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)

Two analog uridine triphosphates tethering additional functionality, one a primary amino group and the second a mercapto group, were prepared and tested for their compatibility with in vitro RNA selection procedures. 5-(3-Aminopropyl)uridine triphosphate (UNH(2)) as a uridine substitute was a more effective substrate for T7 RNA polymerase than 5-(2-mercaptoethyl)uridine triphosphate (USH). However, both functioned in transcription assays of 100 nt templates to generate RNA transcripts in quantities sufficient to initiate RNA selection procedures. Transcription of RNA pools with T7 RNA polymerase and UNH(2) or USH occurred with efficiencies of 43 and 29%, respectively, of the values obtained for native UTP transcription. In addition, the transcribed RNA containing roughly 25% UNH(2) residues exhibited better substrate properties for SuperScript(TM) II RNase H reverse transcriptase than did RNA transcripts containing approximately 25% of the USH analog. With either analog, both transcription and reverse transcription proceeded with high fidelity for insertion of the analog residue.
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PMID:Expanding the structural and functional diversity of RNA: analog uridine triphosphates as candidates for in vitro selection of nucleic acids. 1095

Renin secretion can be stimulated by ATP via purinergic P2Y receptors. ATP is a cotransmitter with norepinephrine and is released from the cytosol during cell damage. Such release could account for the de novo renin expression seen in the proximal tubule in renal disease and in myocardial infarct borders. Whereas most P2Y purinoceptor subtypes utilize phosphoinositide signal-transduction pathways, the effector mechanisms of the subtype P2Y(11) also involve increases in cAMP, a well-known renin secretagogue and stimulus to renin production. The present study tested the effect of ATP on human renin gene (REN) promoter activity and the role of P2Y(11). By means of reverse transcriptase-polymerase chain reaction, we found that renin-expressing Calu-6 cells express P2Y(11) mRNA. Expression was also detected in the brain, kidney, testis, muscle, liver, and spleen. We made a novel cell line (Calu-6/P2Y11) in which P2Y(11) cDNA, under the control of a strong promoter, was stably integrated into genomic DNA. These cells produced P2Y(11) mRNA during culture. Treatment of Calu-6/P2Y11 cells with 1 mmol/L ATP caused a 3-fold increase in renin mRNA and protein over 36 hours. Transient transfection of Calu-6/P2Y11 cells with constructs containing 896 bp of human REN 5'-flanking DNA linked to the luciferase reporter gene led to a 5.8+/-0.6-fold increase (mean+/-SEM) in reporter activity in response to ATP (P=0.0015). In contrast, UTP produced only a 1.4+/-0.1-fold increase (P=0.016). For ADP, it was 1.7+/-0.1-fold (P=0.011). The response profile was ATP>ADP>AMP=adenosine=0, consistent with a P2Y(11) effect. Mutation of the cAMP response element (CRE) located at -222 in the REN promoter DNA abolished the effect of ATP. Furthermore, ATP induced a rapid, time-dependent increase in the phosphorylation of CRE binding protein (CREB) and activating transcription factor-1. These data implicate a cAMP pathway in mediation of the P2Y(11) effect. In conclusion, we have made a novel cell line that overexpresses the P2Y(11) purinoceptor. Stimulation of these cells by ATP activates a cAMP signal-transduction pathway that phosphorylates CREB and stimulates renin promoter activity via the CRE at -222. The data raise the possibility of a contribution of ATP/P2Y(11) effects to sympathetic stimulation of renin, as well as to responses in renin seen after tissue damage, such as in kidney disease and myocardial infarction.
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PMID:Capacity for purinergic control of renin promoter via P2Y(11) receptor and cAMP pathways. 1111 31

Apical ATP, ATP, UTP and UDP evoked transient increases in short circuit current (I(SC), a direct measure of transepithelial ion transport) in confluent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors. Experiments using cells grown on glass coverslips showed that ATP and UTP consistently increased intracellular free calcium ([Ca(2+)](i)) whilst sensitivity to UDP was variable. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. Messenger RNA transcripts corresponding to the P2Y(2), P2Y(4) and P2Y(6) receptors genes were detected in cells grown on Transwell membranes by the reverse transcriptase - polymerase chain reaction. Identical results were obtained for cells grown on glass. Experiments in which I(SC) and [Ca(2+)](i) were monitored simultaneously in cells on Transwell membranes, confirmed that apical ATP and UTP increased both parameters and showed that the UDP-evoked increase in I(SC) was accompanied by a [Ca(2+)](i)-signal. Ionomycin consistently increased [Ca(2+)](i) in such polarized cells but caused no discernible change in I(SC). However, subsequent application of apical ATP or UTP evoked a small rise in I(SC) but no rise in [Ca(2+)](i). UDP evoked no such response. As well as evoking increases in [Ca(2+)](i), the ATP/UTP-sensitive receptors present in Caco-2 cells thus allow direct control over ion channels in the apical membrane. The UDP-sensitive receptors, however, appear to simply evoke a rise in [Ca(2+)](i).
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PMID:Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells. 1113 43

