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)

Dissemination of lymph nodes is a known prognostic factor in colorectal carcinoma. Micrometastases in lymph nodes can be missed when studied by routine techniques. We analyzed 162 lymph nodes from 30 patients with colonic carcinoma and using routine techniques, they were classified as follows: two Dukes A; nineteen Dukes B; and nine Dukes C. A patient with benign colon disease served as negative control. Lymph nodes were all sectioned in halves, with one of the halves stored in liquid nitrogen for molecular biology examination by carcinoembryonic antigen expression. The other formalin-fixed and paraffin embedded halves were saved for both pathologic and immunohistochemical examination. For Dukes A and Dukes B tumors, reverse transcriptase-polymerase chain reaction (RT-PCR) had a 50% higher sensitivity in the detection of micrometastases. The expression of carcinoembryonic antigen (CEA) was detected in all Dukes C cases, which were considered as positive controls. These results showed that RT-PCR has a higher sensitivity in the detection of micrometastases than routine techniques, including immunohistochemistry.
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PMID:[Detection of micrometastases of colorectal carcinoma in lymph nodes]. 1462 49

The activated sludge process relies on the formation of strong microbial flocs. The knowledge about dominant floc-forming bacteria is at present very limited, especially from a phylogenetic perspective. In this study, numerous microcolonies in the activated sludge flocs were found to be targeted by a Betaproteobacteria-group-specific oligonucleotide probe using fluorescence in situ hybridization (FISH). Some of these were micromanipulated and further identified by reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing to belong to the Aquaspirillum genus in the Neisseriaceae family. A specific oligonucleotide probe, Aqs997, was designed to target the identified bacteria. A survey in nine different wastewater treatment plants with nutrient removal (WWTP) showed a high abundance of bacteria hybridizing to the oligonucleotide probe developed. Microautoradiography (MAR) combined with FISH on activated sludge incubated with radiolabelled substrate showed uptake of substrate with oxygen, nitrate and nitrite as electron acceptor demonstrating a denitrifying potential of the bacteria investigated. The Aquaspirillum-related bacteria seemed to be abundant denitrifiers in WWTPs with nitrogen removal and they were particularly numerous in plants mainly receiving domestic wastewater, where they constituted up to 30% of all bacteria.
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PMID:Micromanipulation and further identification of FISH-labelled microcolonies of a dominant denitrifying bacterium in activated sludge. 1504 20

Regulation of medium-chain-length polyhydroxyalkanoate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida was studied conducting PHA accumulation experiments and transcriptional analysis of PHA biosynthesis genes with wild type strains and rpoN-negative mutants. In P. putida PHA accumulation was RpoN-independent, whereas in P. aeruginosa PHA accumulation was RpoN-dependent. Transcriptional analysis applying reverse transcriptase-polymerase chain reaction showed strong induction of phaG, encoding the transacylase, under nitrogen starvation in P. putida KT2440 and the respective rpoN-negative mutant, indicating an RpoN-independent regulation of phaG. No transcription of phaG and no PHA accumulation was detected in the rpoN-negative mutant of P. aeruginosa neither from gluconate nor from octanoate as carbon source. Alginate-overproducing mutant P. aeruginosa FRD1 showed strongly decreased PHA accumulation from gluconate but no difference in phaC1 (encoding the PHA synthase) transcription, indicating that alginate biosynthesis competes with PHA biosynthesis regarding acetyl-CoA as precursor for both biopolymers. Transcription of phaF and phaI-F was nitrogen independent.
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PMID:Regulation of polyhydroxyalkanoate biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa. 1526 31

An HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz is proposed here for the simultaneous analysis of the new HIV protease inhibitor atazanavir (ATV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection. After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl) with clozapine (internal standard) is diluted 1 + 1 with phosphate buffer pH 7 and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H(3)PO(4) neutralised with NaOH to pH 7. ATV is eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl MeOH/H(2)O 50/50. A 40 microl volume is injected onto a Nucleosil 100-5 microm C18 AB column. ATV is analysed by UV detection at 201 nm using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.14. The mobile phase also contains 0.02% sodium heptanesulfonate, enabling an excellent separation of ATV from the other HIV protease inhibitors (PIs) amprenavir, indinavir, saquinavir, ritonavir, lopinavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. The calibration curves are linear up to 10 microg/ml, with a lower limit of quantification of 0.2 microg/ml. The mean absolute recovery of ATV is 96.4 +/- 3.2%. The method is precise with mean inter-day CVs within 1.1-6.1%, and accurate (range of inter-day deviations +0.3 to +2.3%). The method has been validated and is currently applied to the monitoring of ATV in HIV patients.
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PMID:Determination of the new HIV-protease inhibitor atazanavir by liquid chromatography after solid-phase extraction. 1535 4

