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)

Recently, several class-related adverse events have been recognized with antiretroviral drugs. For nucleoside analogue reverse transcriptase inhibitors. (NRTI), lactic acidosis with hepatomegaly and hepatic steatosis have been reported. These appear to occur at a low frequency, but with a high fatality rate. We report a case of fatal lactic acidosis in a patient with acquired immunodeficiency syndrome (AIDS) treated with stavudine (d4T), lamivudine (3TC) and indinavir (IDV). A 48-year-old male AIDS patient was admitted with complaints of general fatigue and dyspnea. His medications at presentation included d4T, 3TC and IDV. Physical examination demonstrated icteric sclerae and abdominal tenderness with hepatomegaly. Laboratory data demonstrated a severe metabolic acidosis with an anion gap due to lactate accumulation. Despite intensive treatment, cardiorespiratory arrest occurred and this could not be resuscitated.
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PMID:[Fatal lactic acidosis in a patient with acquired immunodeficiency syndrome treated with stavudine, lamivudine and indinavir]. 1065 86

Interactions between the nucleocapsid protein (NC) and reverse transcriptase of HIV-1 have been shown to promote the initiation of reverse transcription. We assayed the effect of NC on later events, using a strand transfer system with donor and acceptor HIV RNA templates and found that the presence of NC resulted in increased synthesis of full-length strand-transferred (FLST) DNA. This effect also occurred with mutated forms of NC that lacked both zinc fingers, or that contained a point mutation (histidine-->cysteine) at amino acid 23. In contrast, NC-derived proteins containing only the proximal or distal zinc fingers, or lacking the N- and C-termini, were all unable to catalyze the synthesis of FLST DNA. Band-shift assays using both the mutated and wild-type forms of these proteins revealed that all the NC proteins promoted strand association between (-) strong-stop DNA [(-)ssDNA] and acceptor RNA. The zinc finger motifs were dispensable for full-length processive reverse transcription, and the N- and C-termini were required; however, all NC domains were dispensable for association of (-)ssDNA and acceptor RNA. This suggests that annealing is a less stringent reaction than DNA polymerization.
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PMID:The effect of mutations in the HIV-1 nucleocapsid protein on strand transfer in cell-free reverse transcription reactions. 1073 91

The root-hypocotyl of Arabidopsis produces a relatively large amount of secondary vascular tissue when senescence is delayed by the removal of inflorescences, and plants are grown at low population density. Peptidase zymograms prepared from isolated xylem and phloem revealed the existence of distinct proteolytic enzyme profiles within these tissues. cDNA libraries were constructed from isolated xylem and bark of the root-hypocotyl and screened for cDNAs coding for cysteine, serine, and aspartic peptidases. Three cDNAs, two putative papain-type cysteine peptidases (XCP1 and XCP2) and one putative subtilisin-type serine peptidase (XSP1), were identified from the xylem library for further analysis. Using RNA gel blots it was determined that these peptidases were expressed in the xylem and not in the bark. Quantitative reverse transcriptase-polymerase chain reaction confirmed the RNA gel-blot results and revealed high levels of XCP1 and XCP2 mRNA in stems and flowers of the infloresence. A poly-histidine-tagged version of XCP1 was purified from Escherichia coli by denaturing metal-chelate chromatography. Following renaturation, the 40-kD recombinant XCP1 was not proteolytically active. Activation was achieved by incubation of recombinant XCP1 at pH 5.5 and was dependent on proteolytic processing of the 40-kD inactive polypeptide to a 26-kD active peptidase.
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PMID:Exploiting secondary growth in Arabidopsis. Construction of xylem and bark cDNA libraries and cloning of three xylem endopeptidases. 1088 67

The tissue or glandular kallikreins (KLK) are members of a highly conserved multigene family encoding serine proteases that are central to many biological processes. The rodent KLK families are large, highly conserved and clustered at one locus. The human KLK gene family is clustered on chromosome 19q13.3-13.4, and until recently consisted of just three members. However, recent studies have identified up to 11 new members of the KLK family that are less conserved than their rodent counterparts. Using a Southern blot and sequence analysis of 10 BACs and cosmids spanning approximately 400 kilobases (kb) either side of the original KLK 60-kb locus, we demonstrated that these genes also lie adjacent to this. We have also clarified the position of several microsatellite markers in relation to the extended KLK locus. Moreover, from Southern blot analysis of the cosmids and BACs with a degenerate oligonucleotide probe to the histidine-encoding region of serine proteases, we have shown that there are no other serine protease genes approximately 400 kb centromeric and 220 kb telomeric of the extended locus. We performed an extensive analysis of the expression patterns of these genes by poly(A)(+) RNA dot blot and reverse transcriptase-polymerase chain reaction analysis, and demonstrated a diverse pattern of expression. Of interest are clusters of genes with high prostate (KLK2-4) and pancreatic (KLK6-13) expression suggesting evolutionary conservation of elements conferring tissue specificity. From these findings, it is likely that the human KLK gene family consists of just 14 clustered genes within 300 kb and thus is of a comparable size to the rodent families (13-24 genes within 310 and 480 kb, respectively). In contrast to the rodent families, the newest members of the human KLK family are much less conserved in sequence (23-44% at the protein level) and appear to consist of at least four subfamilies. In addition, like the rat, these genes are expressed at varying levels in a diverse range of tissues although they exhibit quite distinct patterns of expression.
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PMID:Tissue-specific expression patterns and fine mapping of the human kallikrein (KLK) locus on proximal 19q13.4. 1096 73

