke, Schistosoma japonicum, is described. SjR2 is approximately 3.9 kb in length and is constituted of a single open reading frame encoding a polyprotein with apurinic/apyrimidinic endonuclease and reverse transcriptase domains. The open reading frame is bounded by 5'- and 3'-terminal untranslated regions and, at its 3'-terminus, SjR2 bears a short (TGAC)(3) repeat. Phylogenetic analyses based on conserved domains of reverse transcriptase or endonuclease revealed that SjR2 belonged to the RTE clade of non-long terminal repeat retrotransposons. Further, SjR2 was homologous, but probably not orthologous, to SR2 from the African blood fluke, Schistosoma mansoni; this RTE-like family of non-long terminal repeat retrotransposons appears to have arisen before the divergence of the extant schistosome species. Hybridisation analyses indicated that approximately 10,000 copies of SjR2 were dispersed throughout the S. japonicum chromosomes, accounting for up to 14% of the nuclear genome. Messenger RNAs encoding the reverse transcriptase and endonuclease domains of SjR2 were detected in several developmental stages of the schistosome, indicating that the retrotransposon was actively replicating within the genome of the parasite. Exploration of the coding and non-coding regions of SjR2 revealed two notable characteristics. First, the recombinant reverse transcriptase domain of SjR2 expressed in insect cells primed reverse transcription of SjR2 mRNA in vitro. By contrast, recombinant SjR2-endonuclease did not appear to cleave schistosome or plasmid DNA. Second, the 5'-untranslated region of SjR2 was >80% identical to the 3'-untranslated region of a schistosome heat shock protein-70 gene (hsp-70) in the antisense orientation, indicating that SjR2-like elements were probably inserted into the non-coding regions of ancestral S. japonicum HSP-70, probably after the species diverged from S. mansoni.
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PMID:Reverse transcriptase activity and untranslated region sharing of a new RTE-like, non-long terminal repeat retrotransposon from the human blood fluke, Schistosoma japonicum. 1211 99


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