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)

Gag p19 protein (MA) of the transformation-defective Rous sarcoma virus mutant, tdPH2010, has a point mutation at nucleotide 376 (G to A) that results in an amino-acid change at residue 126 of p19 (Glu to Lys). This single amino-acid change is the cause of the aberrantly fast migration of this protein on SDS-polyacrylamide gels. To study the biological significance of the mutation, we introduced this mutation into a transformation-defective derivative of the molecularly cloned Rous sarcoma virus, SRA2, and examined its effect on virus replication. The virus possessing the mutation in its gag p19 gene had 50% slower replication as measured by the amount of reverse transcriptase as well as gag p27 protein (CA) in the culture media. Glu at position 126 appears to be important for efficient production of Rous sarcoma virus in vitro.
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PMID:A single amino-acid substitution in gag p19 protein (MA) of Rous sarcoma virus suppresses virus production from infected cells. 960 59

A novel lectin has been purified from the fruiting bodies as well as cultured mycelia of the edible mushroom Volvariella volvacea. The lectin, designated as VVL, was a homodimeric protein with a molecular weight of 32 kDa as demonstrated by gel filtration and SDS-PAGE. VVL had no carbohydrate moiety, and its hemagglutinating activity was inhibited by thyroglobulin but not by simple carbohydrates such as monomeric or dimeric sugars. The immunomodulatory activity of VVL was demonstrated by its potent stimulatory activity toward murine splenic lymphocytes. VVL was also found to markedly enhance the transcriptional expression of interleukin-2 and interferon-gamma by reverse transcriptase-polymerase chain reaction. As revealed by its N-terminal amino acid sequence, VVL possessed a molecular structure distinct from other immunomodulatory proteins previously reported in the same fungus.
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PMID:A novel lectin with potent immunomodulatory activity isolated from both fruiting bodies and cultured mycelia of the edible mushroom Volvariella volvacea. 963 63

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid beta-peptide (Abeta) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem. 69, 1580-1591]. The present study was to test the hypothesis that the caffeine/Abeta responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd=9.9+/-1.6 nM, Bmax=25+/-4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]i increased 2.8-fold over baseline following applications of 5-15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]i were observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the beta-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.
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PMID:Expression of ryanodine receptors in human embryonic kidney (HEK293) cells. 969 5

Administration of delavirdine, an HIV-1 reverse transcriptase inhibitor, to rats or monkeys resulted in apparent loss of hepatic microsomal CYP3A and delavirdine desalkylation activity. Human CYP3A catalyzes the formation of desalkyl delavirdine and 6'-hydroxy delavirdine, an unstable metabolite, while CYP2D6 catalyzes only desalkyl delavirdine. CYP2D6 catalyzed desalkyl delavirdine formation was linear with time (up to 30 min) but when catalyzed by cDNA expressed CYP3A4 or human liver microsomes the reaction rate declined progressively with time. Coincubation with triazolam showed that delavirdine caused a time- and NADPH-dependent loss of CYP3A4 activity in human liver microsomes as measured by triazolam 1'-hydroxylation. The catalytic activity loss was saturable and was characterized by a Ki of 21.6 +/- 8.9 microM and a kinact of 0.59 +/- 0.08 min-1. An apparent partition ratio of 41 was determined with cDNA expressed CYP3A4, based on the substrate depletion method. Incubation of [14C]delavirdine with microsomes from several species resulted in irreversible association with an approximately 50 kDa protein, as demonstrated by SDS-PAGE/autoradiography. Binding to the protein was NADPH dependent, glutathione insensitive, proportional to the level of CYP3A expression and was inhibited by ketoconazole, a specific CYP3A inhibitor. NADPH-dependent irreversible binding to human and rat total microsomal protein was demonstrated following exhaustive extraction of microsomal protein. Binding was decreased in the presence of glutathione and appeared to be related to expression level of CYP3A. These results suggest that delavirdine can inactivate CYP3A and has the potential to slow the metabolism of coadministered CYP3A substrates.
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PMID:Microsomal metabolism of delavirdine: evidence for mechanism-based inactivation of human cytochrome P450 3A. 976 59