The D-amino acid oxidase cDNA gene (daao) of Trigonopsis variabilis was prepared by reverse transcriptase-polymerase chain reaction (PCR) and cloned into Escherichia coli expression vector, pTrc99A, under the control of tac promoter. Expression of daao gene significantly affected the growth and morphology of E. coli. The highest D-amino acid oxidase (DAAO) activity was 705 U (mg of protein)(-)(1), which was about 12-fold higher than that of D-alanine-induced T. variabilis. The DAAO protein exhibited activity on native-PAGE and had a M(r)value of 39.3 kDa. We also constructed an expression plasmid, pKm-DAAO, in which kanamycin instead of ampicillin was used as the selective marker. High-performance liquid chromatography (HPLC) analysis demonstrated that cephalosporin C could be converted to 7-glutarylcephalosporanic acid by cell-free extract of E. coli harboring pKm-DAAO. Four inactive DAAO mutants were obtained by error-prone PCR. Sequence analysis of these four DAAO mutants indicated the occurrence of mutations at Val-167, Pro-291, Pro-309, and Ala-343 residues. The His(6)-tagged DAAOs were expressed in E. coli and purified by nickel ion affinity chromatography. The results showed that all DAAO mutants lost their enzymatic activities and characteristic adsorption spectra for flavoenzyme. Based on the crystal structure of a homologous protein, pig DAAO, it is suggested that these four residues may play essential structural roles in DAAO conformation, thereby influencing DAAO's catalytic activity.
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PMID:Expression of Trigonopsis variabilis D-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants. 1097 70

The authors had previously mapped a new locus-DFNA17, for nonsyndromic hereditary hearing impairment-to chromosome 22q12.2-q13. 3. DFNA17 spans a 17- to 23-cM region, and MYH9, a nonmuscle-myosin heavy-chain gene, is located within the linked region. Because of the importance of myosins in hearing, MYH9 was tested as a candidate gene for DFNA17. Expression of MYH9 in the rat cochlea was confirmed using reverse transcriptase-PCR and immunohistochemistry. MYH9 was immunolocalized in the organ of Corti, the subcentral region of the spiral ligament, and the Reissner membrane. Sequence analysis of MYH9 in a family with DFNA17 identified, at nucleotide 2114, a G-->A transposition that cosegregated with the inherited autosomal dominant hearing impairment. This missense mutation changes codon 705 from an invariant arginine (R) to histidine (H), R705H, within a highly conserved SH1 linker region. Previous studies have shown that modification of amino acid residues within the SH1 helix causes dysfunction of the ATPase activity of the motor domain in myosin II. Both the precise role of MYH9 in the cochlea and the mechanism by which the R705H mutation leads to the DFNA17 phenotype (progressive hearing impairment and cochleosaccular degeneration) remain to be elucidated.
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PMID:Human nonsyndromic hereditary deafness DFNA17 is due to a mutation in nonmuscle myosin MYH9. 1102 10