Osteocytes, the most abundant bone cell type with important roles in tissue maintenance and pathological aberrations such as observed in bone metastases, are enclosed within a highly compact, calcified extracellular matrix. This location complicates analysis in native bone, with the consequence that despite their importance their in vivo molecular physiology is only poorly understood. We have examined the possibility of isolating osteocyte RNA for transcript profiling from native, frozen bone instead of employing the formalin-fixed, paraffin-embedded, decalcified version routinely used in histology, providing chemically modified and highly disintegrated RNAs. Bone tissue was tape-assisted cryosectioned and fixed to glass slides by support of UV-flash-triggered adhesive polymerization followed by quick hematoxylin-eosin staining to generate a guidance image for microdissection. Using an UVa-nitrogen laser, matrix-enclosed osteocytes were either excised and catapulted into RNA preparation vials or freed of accompanying nonosteocyte cellular material. The influences of bone sectioning, staining, and osteocyte capturing procedures on the prepared osteocyte RNAs were analyzed and the method was optimized accordingly. The obtained osteocyte RNAs showed the expected expression pattern of marker genes (reverse transcriptase-polymerase chain reaction), and, following conversion into fluorescent-labeled cDNAs, led to transcript profiles (cDNAchips; 2600 genes) with scatter-graph geometries indicating suitability for high-confidence evaluation. With the approach described here we introduce a methodological way for the characterization of the in vivo molecular physiology of osteocytes by functional genomics.
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PMID:High-quality RNA preparation for transcript profiling of osteocytes from native human bone microdissections. 1555 65

Nitric oxide (NO) derived from inducible NO synthase has been implicated in cardiac rejection. However, little is known about the role of the reactive nitrogen species peroxynitrite. We examined the protective actions of a peroxynitrite decomposition catalyst, WW85, in an experimental model of acute cardiac rejection. Heterotopic, abdominal transplantation of rat donor hearts was performed. Groups included isografts, allografts, or allografts treated with WW85, cyclosporine, or cyclosporine + WW85. We determined graft survival, histological rejection, and graft function (by in situ sonomicrometry). Intragraft biochemical analysis of cytokines and proapoptotic and antiapoptotic gene expression using reverse transcriptase-polymerase chain reaction were determined. Treatment with WW85 or cyclosporine alone prolonged graft survival, improved graft function, and decreased histological rejection. Graft survival was further significantly (P < 0.001) enhanced by combination treatment. A decrease was also shown in nitrotyrosine, poly(ADP-ribose) polymerase (PARP) activation, and lipid peroxide formation by WW85 that was potentiated when given in combination with cyclosporine. Benefits could not be ascribed to changes in intragraft myeloperoxidase activity. Only combination therapy produced significant decreases in inflammatory cytokine gene expression, suggesting that WW85 acted primarily downstream of these stimuli. In general, WW85 had no direct action on expression of the proapoptotic gene, Fas ligand; however, WW85 given alone or with cyclosporine enhanced expression of antiapoptotic genes Bcl-2 and Bcl-xL. Collectively, these findings suggest a protective action of the peroxynitrite decomposition catalyst WW85 on graft rejection that is independent of any action on leukocyte sequestration and cytokine gene expression. Rather, effects seem to be downstream on decreased protein nitration, decreased lipid peroxidation, and decreased PARP activation.
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PMID:Protective mechanisms of a metalloporphyrinic peroxynitrite decomposition catalyst, WW85, in rat cardiac transplants. 1578 53

We examined the effect of Buthus martensi Karsch (BMK) extract on IL-1beta-induced production of nitrogen oxide (NO) in primary human osteoarthritis (OA) chondrocytes. The cells were treated with BMK (10 microg/ml) and IL-1beta (2 ng/ml) for different periods, and inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The cytotoxicity of BMK on human OA chondrocytes was very low (IC50 > 250 microg/ml) as measured by the XTT assay method. Production of NO was determined as nitrite in culture supernatant. Human chondrocytes cotreated with BMK produced significantly less NO compared with chondrocytes stimulated with IL-1beta alone. Activation and translocation of and NF-(kappa)B DNA binding activity were determined by Western blotting and specific enzyme-linked immunosorbent assay. The inhibition of NO production correlated with the suppression of induction and expression of nuclear factor-kappaB (NF-(kappa)B) and activation protein-1 (AP-1)-dependent gene. BMK inhibited the activation and translocation of NF-(kappa)B to the nucleus, indicating that BMK inhibits the IL-1beta-induced production of NO in human chondrocytes by interfering with the activation of NF-(kappa)B through a novel mechanism. In addition, BMK reduced prostaglandin E2 (PGE2) production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) or cyclooxygenase-1 (COX-1) was observed. Our data, therefore, suggest that BMK may be a therapeutically effective inhibitor of IL-1beta-induced inflammatory effects that are dependent on NF-(kappa)B activation in human OA chondrocytes. The results indicate that BMK exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and PGE2 production, which could be due to a decreased expression of iNOS and COX-2 through the transcription factors NF-(kappa)B and AP-1.
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PMID:Inhibitory effect of Buthus martensi Karsch extracts on interleukin-1beta-induced expression of nitric oxide (NO) synthase and production of NO in human chondrocytes and LPS-induced NO and prostaglandin E2 production in mouse peritoneal macrophages. 1596 82

