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)

In oviparous females, the synthesis of the yolk precursor vitellogenin is an important step in ovarian maturation and oocyte development. In decapod Crustacea, including the red-claw crayfish (Cherax quadricarinatus), this reproductive process is regulated by inhibitory neurohormones secreted by the endocrine X-organ-sinus gland (XO-SG) complex. In males, the C. quadricarinatus vitellogenin gene (CqVg), although present, is not expressed under normal conditions. We show here that endocrine manipulation by removal of the XO-SG complex from male animals induced CqVg transcription. The CqVg gene was expressed differentially during the molt cycle in these induced males: no expression was seen in the intermolt stages, but expression was occasionally detected in the premolt stages and always detected in the early postmolt stages. Relative quantitation with a real-time reverse transcriptase-polymerase chain reaction showed that expression of CqVg in induced early postmolt males was an order of magnitude lower than that in reproductive females, a finding that was consistent with RNA in situ hybridization results. The SDS-PAGE of high-density lipoproteins from the hemolymph of endocrinologically induced early postmolt males did not show the typical vitellogenin-related polypeptide profile found in reproductive females. On the other hand, removal of the XO-SG complex from intersex individuals, which are chromosomally female but functionally male and possess an arrested female reproductive system, induced the expression, translation, and release of CqVg products into the hemolymph, as was the case for vitellogenic females. The expression of CqVg in endocrinologically manipulated molting males and intersex animals provides an inducible model for the investigation and understanding of the endocrine regulation of CqVg expression and translation in Crustacea as well as the relationship between the endocrine axes regulating molt and reproduction.
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PMID:Expression of the reproductive female-specific vitellogenin gene in endocrinologically induced male and intersex Cherax quadricarinatus crayfish. 1574 19

A novel protein, named BAS-AH, was purified and characterized from the skin of the toad Bufo andrewsi. BAS-AH is a single chain protein and the apparent molecular weight is about 63 kDa as judged by SDS-PAGE. BAS-AH was determined to bind heme (0.89 mol heme/mol protein) as determined by pyridine haemochrome analysis. Fifty percentage cytotoxic concentration (CC50) of BAS-AH on C8166 cells was 9.5 microM. However, at concentrations that showed little effect on cell viability, BAS-AH displayed dose dependent inhibition on HIV-1 infection and replication. The antiviral selectivity indexes (CC50/EC50) were 14.4 and 11.4, respectively, corresponding to the measurements of syncytium formation and HIV-1 p24 antigen expression. BAS-AH also showed an inhibitory effect on the activity of recombinant HIV-1 reverse transcriptase (IC50 = 1.32 microM). The N-terminal sequence of BAS-AH was determined to be NAKXKADVIGKISILLGQDNLSNIVAAM, which exhibited little identity with other known anti-HIV-1 proteins. BAS-AH is devoid of antibacterial, proteolytic, trypsin inhibitory activity, l-amino acid oxidase activity and catalase activity.
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PMID:A novel heme-containing protein with anti-HIV-1 activity from skin secretions of Bufo andrewsi. 1615 59

A 67-kDa hemagglutinin composed of two identical subunits was purified from Phaseolus vulgaris cv. 'Dark Red Kidney Bean'. It was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. The hemagglutinin was highly purified after the two aforementioned chromatographic steps as revealed by a single peak in gel filtration on Superdex 75 and a single band in SDS-PAGE. The hemagglutinating activity was stable between 25 degrees C and 70 degrees C, and between pH 4 and pH 11, and in the presence of a variety of divalent metal chlorides at 500 mM concentration. The activity was reduced by 50% at 80 degrees C, and also when the pH was lowered to 3 or elevated to 12. The activity was reduced by 75% in the presence of 250 mM KCl or NaCl. A variety of sugars tested failed to inhibit the hemagglutinating activity of the hemagglutinin. Although the hemagglutinin exhibited mitogenic activity toward murine splenocytes, it had no effect on the activity of HIV-1 reverse transcriptase or mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. It exerted an antiproliferative activity on leukemia L1210 cells.
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PMID:A hemagglutinin with mitogenic activity from dark red kidney beans. 1694 95

The nucleocapsid (N) gene of human coronavirus strain OC43 (HCoV-OC43) was amplified by reverse transcriptase-polymerase chain reaction, and cloned in pENTR/D-TOPO plasmid. This plasmid containing the N gene was recombined with in a BaculoDirect baculovirus DNA designed in order to express N protein in fusion with a C-terminal polyhistidine tag containing V5 epitope. Sf21 cells were transfected with recombinant baculovirus DNA. Recombinant N protein was extracted from infected cells, analysed by SDS-PAGE and Western blot, and purified by Ni2+ affinity procedure. Sera from 100 healthcare workers and five 2-3-year-old children were tested in a Western blot assay using the purified recombinant N protein. All of the sera from adults and two of the sera from children have a positive result.
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PMID:Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43. 1707 26