A novel milk protein, which is secreted only in the early stage of lactation, has been identified in the whey fraction of milk from the tammar wallaby (Macropus eugenii). The amino acid sequence currently available suggests the protein comprises 71 amino acids. The protein migrates at 18 kDa when analysed by SDS polyacrylamide gel electrophoresis but has a calculated molecular weight of 8 kDa. A partial cDNA clone of 153 bp has been isolated by reverse transcriptase PCR. Northern analysis of mammary gland RNA extracted from various stages throughout the entire lactation period showed a messenger RNA transcript of approximately 500 bp present only in the first third of lactation. The protein shares 74.5% similarity at the amino acid level with early lactation protein (ELP) from the brush-tailed possum (Trichosurus vulpecula) and 37% with bovine colostrum trypsin inhibitor, a member of the Kunitz family of protease inhibitors. We hypothesise that the expression of this gene may be controlled by changes in the sucking patterns of the dependent pouch young.
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PMID:Developmentally-regulated expression of a putative protease inhibitor gene in the lactating mammary gland of the tammar wallaby, Macropus eugenii. 978 13

Our aim was to investigate the collagenolytic potential and localization of matrix metalloproteinase-2 (MMP-2) in relation to its regulatory proteins membrane type MT1-MMP and tissue inhibitor of metalloproteinases-2 (TIMP-2) in rheumatoid arthritis (RA). For this purpose, we have used purification of MMP-2, MMP-8, MMP-9 and interstitial type I, II and III collagens; SDS-PAGE/densitometric collagenase activity assay; zymography; Western blotting; reverse transcriptase polymerase chain reaction; in situ hybridization; and immunofluorescence, ABC, ABC-APAAP double immunostainings. MMP-2 degraded human type II collagen almost as effectively as MMP-8, whereas MMP-9 did not cleave type II collagen. In synovial tissue, MT1-MMP, TIMP-2 and MMP-2 were found in synovial lining in fibroblast- and macrophage-like cells, in stromal cells and in vascular endothelium. MT1-MMP, TIMP-2 and MMP-2 were strongly expressed in the pannocytes of the invasive pannus at the interface, but staining was weak and/or there were few positive cells both "above" and "below" the soft-to-hard tissue (cartilage and/or bone) interface. Rheumatoid synovial tissue extract contained proteolytically active 62/59 kDa MMP-2 and 43 kDa MT1-MMP, but no free TIMP-2. These results indicate that components of the ternary MT1-MMP/TIMP-2/MMP-2 complex are coexpressed in the normal synovial lining and in its pathological extension on the hyaline articular cartilage. MMP-2 may participate in the remodeling of the normal lining and also seems to be localized/focalized to pannocytes at a site critical for tissue destruction in arthritis.
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PMID:New collagenolytic enzymes/cascade identified at the pannus-hard tissue junction in rheumatoid arthritis: destruction from above. 992 52

Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A.
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PMID:Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity. 1021 88

Quantifying bcr/abl fusion transcripts in chronic myelogenous leukemia is thought to serve as a powerful parameter for monitoring the kinetic nature of this clonal disease in vivo and in vitro. Recently, we demonstrated the technical advantages as well as the clinical relevance of quantitating bcr/abl fusion mRNA using the 5-nuclease assay and a real-time fluorescence reverse transcriptase-PCR (RT-PCR) detection system (ABI PRISM 7700 SDS). Meanwhile, another technique was introduced (LightCycler technology) that may be used for the same purpose. To investigate whether this method may be an appropriate alternative to the described procedure, we have established bcr/abl LightCycler RT-PCR for major and minor bcr/abl fusion transcripts. We found that, with only minor modifications, TaqMan RT-PCR and fluorescent probe design can be used to obtain comparable results in the LightCycler system. The developed method could quantitate as little as 10 bcr/abl copies per 100 ng cDNA and was as safe and reproducible as the previously described technique. Because reaction efficiency was identical within different bcr/abl major fusions, one single RT-PCR could be established that simultaneously detects b2a3, b2a2, b3a2, and b3a3 fusion RNA with equal specificity and sensitivity. Compared to results generated by the ABI PRISM 7700 SDS, absolute amounts of bcr/abl did not differ significantly, and there was a linear correlation between the respective values. We conclude that TaqMan chemistry can be used in the LightCycler and that both real-time fluorescence PCR detection systems equally fulfill the criteria for the safe and reliable quantitation of bcr/abl fusion RNA in clinical samples. This may be of help for further standardization of quantitative bcr/abl RT-PCR, which, again, is necessary for the comparison of results generated by different investigators.
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PMID:LightCycler technology for the quantitation of bcr/abl fusion transcripts. 1039 61

