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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interleukin-5 (IL-5) is a T-cell derived cytokine which stimulates eosinophil production and activation in human, mice and sheep. IL-5 is active as a growth factor for mouse but not human B cells. The role of IL-5 on ruminant B cells has not been clearly defined. By hybridisation with human IL-5 cDNA, the ovine IL-5 gene was isolated from a liver genomic library. The IL-5 cDNA was obtained by reverse-transcriptase PCR using primers designed from the 5' and 3' coding sequence derived from the ovine IL-5 gene. The sequences of the cDNA shows that there is 79% and 73% nucleotide homology with the human and mouse sequences. The ovine IL-5 cDNA encoded a protein of 132 amino acids and the level of amino acid homology with human and mouse IL-5 is 64% and 56%, respectively. Two cysteine residues are conserved in ovine, human and mouse IL-5. There are two potential N-linked glycoyslation sites in ovine IL-5.
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PMID:Cloning and sequencing of an ovine interleukin-5 cDNA. 898 71

Primary deficiency of merosin is the cause of the classic form of congenital muscular dystrophy (CMD) accompanied by brain white matter abnormalities. We report a female infant with dystrophinopathy who was deficient in merosin in skeletal muscle. The patient had a phenotype of typical CMD and white matter abnormalities on brain MRI. Merosin was greatly reduced in the biopsied skeletal muscle. However, the expression of dystroglycan and syntrophin was also greatly reduced, and the immunoreactivity for the antibodies against the cysteine-rich/C-terminal domains of dystrophin was absent in the sarcolemma. Reverse transcriptase polymerase chain reaction analysis of the dystrophin gene revealed a complete lack of exons 71 through 74. In skeletal muscle, only the mutant gene was expressed. These results suggest that the patient is a symptomatic Duchenne muscular dystrophy carrier with skewed X-inactivation. This patient illustrates for the first time that a dystrophin abnormality can cause a secondary deficiency of merosin in dystrophinopathy. The reduction of merosin may account for the clinical phenotype of CMD and correlate with the white matter abnormalities in our patient.
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PMID:Deficiency of syntrophin, dystroglycan, and merosin in a female infant with a congenital muscular dystrophy phenotype lacking cysteine-rich and C-terminal domains of dystrophin. 927 Jun

Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid-polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.
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PMID:Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease. 937 54

The present study underscores the importance of N-acetyl cysteine (NAC), a potent antioxidant, in inhibiting the induction of NO production by lipopolysaccharides (LPS) and cytokines in peritoneal macrophages, C6 glial cells and primary astrocytes. LPS, interleukin-1 beta (IL-1beta), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) alone or in combinations induced the production of NO to different degrees. NAC when added 2 h earlier to the addition of these stimuli potentially blocked the increase in NO production in macrophages, astrocytes and C6 glial cells. The decrease in NO production by NAC was accompanied by a decrease in inducible nitric oxide synthase (iNOS) activity, in iNOS protein detected by immunoblot analysis with antibodies against iNOS, and in iNOS mRNA determined by reverse-transcriptase coupled polymerase chain reaction (RT-PCR). Time course studies show that inhibition was maximum when NAC was added 2 h prior to the addition of LPS and the degree of inhibition decreased progressively with the increase in time interval when NAC was added after the addition of LPS. In addition to NAC, another antioxidant pyrrolidine dithiocarbamate (PDTC) was also found to inhibit the induction of NO production effectively. Since activation of NF-kappaB is necessary for the induction of iNOS, we examined the effect of NAC on the activation of NF-kappaB. Inhibition of LPS-induced activation of NF-kappaB by NAC in rat peritoneal macrophages suggests that the inhibitory effect of NAC on the induction of iNOS is due to the inhibition of NF-kappaB. Besides NO, NAC also blocked the production of TNF-alpha in rat peritoneal macrophages activated with endotoxin. These results suggest that expression of iNOS and TNF-alpha in macrophages do involve oxygen radicals. The importance of these results in relation to controlling various harmful effects of cytokines released by activated macrophages and glial cells is discussed.
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PMID:N-acetyl cysteine inhibits induction of no production by endotoxin or cytokine stimulated rat peritoneal macrophages, C6 glial cells and astrocytes. 943 12