There are various techniques to detect mRNA in tissue specimens. Among these in situ hybridization is widely applied, and for the detection of small quantities of RNA in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR) has been applied. Furthermore in situ transcription, where signal is produced by direct incorporation of labeled nucleotides during production of a cDNA by reverse transcription, has been shown by a few investigators. We present a non-radioactive in situ reverse transcriptase (in situ RT) protocol which is at least as sensitive as in situ hybridization but avoids probe production and long procedures of preincubation, incubation, and washing. Digoxigenin-labeled UTP is incorporated into a cDNA produced by in situ reverse transcription of mRNA. This method is combined with the fast and sensitive immunogold-silver detection system allowing demonstration of the mRNA within 7 h compared to days in the case of in situ hybridization. Contrary to in situ RT-PCR this new method of in situ RT has no background problems due to non-specific amplification or diffusion of the reaction product.
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PMID:Detection of mRNA by non-radioactive direct primed in situ reverse transcription. 1168 47

The Fas system is involved in the regulation of germ cell apoptosis associated with testicular injury in experimental animals exposed to various insults. We tested the hypothesis that enhanced germ cell apoptosis mediated by the up-regulation of the Fas system and the activation of caspases may be involved in ethanol-induced testicular injury. Adult Wistar rats were fed either ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 12 weeks. Marked Sertoli cell vacuolization and germ cell degeneration were observed in the testes of ethanol-treated rats (ETR) by both light and electron microscopy. Enhanced apoptosis of germ cells in ETR was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labelling (TUNEL) method, transmission electron microscopy, and was associated with elevated activity of caspase-3, -8 and -9. The expression levels of the Fas ligand (FasL) in Sertoli cells and of both Fas and caspase-3 in germ cells of ETR detected immunohistochemically were higher than those of the control testes. Furthermore, reverse transcriptase-polymerase chain reaction analysis demonstrated an increase in both Fas and FasL mRNA levels in ETR. Fas system up-regulation and the elevated activity of caspases in the testes of ETR may be a reflection of ethanol-induced testicular injury resulting in enhanced germ cells apoptosis, which may be involved in infertility associated with alcohol abuse.
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PMID:Involvement of Fas system and active caspases in apoptotic signalling in testicular germ cells of ethanol-treated rats. 1203 Oct 44

Human airway epithelia express Ca2+-activated Cl- channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K+ channels in CaCC-mediated Cl- secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K+ channels, and by clotrimazole, an inhibitor of Ca2+-activated hSK4 K+ channels. The K+ channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl- secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2+-activated Cl- secretion in native human airway epithelia requires activation of Ca2+-dependent basolateral K+ channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl- secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.
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PMID:Modulation of Ca2+-activated Cl- secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia. 1261 94

A convincing body of evidence indicates that estrogen has significant immunomodulatory properties, including induction of thymic involution. However, it is unclear whether or not estrogen induces thymic involution by triggering apoptosis depended on Fas-FasL interactions. In the present study, estradiol-17beta (E(2)) was used to treat rats by gavages at 10, 1, 0.1, 0.01, and 0 ng/kg/day, respectively. Atrophy of thymus was determined by in situ morphological examination. Apoptotic cells were identified by terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labeling (TUNEL) assay. A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze Fas and FasL mRNA levels. The results showed that E(2) induced thymic atrophy, increased the rates of apoptotic death, and enhanced the Fas/FasL mRNA levels. These findings suggested that Fas/FasL-mediated apoptosis involved in the induction of thymic atrophy by E(2).
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PMID:Thymic atrophy via estrogen-induced apoptosis is related to Fas/FasL pathway. 1499 13