Bladder filling depends upon the coordinated control of a storage chamber, the bladder body, and its outlet, the bladder base and urethra. Bladder emptying results from development of force in the bladder body and relaxation of the outlet. Muscle strips from bladder body reveal phasic characteristics, whereas the strips from urethral wall are tonic. To determine whether the compositions of myosin heavy chain (MHC) isoforms and the level of myosin light chain (MLC) phosphorylation contribute to the regional variation in the contractile states of the bladder smooth muscle, we analyzed the levels of MLC phosphorylation and the expression of myosin isoforms in smooth muscle tissues from different regions of the urinary bladder. Strips of bladder from the dome, mid body, base of the bladder and urethra were removed and analyzed for the levels of MLC phosphorylation at the resting tone. The expression of MHC isoforms that differ in the C-terminus (SM1 and SM2) and in the N-terminal region (SM-A and SM-B), formed by alternative splicing of the pre-mRNA at either the 3' end or the 5' end, respectively, was analyzed. The expression of these isoforms was characterized at the mRNA and protein levels using reverse transcriptase-polymerase chain reaction (RT-PCR), SDS-PAGE, and Western blotting. The levels of MLC phosphorylation were 35.5 +/- 4.6, 24.7 +/- 2.2, 13.6 +/- 2.1, and 12.8 +/- 2.7 for dome, mid bladder body, base and urethra respectively. Almost 100% of the MHC mRNA in the dome, mid bladder body, and base contains a 7-amino acid insert near the ATP-binding region, whereas the MHC in the urethral smooth muscle is only 81% inserted. Prior studies have shown that inserted myosin has a two-fold higher actin-activated ATPase activity compared to the myosin isoform that lacks the insert, and the maximum velocity of shortening of smooth muscle containing this insert is high compared to muscle that do not contain the insert. The expression of SM1 and SM2 were not significantly different. Our data suggests the presence of a high degree of inserted myosin and LC20 phosphorylation in the bladder dome and mid-body helps to facilitate rapid force development and emptying. Non-inserted myosin and the low level of MLC phosphorylation in the urethra may contribute to slowly or non-cycling myosin cross bridges and the maintenance of a tonic or contracted state during bladder filling.
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PMID:Myosin light chain phosphorylation at resting level and the composition of myosin isoforms in the bladder body and urethra. 1057 76

The acute phase response (APR) has a long evolutionary history, but it remains to be characterized fully in lower vertebrates. To study the acute phase proteins of a teleost, rainbow trout (Oncorhynchus mykiss), we induced an APR by injecting Vibrio bacterin emulsified in FIA. In samples taken over the next 3 weeks, the total plasma protein profile changed consistently as seen in one and two-dimensional SDS PAGE. One 18.1 kD upregulated protein was isolated from 2D gels and an N-terminal sequence obtained. Using reverse transcriptase-PCR, a 700 bp cDNA sequence was amplified. The sequence is 53% similar at the amino acid level with rat precerebellin (regions aa 42-184 from trout and aa 89-224 precerebellin), and 46% similar with the globular portion of the human B chain of the first complement component C1q. However, it lacks the collagen portion of C1q with its characteristic Gly-X-Y repeats. The isolated protein seems to be involved in the inflammatory response but its physiological function is unknown.
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PMID:A precerebellin-like protein is part of the acute phase response in rainbow trout, Oncorhynchus mykiss. 1083 94


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