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)

Despite advances made in its therapeutic management, human immunodeficiency virus (HIV) infection has remained an intractable problem, and complete eradication of the virus an unrealized goal. Experience in the clinical application of combination therapy using a variety of reverse transcriptase and protease inhibitors have revealed a number of challenges, in spite of the observed albeit temporary success in reduction of patient viral loads. Problems with current protocols include poor patient compliance, and the presence of latent reservoirs of virus that ultimately result in the appearance of phenotypic resistance. These considerations necessitate continued research and development into alternative strategies to circumvent the aforementioned problems. One approach to minimizing and/or eliminating the appearance of escape mutants and latent viral reservoirs is the targeting of essential and mutationally intolerant enzymes such as the nucleocapsid protein, which contains two highly conserved zinc knuckles. Concerns have been raised regarding the targeting of this protein, since the ubiquitous occurrence of important mammalian zinc finger proteins implies that drug specificity towards the nucleocapsid protein may be difficult to attain. In this review, strong evidence supporting the hypothesis that this protein can be targeted to the exclusion of other cellular zinc finger proteins is presented. The effects of small molecule induced abrogation of nucleocapsid protein mediated activities, as well as efforts to develop nucleocapsid protein inhibitors as antiretrovirals are also discussed.
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PMID:The HIV-1 nucleocapsid zinc finger protein as a target of antiretroviral therapy. 1557 99

The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2'-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.
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PMID:Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins. 1565 Jan 79

During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer. To gain insights into specific interactions between HIV-1 NC and RNA and the influence of NC on R folding, we investigated the secondary structures of R in two natural contexts, namely at the 5' or 3' end of RNAs representing the terminal regions of the genome, and in two ectopic internal positions that also support efficient minus-strand transfer. To investigate the roles of NC zinc fingers and flanking basic domains in the NC/RNA interactions, we used NC mutants. Analyses of the viral RNA/NC complexes by chemical and enzymatic probings, and gel retardation assays were performed under conditions allowing ss-cDNA transfer by reverse transcriptase. We report that NC binds the TAR apical loop specifically in the four genetic contexts without changing the folding of the TAR hairpin and R region significantly, and this requires the NC zinc fingers. In addition, we show that efficient annealing of cTAR DNA to the 3' R relies on sequence complementarities between TAR and cTAR terminal loops. These findings suggest that the TAR apical loop in the acceptor RNA is the initiation site for the annealing reaction that is chaperoned by NC during the minus-strand transfer.
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PMID:Specific interactions between HIV-1 nucleocapsid protein and the TAR element. 1585 44

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.
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PMID:Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis. 1585 48

Highly active antiretroviral therapy (HAART) dramatically changed the course of HIV infection. Currently, this therapy involves the use of agents from at least two distinct classes of antivirals: a protease inhibitor (PI) in combination with two nucleoside/nucleotide reverse transcriptase inhibitors (N(t)RTIs), or a non-nucleoside reverse transcriptase inhibitor (NNRTI) in combination with NRTIs. Recently, the third family of antivirals started to be used clinically, with the advent of enfuvirtide, the first fusion inhibitor (FI). Several pharmacological agents are available form these classes of antivirals, NRTIs, NNRTIs, PIs and FIs, which will be briefly reviewed here. Some more agents are in advanced clinical evaluation or have recently been approved (such as tenofovir, a NtRTI; atazanavir, a PI; tipranavir, another PI), mainly against drug-resistant viruses. Compounds inhibiting HIV integrase, the third enzyme of HIV, are also available ultimately, with several such derivatives in clinical trials (L-731, 988 and S-1360). Another approach to inhibit the growth of retroviruses, including HIV, targets the ejection of zinc ions from critical zinc finger viral proteins, which has as a consequence the inhibition of viral replication in the absence of mutations leading to drug resistance phenotypes. All steps in the process of HIV entry into the cell may be targeted by specific compounds that might be developed as novel types of antiretrovirals. Thus, inhibitors of the gp120-CD4 interaction have been detected (zintevir, FP-21399 and BMS-378806 in clinical trials). Small molecule chemokine antagonists acting as HIV entry inhibitors also were described in the last period, which interact both with the CXCR4 coreceptor (such as AMD3100; AMD3465; ALX40-4C; T22, T134 and T140), or which are antagonist of the CCR5 coreceptor (TAK-779, TAK-220, SCH-C, SCH-D, E913, AK-602 and NSC 651016 in clinical trials), together with new types of fusion inhibitors possessing the same mechanism of action as enfuvirtide (such as T1249). Compounds interacting with Tat/Tar have also been detected which inhibit HIV replication in low micromolar range (EM2487, tamacrazine, CGP 64222 or CGA 137053 among others). Unexploited viral and cellular targets (such as the maturation process-with a first potent compound available, PA-457; the cellular proteins Tsg101, APOBEC3G, or the viral ones Vif, Rev or RNase H) are also presented, together with recently emerged approaches for eradication of HIV reservoirs. A review on the pharmacology and interactions of these agents with other drugs is presented here, with emphasis on how these pharmacological interferences may improve the clinical use of antivirals, or how side effects due to these drugs may be managed better by taking them into account.
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PMID:Highly active antiretroviral therapy: current state of the art, new agents and their pharmacological interactions useful for improving therapeutic outcome. 1589 77