The gene, xynB, from Streptomyces olivaceoviridis A1 encoding xylanase, XYNB, with a high specific activity for xylan, was transformed into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens. The integration of xynB into genomic DNA was confirmed by PCR and reverse transcriptase-PCR. The gene was expressed under the control of a constitutive double cauliflower mosaic virus (CaMV) 35S promoter. Both SDS-PAGE and western blot analysis showed high levels of expression of the 21 kDa and 31 kDa XYNB proteins in transgenic potato plants transformed by the binary vectors pBinXy and signal peptide contained pBinSPXy, respectively. The recombinant XYNB protein was present at up to 5% of total soluble leaf protein in the cytoplasm. In transgenic leaf and tuber extracts, xylanase activity was up to 87 micromol min(-1) g(-1) fresh leaf (9.7 micromol min(-1) mg(-1) total soluble protein). The xylanase was stable at 60 degrees C and 70 degrees C in buffers (pH 5.2) for 5 min. Furthermore, the xylanase enzymatic activity remained virtually unchanged over several generations of potato. These results demonstrate that the transgenic potato can be used to produce recombinant xylanase with high specific enzyme activity and can potentially be an alternative to present-day xylanase additives to animal feed.
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PMID:Expression of xylanase with high specific activity from Streptomyces olivaceoviridis A1 in transgenic potato plants (Solanum tuberosum L.). 1720 70

RNA isolation from Streptococcus mutans within biofilms is challenging because of the presence of extracellular polysaccharide matrix that interferes with RNA extraction procedures. In an effort to solve this difficult problem, we examined several protocols to extract and purify RNA from S. mutans biofilms. A combination of sonication (three times using a 30-s pulse at 7 W) with washing in phosphate-buffered saline removed most of the extracellular polysaccharides from the biofilms and provided the highest RNA yield. Further homogenization-mechanical cells disruption in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA, and 1% SDS, pH 5.0) and acid phenol/chloroform yielded 547.2+/-23.4 microg RNA/100 mg of biofilm dry weight. An additional acid phenol/chloroform extraction further improved the purification of RNA without significantly affecting the RNA yield. The combination of DNase I in silica gel-based column and recombinant DNase I in solution effectively removed the genomic DNA as determined by real-time quantitative reverse transcriptase PCR (RT-PCR), resulting in 92.0+/-0.6 microg of purified RNA per 100 mg of biofilm dry weight. The complementary DNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results demonstrated a method that yields high-quality RNA from biofilms of S. mutans in sufficient quantity for real-time RT-PCR analyses, and our data have relevance for isolation of RNA from other biofilm-forming microorganisms.
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PMID:Extraction and purification of total RNA from Streptococcus mutans biofilms. 1747 97

Calreticulin (CRT) is a resident protein of the endoplasmic reticulum where it serves as a calcium modulator and chaperone to newly synthesized glycoproteins. In mammals, CRT is a structurally conserved 46 kDa protein that demonstrates anomalous migration at 60 kDa on SDS polyacrylamide gels and can be up-regulated by A23187 and thapsigargin due to the endoplasmic reticulum stress elements (ERSE) in the promoter region of its gene. CRT has numerous proposed functions and has been localized to the surface of PHA-stimulated T lymphocytes. CRT has been identified in mammals, plants and more recently from rainbow trout. Here, we report the cloning of the CRT proximal promoter from rainbow trout which includes elements typical of genes transcribed by RNA polymerase II including a TATA box, an Sp1 binding site, CCAAT boxes and the conservation of promoter stress elements (ERSE) demonstrated to be responsible for calcium modulation in mammals. This report demonstrates that the anomalous 60 kDa gel migration of mammalian CRT is conserved in rainbow trout and that CRT exists primarily as a dimer or oligomer in all tissues tested, excluding muscle and sperm in which it exists as a single polypeptide. Although it contains a potential N-glycosylation site, rainbow trout CRT is not subject to N-type glycosylation. Through the use of reverse transcriptase (RT) PCR along with western blotting, in both primary cultured leukocytes and the macrophage cell line RTS11, this report demonstrates that, unlike mammals, rainbow trout CRT is not strongly up-regulated by the calcium homeostasis antagonists, A23187 and thapsigargin, but is present on the cell surface of PHA-stimulated leukocytes. Taken together, this data suggests that CRT may have an alternative mode of regulation or function in fish.
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PMID:Calreticulin in rainbow trout: a limited response to endoplasmic reticulum (ER) stress. 1749 Sep 7

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerase gene to express the target protein. HBV polymerase expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.
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PMID:High-level production of a functional recombinant hepatitis B virus polymerase in insect cells with a baculovirus expression system. 1764 39

This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing-regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real-time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxR(Vh) mRNA levels in wild-type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxR(Vh) response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxR(Vh) protein also showed less shifts with furanone-treated LuxR(Vh), whereas the LuxR(Vh) concentration was found not to be altered by the furanone (as determined by SDS-PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxR(Vh) unable to bind to the promoter sequences of quorum sensing-regulated genes.
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PMID:The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxR. 1780 74

A third fructan exohydrolase isoform (1-FEHw3) was purified from wheat stems by a combination of ammonium sulfate precipitation, ConA affinity and ion-exchange chromatography. Homogeneity of the preparation was indicated by the presence of a single band (70 kDa) after SDS-PAGE. The enzyme hydrolyzed mainly beta2-1 linkages in fructans and was inhibited by sucrose. A cDNA could be obtained after reverse transcriptase polymerase chain reaction (RT-PCR)-based strategies and screening of a cDNA library. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed that the encoded protein has essentially the same characteristics as the native enzyme. Homology with previously described 1-FEH isoforms from wheat was high (97% identity), and the enzyme showed minor differences to the previously published enzymes. The relative abundance of 1-FEH transcripts in different tissues was investigated by using quantitative RT-PCR.
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PMID:Purification, cloning and functional differences of a third fructan 1-exohydrolase (1-FEHw3) from wheat (Triticum aestivum). 1834 83


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