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)

Glutamine is taken up into the rat hepatoma cell line H4-IIE-C3 by a Na+-dependent transport system which is specific for glutamine, alanine, serine, cysteine and asparagine and does not tolerate substitution of Na+ by Li+. Glutamine transport was relatively weakly inhibited by a 50-fold excess of leucine and was not inhibited by phenylalanine or N -methyl aminoisobutyrate. These general properties are characteristic of the recently identified ASCT/B0 family of transporters. Using a reverse transcriptase PCR-based homology cloning approach, we have characterized a cDNA for a novel member of this transporter family (H4-ASCT2) from H4-IIE-C3 cells. The cDNA encodes a 551-amino acid protein which exhibits similarities of between 75 and 85% with ASCT/B0 transporters previously cloned from other sources. When expressed in Xenopus oocytes, this transporter catalyses Na+-dependent glutamine uptake with characteristics very similar to those of glutamine uptake into the H4-IIE-C3 cells. This newly characterized transporter possesses a number of amino acid sequence differences from ASCT2 clones recently isolated from rat astroglial cells and from normal rat liver. In particular, the loop region between transmembrane helices 3 and 4 from H4-ASCT2 shares less than 60% sequence similarity with ASCT2 from rat liver; furthermore, there are some 25 single amino acid substitutions elsewhere in the H4-ASCT2 sequence compared with that from rat liver. Thus enhanced glutamine uptake in rat hepatoma cells is mediated by the expression of a novel ASCT/B0 transporter isoform rather than by increased expression of the ASCT2 mRNA found in normal rat liver.
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PMID:Identification of a plasma membrane glutamine transporter from the rat hepatoma cell line H4-IIE-C3. 1217 99

Flavonoids are plant pigments that are synthesised from phenylalanine, generally display marvelous colors known from flower petals, mostly emit brilliant fluorescence when they are excited by UV light, and are ubiquitous to green plant cells. The flavonoids are used by botanists for taxonomical classification. They regulate plant growth by inhibition of the exocytosis of the auxin indolyl acetic acid, as well as by induction of gene expression, and they influence other biological cells in numerous ways. Flavonoids inhibit or kill many bacterial strains, inhibit important viral enzymes, such as reverse transcriptase and protease, and destroy some pathogenic protozoans. Yet, their toxicity to animal cells is low. Flavonoids are major functional components of many herbal and insect preparations for medical use, e.g., propolis (bee's glue) and honey, which have been used since ancient times. The daily intake of flavonoids with normal food, especially fruit and vegetables, is 1-2 g. Modern authorised physicians are increasing their use of pure flavonoids to treat many important common diseases, due to their proven ability to inhibit specific enzymes, to simulate some hormones and neurotransmitters, and to scavenge free radicals.
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PMID:The biochemistry and medical significance of the flavonoids. 1245 66

The tryptophan repeat motif of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) is comprised of a cluster of six tryptophan residues at codons 398, 401, 402, 406, 410 and 414 that are highly conserved amongst primate lentiviral RTs. To determine the contributions of each of these residues for HIV-1 RT dimerization, we introduced changes into cloned DNA and tested the mutant subunits for their capacity to mediate heterodimerization in the yeast two-hybrid system. Changes of residue 401 to either leucine or alanine (but not phenylalanine) and residue 414 to leucine resulted in major reductions in beta-galactosidase activity produced from the reporter gene as compared to yeast expressing wild-type p66 bait and p51 prey fusions. Subunit selective mutagenesis revealed that the effect of these mutations was mediated mainly through the p66 subunit. Introduction of tryptophan mutants into the bacterial expression vector pRT6H/NB-PROT showed that RTs containing W401A or W401L substitutions (but not W401F) and W414L were defective for dimerization in vitro. Consistent with their dimerization defect, the W401A, W401L and W414L mutants were devoid of RT activity. Using the yeast two-hybrid system, we identified several second-site suppressors in p66 that restored interaction of the p66W401A bait to the p51W401A prey. The suppressors (T409I, D110G, V372A and I393M) also restored heterodimerization of bacterially expressed W401A subunits. When introduced into the W401A mutant, T409I was able to restore RT activity to 50% of the wild-type level. Examination of the RT structures revealed that K331 in p51 makes multiple hydrogen bond contacts with residues in the p66 loop spanned by W401 and W414. Consistent with this observation, the K331A RT mutant was dimerization-defective. We conclude that mutations at codons 401 and 414 in p66 impair dimerization by altering the proper positioning of structural elements in between these residues that make important contacts with p51.
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PMID:Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization. 1255 8