A 303-bp cDNA of intestinal zinc exporter (ZnT1) was isolated from chicken jejunum by reverse transcriptase-polymerase chain reaction and sequenced, and showed 42% homology to Homo sapiens and Rattus novergicus intestinal ZnT1 genes. This specific probe was used to examine the effect of zinc-methionine (ZnMet) administration on the mRNA expression of ZnT1 and on small intestinal development and functionality. In this study, ZnMet was injected into the naturally consumed amniotic fluid of 17-day-old chicken embryos. The ZnT1 gene showed an approximately 200% increase in its mRNA levels from 48 h post-ZnMet injection, as compared to the control. An analysis of the gene expression of the brush-border enzymes and transporters showed increased mRNA expression of sucrase isomaltase, leucine-aminopeptidase, sodium-glucose cotransporter and Na+K+ATPase transporter (Na+K+ATPase) from 48 h post-ZnMet injection, in comparison to controls. Significant increases (P<.05) in the biochemical activity of the brush-border enzymes and transporters, and in jejunal villus surface area were detected from day of hatch (96 h post-ZnMet injection) as compared to controls. These results suggest that ZnMet administration into prenatal intestine via injection into the amniotic fluid enhances intestinal development and improves its functionality.
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PMID:Changes in chicken intestinal zinc exporter mRNA expression and small intestinal functionality following intra-amniotic zinc-methionine administration. 1593 45

A ribonuclease, with a molecular mass of 9 kDa and an N-terminal sequence resembling the sequence of a fragment of tRNA/rRNA cytosine-C5-methylase and a fragment of a alanyl-tRNA synthetase, was isolated from fresh fruiting bodies of the brown oyster mushroom Pleurotus ostreatus. The ribonuclease was purified using a very simple protocol that comprised ion-exchange chromatography on carboxymethyl (CM)-cellulose and affinity chromatography on Affi-gel blue gel. Subsequent gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis revealed that the ribonuclease was purified after the first two chromatographic steps. The ribonuclease was adsorbed on CM-cellulose and Affi-gel blue gel. The ribonuclease exhibited the highest activity toward poly A, lower activity toward poly C, slight activity toward poly G, and indiscernible activity toward poly U. The enzyme was stimulated upon exposure to 1 microm Mg2+ and 10 microm Zn2+, but was inhibited by the following ions at 10 mm: Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Mn2+, and Fe3+. The ribonuclease required a pH of 8.0 and a temperature of 50-70 degrees C to express maximal activity. It had a Km of 60 microm toward yeast tRNA. It lacked mitogenic and HIV-1 reverse transcriptase inhibiting activities, but exerted antiproliferative activity toward leukemia L1210 cells.
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PMID:A low-molecular mass ribonuclease from the brown oyster mushroom. 1594 90