UDP-sugars, activated forms of monosaccharides, are synthesized through de novo and salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDP-sugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDP-Gal, UDP-glucuronic acid, UDP-l-arabinose, and UDP-xylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5-7.5, and at 45 degrees C in the presence of 2 mm Mg(2+). The apparent K(m) values for Glc 1-phosphate and l-arabinose 1-phosphate were 0.34 and 0.96 mm, respectively. PsUSP cDNA was cloned by reverse transcriptase-PCR. PsUSP appears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues from Arabidopsis and rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed in Escherichia coli catalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDP-sugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.
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PMID:UDP-sugar pyrophosphorylase with broad substrate specificity toward various monosaccharide 1-phosphates from pea sprouts. 1532 66

The epithelial-mesenchymal interactions have an important role in the folliculomorphogenesis and mature hair follicle cycling. The dermal papilla, a dense aggregate of specialized dermis-derived stromal cells located at the bottom of the hair follicle, is a major component of hair, which signals the follicular epithelial cells to prolong the hair growth process. However, to date, little is known about the significance of the specific gene(s) expression in the dermal papilla cells with regard to their aggregative behaviour and hair cycling. In our previous study, the differentially gene-expressed cDNAs library had been determined by means of suppression subtractive hybridization technique between the aggregated human dermal papilla cells and control cells. Among those cDNAs library, the haematopoietic stem/progenitor cell (HSPC)-related gene HSPC016 was found. In this study, the gene HSPC016 was confirmed to express in the human dermal papilla cells by means of in situ hybridization and reverse transcriptase-polymerase chain reaction. In order to rudimentarily elucidate its biological function, a recombinant eucaryotic expressing plasmid pcDNA3.1(+)/HSPC016 was constructed and was transfected into the human dermal papilla cells and 3T3 fibroblast cells by means of Nucleofector(TM) technique (Amaxa, Cologne, Germany). Terminal deoxynucleotidyl transferase-mediated d-UTP nick end Labelling (TUNEL) assay showed that the expression of gene HSPCO16 resulted in the apoptosis of these cells, which suggested that the apoptosis of dermal papilla cells might be associated with the expression of gene HSPC016 in vitro.
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PMID:Apoptosis of the dermal papilla cells of hair follicle associated with the expression of gene HSPCO16 in vitro. 1574 May 94

Ionizing radiation has been reported to promote accelerated or premature senescence in both normal and tumour cell lines. The current studies were designed to characterize the accelerated senescence response to radiation in the breast tumour cell in terms of its dependence on functional p53 and its relationship to telomerase activity, telomere lengths, expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of telomerase) and human telomerase RNA (hTR, the RNA subunit of telomerase), as well as the induction of cytogenetic aberrations. Studies were performed in p53 wild-type MCF-7 cells, MCF-7/E6 cells with attenuated p53 function, MDA-MB231 cells with mutant p53 and MCF-7/hTERT cells with constitutive expression of hTERT. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP assay), telomere lengths by the terminal restriction fragment (TRF) assay, hTR and hTERT expression by reverse transcriptase-polymerase chain reaction (RT-PCR), senescence by beta-galactosidase staining, and apoptosis by TdT-mediated d-UTP-X nick-end labelling (TUNEL assay). Widespread and extensive expression of beta-galactosidase, a marker of cellular senescence, was evident in MCF-7 breast tumour cells following exposure to 10 Gy of ionizing radiation. Radiation did not suppress expression of either hTERT or hTR, alter telomerase activity or induce telomere shortening. Senescence arrest was also observed in irradiated MCF-7/hTERT cells, which have elongated telomeres due to the ectopic expression of the catalytic component of telomerase. In contrast to MCF-7 cells, irradiated MDA-MB231 breast tumour cells and MCF-7/E6 cells failed to senesce and instead demonstrated a delayed apoptotic cell death. Irradiation produced chromosome end associated abnormalities, including end-to-end fusions (an indicator of telomere dysfunction) in MCF-7 cells, MCF-7/hTERT cells, as well as in MCF-7/E6 cells. When cells were maintained in culture following irradiation, proliferative recovery was evident exclusively after senescence while the cell lines which responded to radiation by apoptosis continued to decline in cell number. Accelerated senescence in response to ionizing radiation is p53 dependent and associated with telomer dysfunction but is unrelated to changes in telomerase activity or telomere lengths, expression of hTERT and hTR. In the absence of functional p53, cells are unable to arrest for an extended period, resulting in apoptotic cell death while accelerated senescence in cells expressing p53 is succeeded by proliferative recovery.
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PMID:p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells. 1630 15


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