Polymorphonuclear leukocytes have been shown to use a multitude of effector functions to combat pathogens and tumors, including enzymes, defensins, and toxic products such as oxygen radicals and nitrogen oxides. Recent studies provided evidence for the expression of granzymes (gzms) and perforin (perf) within the cytotoxic arsenal of human neutrophils, the validity of which was questioned by 2 subsequent studies. We have now used cytology, intracellular flow cytometry, enzymatic assays, immunoelectron microscopy, and quantitative reverse transcriptase-polymerase chain reaction to obtain evidence of the presence of gzms and/or perf in mouse Gr-1+ granulocyte populations. The data obtained clearly demonstrate that neither in vitro- nor in vivo-derived mouse granulocytes synthesize gzmA and gzmB or perf, even following infection/immunization with pathogens or pathogen-derived material. A parallel comparable analysis on the expression of gzmB in human neutrophils from 3 healthy control subjects and 4 patients with diverse diseases failed to detect gzmB expression. The data indicate that polymorphonuclear leukocytes from mice and humans lack the 3 cytotoxic effector molecules, gzmA, gzmB, and perf, generally associated with natural killer and cytotoxic T lymphocytes.
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PMID:Quiescent and activated mouse granulocytes do not express granzyme A and B or perforin: similarities or differences with human polymorphonuclear leukocytes? 1599 31

Organic ions are excreted into the urine via the action of organic anion transporters (OATs). In adult kidney, both OAT1 and OAT3, both multispecific transporters, are abundant; OAT1 is a known transporter of para-aminohippurate (PAH) and OAT3 is a known transporter of sulfoconjugated estrogens. The present study was designed to test the hypotheses that the expression of both OAT1 and OAT3 are developmentally regulated and that the expression increases in late gestation. Fetal kidneys were collected at sacrifice of fetal sheep at 80, 100, 120, 130, and 145 days of gestation, as well as 1 day and 1 week after birth (n=4-5 per group). Renal tissue was separated into cortex and medulla and snap-frozen in liquid nitrogen for later extraction of mRNA. The expression levels of OAT1 and OAT3 were measured using real-time reverse transcriptase polymerase chain reaction (RT-PCR), with specific probes and primers designed in our laboratory. Cellular distribution of protein expression was identified using immunohistochemistry with commercially available antisera. The OAT1 and OAT3 mRNA in renal cortex was increased in the more mature animals. At 145 days of gestation, OAT1 mRNA abundance was increased and remained elevated postnatally. Compared with prenatal ages, OAT3 mRNA was increased postnatally. The expression of both transporters was not significantly changed as a function of development in the renal medulla. The protein expression of OAT1 and OAT3 was identified in tubular epithelium in renal cortex, although the immunoreactivity for OAT1 was greater than for OAT3. We conclude that there is a developmental pattern of expression of both OAT1 and OAT3 in ovine renal cortex, and that the pattern of expression suggests that the function of both transporters is likely to be greater starting in late gestation.
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PMID:Ontogeny of expression of organic anion transporters 1 and 3 in ovine fetal and neonatal kidney. 1617 35

The availability of fixed inorganic nitrogen often plays a fundamental role in regulating primary production in both aquatic and terrestrial ecosystems. Because biological nitrogen fixation is an important source of nitrogen in marine environments, the study of N2-fixing microorganisms is of fundamental importance to our understanding of global nitrogen and carbon cycles. Quantitative molecular tools have made it possible to examine uncultivated N2-fixing microorganisms directly in the environment. Currently, we are using quantitative polymerase chain reaction (PCR; Q-PCR) and quantitative reverse transcriptase PCR (Q-RT-PCR) to study the ecology and gene expression of N2-fixing bacteria in aquatic environments. Using these methods, we discovered that specific estuarine diazotrophs have distinct nonrandom distributions and that some diazotrophs in the open ocean have different diel patterns of nifH gene expression. This chapter describes briefly our 5' nuclease assay protocols for Q-PCR and Q-RT-PCR of nifH gene fragments in environmental samples and discusses some important methodological considerations for the quantitative molecular examination of microbes in aquatic environments.
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PMID:Quantitative analysis of nifH genes and transcripts from aquatic environments. 1626 Mar 4


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