Heterologous expression of human LINE-1 ORF2 in yeast yielded a single polypeptide (Mr145 000) which reacted with specific antibodies and co-purified with a reverse transcriptase activity not present in the host cells. Various deletion derivatives of the ORF2 polypeptide were also synthesized. Reverse transcriptase assays using synthetic polynucleotides as template and primer revealed that ORF2 protein missing a significant portion of the N-terminal endonuclease domain still retains some activity. Deletion of the C-terminal cysteine-rich motif reduces activity only a small amount. Three non-overlapping deletions spanning 144 amino acids just N-terminal to the common polymerase domain of the ORF2 protein were analyzed for their effect on reverse transcriptase activity; this region contains the previously-noted conserved Z motif. The two deletions most proximal to the polymerase domain eliminate activity while the third, most-distal deletion had no effect. An inactive enzyme was also produced by substitution of two different amino acids in a highly-conserved octapeptide sequence, Z8, located within the region removed to make the deletion most proximal to the polymerase domain; substitution of a third had no effect. We conclude that the octapeptide sequence and neighboring amino acids in the Z region are essential for reverse transcriptase activity, while the endonuclease and cysteine-rich domains are not absolutely required.
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PMID:The human LINE-1 reverse transcriptase:effect of deletions outside the common reverse transcriptase domain. 967 14

Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.
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PMID:Mapping of the feline calicivirus proteinase responsible for autocatalytic processing of the nonstructural polyprotein and identification of a stable proteinase-polymerase precursor protein. 1040 Jul 60

Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by immunoprecipitation with respective antibodies. The expression of viral proteins in the transfected cells was also demonstrated by immunofluorescence microscopy. Viral replication was detected in the transfected cells up to 33 days posttransfection (six passages). The culture supernatant from the transfected cells was able to produce HEV infection in a rhesus monkey (Macaca mulatta) following intravenous injection, indicating the generation of viable HEV particles following transfection of cells with in vitro-synthesized genomic RNA. This transient cell culture model using in vitro-transcribed RNA should facilitate our understanding of HEV biology.
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PMID:The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious. 1066 75

Selenocysteine lyase (SCL) (EC 4.4.1.16) is a pyridoxal 5'-phosphate-dependent enzyme that specifically catalyzes the decomposition of L-selenocysteine to L-alanine and elemental selenium. The enzyme was proposed to function as a selenium delivery protein to selenophosphate synthetase in selenoprotein biosynthesis (Lacourciere, G. M., and Stadtman, T. C. (1998) J. Biol. Chem. 273, 30921-30926). We purified SCL from pig liver and determined its partial amino acid sequences. Mouse cDNA clones encoding peptides resembling pig SCL were found in the expressed sequence tag data base, and their sequences were used as probes to isolate full-length mouse liver cDNA. The cDNA for mouse SCL (mSCL) was determined to be 2,172 base pairs in length, containing an open reading frame encoding a polypeptide chain of 432 amino acid residues (M(r) 47, 201). We also determined the sequence of the N-terminal region of putative human SCL. These enzymes were shown to be distantly related in primary structure to NifS, which catalyzes the desulfurization of L-cysteine to provide sulfur for iron-sulfur clusters. The recombinant mSCL overproduced in Escherichia coli was a homodimer with the subunit M(r) of 47,000. The enzyme was pyridoxal phosphate-dependent and highly specific to L-selenocysteine (the k(cat)/K(m) value for L-selenocysteine was about 4,200 times higher than that for L-cysteine). Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that mSCL is cytosolic and predominantly exists in the liver, kidney, and testis, where mouse selenophosphate synthetase is also abundant, supporting the view that mSCL functions in cooperation with selenophosphate synthetase in selenoprotein synthesis. This is the first report of the primary structure of mammalian SCL.
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PMID:cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase. Candidate for selenium delivery protein in selenoprotein synthesis. 1069 12

Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.
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PMID:Identification of mouse hepatitis virus papain-like proteinase 2 activity. 1093 99

The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a threefold reduction in polymerase activity. Deletion of an additional one (the Thr-1236 amino terminus) or two (the Ala-1237 amino terminus) amino acids produced derivatives that were 7- and 175-fold, respectively, less active than Pro-Pol. FCV proteinase-dependent processing of Pro-Pol in the interdomain region preceding Val-1235 was not observed in the presence of a catalytically active proteinase; however, processing within the polymerase domain was observed. Inactivation of proteinase activity by changing the catalytic cysteine-1193 to glycine permitted the production and purification of intact Pro-Pol. Biochemical analysis of Pro-Pol showed that this enzyme has properties expected of a replicative polymerase, suggesting that Pro-Pol is an active form of the FCV RdRP.
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PMID:Proteinase-polymerase precursor as the active form of feline calicivirus RNA-dependent RNA polymerase. 1115 94


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