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)

Brevican is a member of the aggrecan/versican family of proteoglycans. In contrast to the other family members, brevican occurs both as soluble isoforms secreted into the extracellular space and membrane-bound isoforms which are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety. Expression of both variants, which are encoded by two differentially processed transcripts from the same gene, is confined to the nervous system. In the current study, we have used in situ hybridization to examine the cellular sites of synthesis for both mRNAs during postnatal development of the rat brain. Whereas the 3.6-kb transcript encoding secreted brevican displays a widespread distribution in grey matter structures, including cerebellar and cerebral cortex, hippocampus and thalamic nuclei with silver grains accumulating over neuronal cell bodies, the smaller transcript (3.3 kb) encoding GPI-anchored isoforms appears to be largely confined to white matter tracts and diffusely distributed glial cells. This expression pattern is further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments with RNA from different glial cell cultures, and by biochemical data demonstrating that the crude membrane fraction from isolated optic nerve contains high amounts of phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive brevican immunoreactivity. During ontogenetic development, both brevican transcripts are generally up-regulated. However, the expression of glypiated brevican is delayed by about 1 week, compared with the expression of the secreted isoform. This late appearance of GPI-linked brevican, its predominant expression in glial cells and its tight association with brain myelin fractions suggest a functional role in neuroglia.
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PMID:Transcripts for secreted and GPI-anchored brevican are differentially distributed in rat brain. 975 Nov 35

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

Lymphocyte function associated antigen-3 (LFA-3) is a cell surface glycoprotein involved in antigen independent T-cell activation and proliferation. The expression and function of LFA-3 at the blood-brain barrier were studied in an in vitro model consisting of primary cultures of human brain microvessel endothelial cells (HBMEC). Surface expression of LFA-3 was detected by immunogold silver staining and the presence of RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Unstimulated HBMEC in primary culture constitutively express LFA-3 on their surface. Expression is only marginally upregulated following stimulation with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma). Similarly, LFA-3 RNA is present constitutively in unstimulated HBMEC with minimal increase after co-incubation with TNF-alpha and IFN-gamma. The function of LFA-3 as a costimulatory molecule on HBMEC was investigated by incubating purified CD4+ T-lymphocytes with resting or IFN-gamma treated HBMEC monolayers. Proliferation of alpha-CD3 activated CD4+ T-cells was significantly increased upon incubation with resting or activated endothelial cells. Monoclonal antibodies to LFA-3 consistently blocked the proliferative response by 64-76%. The ability of the cerebral endothelium to express LFA-3 and provide secondary signals for T-cell proliferation suggests that cerebral EC may be important in the initiation of inflammatory responses in the human central nervous system.
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PMID:Expression and function of lymphocyte function associated antigen-3 (LFA-3) at the blood-brain barrier. 1009 37

The effect of 44 different metal ions (Ag+, Al3+, As(O-)2, Au3+, Ba2+, Be2+, Bi3+, Cd2+, Ce3+, CO2+, Cr(O2-)4, Cr3+, Cs+, Cu2+, Fe3+, Fe2+, Ga3+, Ge4+, Hg2+, Ir4+, La3+, Li+, Mn2+, MO6+, Ni2+, OS4+, Pb2+, Pt4+, Rb+, Rh3+, Sb5+, Se(O2-)4, Se(O2-)3, Sn2+, Sr2+, Th4+, T1+, U(O2+)2, V(O-)3, VO2+, W(O2-)4, Y3+, Zn2+, and Zr4+) on the activity of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV-1) was investigated in vitro. For this study, the RT activity assay was carried out by means of an enzyme-linked immunosorbent assay (ELISA) kit, using the template/primer hybrid poly(A) oligo(dT)15, which required some modifications: (1) possible interfering metal chelators (such as EDTA) in the original lysis buffer were avoided, and a new buffer (50 mM Tris-NO3, pH 7.8) was used throughout; (2) an amount of 2 ng of RT per well was considered to be optimal after checking the linearity of the reaction with increasing amounts of enzyme; (3) an incubation temperature of 37 degrees C and an incubation time of 1 h were chosen after preliminary studies in a wide range of temperature and time. At an incubation temperature > or = 40 degrees C, there was a dramatic loss of enzymatic activity. In addition, when RT alone was preincubated for 1 h at 5 degrees C, 25 degrees C, and 37 degrees C, there was a large (83%) loss of activity at 37 C as compared to that at 5 degrees C. These results are indicative of enzyme thermolability, which is higher in the absence of substrates. The effect of metal ions on RT activity was tested using two different metal salt concentrations (10(-4) M and 10(-5) M). Under such experimental conditions, the presence of five metal ions (Pt4+, Ag+, Rh3+, Zn2+, and Hg2+) decreased the RT activity in a dose-response fashion. The observed order of effectiveness with respect to inhibition was Pt4+ > Ag+ > Rh3+ > Zn2+ = Hg2+. Estimated mean inhibitory concentrations (IC50) were 7.8 microM for (NH4)2PtCl6, 14.1 microM for AgNO3, 46.8 microM for RhCl3, 53.7 microM for Zn(SO)4, and 56.2 microM for Hg(NO3)2. Because these data are of the same order of magnitude as the corresponding values related to other RT inhibitors used in anti-AIDS therapy, metal compounds or their derivatives could give an interesting contribution in the development of new RT inhibitors for clinical use.
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PMID:Effects of trace metal compounds on HIV-1 reverse transcriptase: an in vitro study. 1032 22