Novel metallothionein (MT) complementary DNA and genomic sequences were isolated from a cartilaginous shark species, Scyliorhinus torazame. The full-length open reading frame (ORF) of shark MT cDNA encoded 68 amino acids with a high cysteine content (29%). The genomic ORF sequence (932 bp) of shark MT isolated by polymerase chain reaction (PCR) comprised 3 exons with 2 interventing introns. Shark MT sequence shared many conserved features with other vertebrate MTs: overall amino acid identities of shark MT ranged from 47% to 57% with fish MTs, and 41% to 62% with mammalian MTs. However, in addition to these conserved characteristics, shark MT sequence exhibited some unique characteristics. It contained 4 extra amino acids (Lys-Ala-Gly-Arg) at the end of the beta-domain, which have not been reported in any other vertebrate MTs. The last amino acid residue at the C-terminus was Ser, which also has not been reported in fish and mammalian MTs. The MT messenger RNA levels in shark liver and kidney, assessed by semiquantitative reverse transcriptase PCR and RNA blot hybridization, were significantly affected by experimental exposures to heavy metals (cadmium, copper, and zinc). Generally, the transcriptional activation of shark MT gene was dependent on the dose (0-10 mg/kg body weight for injection and 0-20 microM for immersion) and duration (1-10 days); zinc was a more potent inducer than copper and cadmium.
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PMID:Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and expression analysis. 1597 34

Endometrial stromal tumors are rare uterine neoplasms including benign stromal nodules, low-grade endometrial stromal sarcomas (ESS), and undifferentiated endometrial sarcomas (UES), the latter representing the most aggressive form. Morphological characteristics and cytogenetic abnormalities are heterogeneous, making diagnosis difficult. Recently, a gene fusion on chromosome 7 that includes two zinc-finger genes (JAZF1 and JJAZ1) has been discovered in these tumors. Hitherto only 31 cases, described by three different research groups, have shown JAZF1/JJAZ1 fusion in approximately 50% of all analyzed low-grade ESSs whereas it is less frequent in UESs. In this study we analyzed 20 ESS and 2 UES cases using two-step reverse transcriptase-polymerase chain reaction optimized for formalin-fixed, paraffin-embedded tissue. In our subset of samples, the JAZF1/JJAZ1 fusion transcript occurred in 80% of analyzed ESS cases and in none of two UES cases. In comparison to published data, our results identified the JAZF1/JJAZ1 gene fusion more frequently in endometrial stromal tumors than hitherto presumed. This cytogenetic abnormality was not present in normal endometria, leiomyomas, or leiomyosarcomas or in lung, gastric, or hepatic carcinomas, indicating its specificity for endometrial stromal tumors. In combination with other established methods, accurate reverse transcriptase-polymerase chain reaction analysis of JAZF1/JJAZ1 gene fusion may be useful in diagnosing difficult or unusual ESS/UES cases.
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PMID:JAZF1/JJAZ1 gene fusion in endometrial stromal sarcomas: molecular analysis by reverse transcriptase-polymerase chain reaction optimized for paraffin-embedded tissue. 1604 11

Caffeic acid phenethyl ester (CAPE) derived from honeybee propolis has been used as a folk medicine. Recent study also revealed that CAPE has several biological activities including antioxidation, anti-inflammation and inhibition of tumor growth. The present study investigated the effect of CAPE on tumor invasion and metastasis by determining the regulation of matrix metalloproteinases (MMPs). Matrix metalloproteinases, which are zinc-dependent proteolytic enzymes, play a pivotal role in tumor metastasis by cleavage of extracellular matrix (ECM) as well as nonmatrix substrates. On this line, we examined the influence of CAPE on the gene expression of MMPs (MMP-2, MMP-9, MT1-MMP), tissue inhibitor of metalloproteinase-2 (TIMP-2) and in vitro invasiveness of human fibrosarcoma cells. Dose-dependent decreases in MMP and TIMP-2 mRNA levels were observed in CAPE-treated HT1080 human fibrosarcoma cells as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Gelatin zymography analysis also exhibited a significant down-regulation of MMP-2 and MMP-9 expression in HT1080 cells treated with CAPE compared to controls. In addition, CAPE inhibited the activated MMP-2 activity as well as invasion, motility, cell migration and colony formation of tumor cells. These data therefore provide direct evidence for the role of CAPE as a potent antimetastatic agent, which can markedly inhibit the metastatic and invasive capacity of malignant cells.
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PMID:Inhibitory effects of caffeic acid phenethyl ester on cancer cell metastasis mediated by the down-regulation of matrix metalloproteinase expression in human HT1080 fibrosarcoma cells. 1621 27


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