Cadherins, calcium-dependent cell adhesion molecules, play crucial roles, not only in the maintenance of tissue integrity, but also in the regulation of many aspects of cell behavior. We investigated the expression of "classic" E-, N- and P-cadherins in bone marrow-derived cultured mast cells (BMMC) and peritoneal mast cells (PMC) from mice. Flow cytometric analysis and immunocytochemical staining indicated that E-cadherin was expressed on the cell surface of BMMC and also at lower levels on PMC. N-cadherin was also expressed on the surface of BMMC, but not of PMC, whereas P-cadherin expression was seen in neither cell type. Significant expression of E- and N-cadherin mRNA was observed in BMMC by reverse transcriptase-polymerase chain reaction (RT-PCR), but PMC expressed only E-cadherin mRNA. Western blotting analysis indicated expression of alpha- and beta-catenins and p120-catenin (or p120 cas) in BMMC, whereas PMC showed less intense expression of alpha- and beta-catenins with high levels of p120 expression. Analyses of beta-catenin or E-cadherin immunoprecipitates from BMMC lysate revealed that alpha-catenin, beta-catenin, and E-cadherin were co-precipitated, suggesting that E-cadherin and catenins form a complex in mast cells. Addition of a blocking antibody of homophilic E-cadherin interactions, or a synthetic E-cadherin-binding decapeptide containing the histidine-alanine-valine (HAV) sequence in methylcellulose cultures of gut intraepithelial mononuclear cells or BMMC, significantly suppressed the clonal growth of mast cells. Furthermore, the blocking antibody or synthetic decapeptide significantly suppressed BMMC adhesion to E-cadherin-expressing F9 cell monolayers. These results indicated that E-cadherin and associated cytoplasmic proteins in mast cells might be involved in the regulation of certain stages of mast cell differentiation and cell-cell interactions.
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PMID:E-cadherin and cadherin-associated cytoplasmic proteins are expressed in murine mast cells. 1104 74

Lactic acidosis and hepatic steatosis caused by mitochondrial toxicity of nucleoside reverse transcriptase inhibitors (NRTI) is a rare cause of liver disease with a high mortality rate. This report describes a male, HIV-positive patient with a 4-week history of nausea, vomiting and abdominal pain. His medication consisted of prednisone 5 mg od (because of auto-immune thrombocytopenia), didanosine (for 2 years) and stavudine (for 3 months). Laboratory studies showed cholestasis and elevation of aminotransferases. Lactic level was not measured. Liver biopsy revealed steatosis and cholestatic hepatitis. In the absence of other causes of liver disease a probable diagnosis of stavudine-induced hepatic toxicity was made. After discontinuation of NRTI, he recovered completely. Because lactic acidosis had not been confirmed, stavudine was restarted and within 1 week the lactate level increased significantly. Therefore stavudine was discontinued again. One year later the patient is doing well on a double protease inhibitor regimen. In conclusion, clinicians treating patients with NRTI should be aware of the risk of lactic acidosis and hepatic steatosis. When this is suspected, all NRTI must be stopped. The diagnosis can be made when elevated lactate levels and hepatic steatosis are present in the absence of other causes of liver disease.
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PMID:Hepatic steatosis and lactic acidosis caused by stavudine in an HIV-infected patient. 1106 65

The medically significant, obligate intracellular pathogen Chlamydia trachomatis replicates within vacuoles termed inclusions. A developmental cycle is initiated after entry into a host cell and is manifested by the transformation of infectious elementary bodies (EBs) to larger, non-infectious reticulate bodies (RBs). Analysis of the C. trachomatis genome has revealed that chlamydiae possess genes that may encode a type III secretion apparatus. In other Gram-negative pathogens, the type III secretion mechanism is used to target virulence factors directly to the host cell cytoplasm and is essential for full virulence. To evaluate the possibility of a functional type III secretion mechanism in C. trachomatis, we initially focused on a locus containing genes encoding products with similarity to chaperones (Scc1), secretion pore components (Cds1 and Cds2) and secreted proteins (CopN) from other type III systems. Gene expression was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA extracted from infected HeLa cell monolayers at 2, 6, 12 and 20 h after infection and normalized for the number of C. trachomatis genomes present. Message was detected for Scc1 at all times, whereas message for all other tested genes was detected in significant amounts at 12 h and 20 h. Immunoblot analysis with Scc1- and CopN-specific antibodies revealed that CopN and Scc1 were present in EBs, RBs and whole-culture extracts harvested 20 h after infection. CopN is homologous to the secreted protein YopN of Yersinia sp., and analysis of monolayers 20 h after infection via indirect immunofluorescence showed specific labelling of inclusion membranes when probed with CopN-specific antibodies but not with Scc1-specific antibodies. His-tagged CopN and a chlamydial cytoplasmic control protein (NrdB) were expressed in Yersinia enterocolitica containing or lacking the virulence plasmid pYV. CopN, but not NrdB, was secreted by Y. enterocolitica in a Ca2+- and pYV-dependent fashion. These data indicate that components of the putative type III apparatus of C. trachomatis are expressed and that at least one of these products is secreted by chlamydiae to the inclusion membrane. The observation that CopN is also secreted by the Yersinia type III apparatus provides support for the notion that chlamydiae secrete proteins via a type III mechanism.
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PMID:Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism. 1112 78

The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.
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PMID:cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome P450. 1116 Aug 76


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