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.
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PMID:A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.). 1038 1

Reverse transcription (RT)-PCR is a valuable tool widely used for analysis of gene expression. In bacteria, RT-PCR is helpful beyond standard protocols of northern blot RNA/DNA hybridization (to identify transcripts) and primer extension (to locate their start points), as these methods have been difficult with transcripts that are low in abundance or unstable, similar to long multi-gene operons. In this report, RT-PCR is adapted to analyze transcripts that form long multi-gene operons--where they start and where they stop. The transcripts can also be semiquantitated to follow the expression of genes under different growth conditions. Examples using RT-PCR are presented with two different multi-gene systems for metal cation resistance to silver and mercury ions. The silver resistance system [9 open reading frames (ORFs); 12.5 kb] is shown by RT-PCR to synthesize three nonoverlapping messenger RNAs that are transcribed divergently. In the mercury resistance system (8 ORFs; 6.3 kb), all the genes are transcribed in the same orientation, and two promoter sites produce overlapping transcripts. For RT-PCR, reverse transcriptase enzyme is used to synthesize first-strand cDNA that is used as a template for PCR amplification of single-gene products, from the beginning, middle or end of long multi-gene, multi-transcript gene clusters.
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PMID:RT-PCR: characterization of long multi-gene operons and multiple transcript gene clusters in bacteria. 1057 45

Nuclear and cytoplasmic proteins of human female breast cancer were analysed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Oestrogen receptor and progesterone receptor expression was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. The electropherograms were developed by silver nitrate staining and quantitative analysis was carried out by video densitometer using the software Gel-Pro Analyzer. Nuclear and cytoplasmic proteins of breast carcinomas and normal tissue differed both qualitatively and quantitatively. Nuclear polypeptides of 108, 53 and 48 kD as well as the 36 kD cytoplasmic polypeptide were specific for tumour samples, while the 51 kD nuclear polypeptide was detected only in normal tissue. Quantitative differences in band density were noted in the 32 kD nuclear polypeptide. This polypeptide was expressed in greatest concentration in infiltrating ductal carcinomas which also indicated the greatest oestrogen receptor gene expression. This relationship appeared to be statistically significant (p < 0.005). No correlations were evident between the 32 kD protein expression and the progesterone receptor gene expression in any of the tissue types examined, nor between the 32 kD protein and the patient's age or tumour grade.
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PMID:Female breast carcinomas: nuclear and cytoplasmic proteins versus steroid receptors. 1075 81

The present study focuses on the detection of differentially expressed genes in migrating (healing) and nonmigrating (normal) corneal epithelium on agarose gel using a modified procedure of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). Rabbit corneal epithelial organ cultures were used to obtain nonmigrating and migrating samples. RNA was extracted using Trizol LS reagent. PCR was modified in order to allow detection of amplified products on 3% agarose gel with ethidium bromide staining. Products were also resolved on 6% denaturing polyacrylamide-urea gels and observed by silver staining. Agarose gels showed two prominent bands that were heavily expressed in the 458 bp and 587 bp region of the nonmigrating samples. In addition light bands were visible in the region corresponding to 234 bp and 450 bp. In the migrating samples, two light bands were visible in the region of 267 bp and 300 bp. Eight amplicons, six in the nonmigrating corneal epithelial sample and two in the migrating corneal epithelial samples, were also found to be differentially expressed when products were run on 6% denaturing polyacrylamide-urea gels. Thus, DDRT-PCR products can be detected on agarose gels and prove very helpful and economical in the initial studies of DDRT-PCR.
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PMID:DDRT-PCR: use of agarose gels for detection of amplified products. 1093 Apr 75

Between 1981 and 1998, 37 cases of rabies were diagnosed in human beings in the United States. Information directly linking the cause of infection to animal bite was available for only eight of these cases. Indirect incrimination of the vector by analysis of cDNA sequences obtained by reverse transcriptase polymerase chain reaction of samples indicated that for all cases (12/12) believed to have been acquired in foreign countries, variants of the rabies virus (VRVs) associated with dogs (7/12 involved known bite histories) were the cause of the rabies infections. In contrast, VRVs associated with bats (bat-associated VRVs or BAVs) were implicated as the cause of 88% (22/25) of infections believed to have been acquired within the United States (1/22 involved known bite histories). Sequence analyses revealed that a single BAV (Ln/Ps), associated with rabid silver-haired (Lasionycteris noctivagans) and Eastern pipistrelle (Pipistrellus subflavus) bats, was implicated in 73% (16/22) of bat-associated infections. Silver-haired bats are predominantly solitary and migratory. Eastern pipistrelle bats may occur individually or in small clusters. Both species are only infrequently submitted for rabies testing. Unrecognized bites and unique properties of the Ln/Ps BAV may explain its association with the majority of rabies infections in human beings in the United States.
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PMID:Mammalian reservoirs and epidemiology of rabies diagnosed in human beings in the United States, 1981-1998. 1119 44

Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by "semiquantitative" reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower.
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PMID:Differential expression of 1-aminocyclopropane-1-carboxylate synthase genes during orchid flower senescence induced by the protein phosphatase inhibitor okadaic acid. 1135 Oct 88


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