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti-endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation-blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C-based treatment of severe sepsis.
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PMID:Expression and function of the endothelial protein C receptor in human neutrophils. 1271 92

[Arg(6),D-Trp(7,9),N(me)Phe(8)]-substance P (6-11) (SP-G) is a novel anticancer agent that has recently completed phase I clinical trials. SP-G inhibits mitogenic neuropeptide signal transduction and small cell lung cancer (SCLC) cell growth in vitro and in vivo. Using the SCLC cell line series GLC14, 16 and 19, derived from a single patient during the clinical course of their disease and the development of chemoresistance, it is shown that there was an increase in responsiveness to neuropeptides. This was paralleled by an increased sensitivity to SP-G. In a selected panel of tumour cell lines (SCLC, non-SCLC, ovarian, colorectal and pancreatic), the expression of the mitogenic neuropeptide receptors for vasopressin, gastrin-releasing peptide (GRP), bradykinin and gastrin was examined, and their sensitivity to SP-G tested in vitro and in vivo. The tumour cell lines displayed a range of sensitivity to SP-G (IC(50) values from 10.5 to 119 microM). The expression of the GRP receptor measured by reverse transcriptase-polymerase chain reaction, correlated significantly with growth inhibition by SP-G. Moreover, introduction of the GRP receptor into rat-1A fibroblasts markedly increased their sensitivity to SP-G. The measurement of receptor expression from biopsy samples by polymerase chain reaction could provide a suitable diagnostic test to predict efficacy to SP-G clinically. This strategy would be of potential benefit in neuropeptide receptor-expressing tumours in addition to SCLC, and in tumours that are relatively resistant to conventional chemotherapy.
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PMID:Increased gastrin-releasing peptide (GRP) receptor expression in tumour cells confers sensitivity to [Arg6,D-Trp7,9,NmePhe8]-substance P (6-11)-induced growth inhibition. 1277 99

The Avr9 peptide elicitor from the fungus Cladosporium fulvum, the bacterial pathogen Pseudomonas syringae pathovar tomato carrying the avirulence gene avrPto (Pst (avrPto)), and the organophosphorous insecticide fenitrothion induce resistance-related responses in tomato lines carrying the Cf-9, Pto, and Fen genes, respectively. These responses were associated with synthesis of p-coumaroyloctopamine and p-coumaroylnoradrenaline, a novel compound for plants. In susceptible near isogenic tomato lines (Cf-0, pto, fen) and wounded tomato leaves, the levels of these compounds were reduced or undetectable. The elevated levels of p-coumaroyloctopamine and p-coumaroylnoradrenaline were accompanied by elevated mRNA levels of genes encoding phenylalanine ammonia lyase, p-coumarate CoA ligase, and hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT), enzymes that are involved in the hydroxycinnamic acid amide biosynthesis. Southern hybridization indicated that THT is encoded by a multigene family in tomato. Four different THT full-length cDNAs were derived by reverse transcriptase-PCR using degenerate primers based on potato and tobacco THT sequences. Transcripts for all four homologs were present in unchallenged tomato leaves, but only tomTHT1-3 was highly expressed following challenge with Pst (avrPto). Furthermore, tomTHT1-3 showed a more substantial and rapid induction in the incompatible interaction than in the compatible interaction. The cDNAs tomTHT1-3, tomTHT7-1, and tomTHT7-8 encoded proteins with a high degree of amino acid sequence homology, although the recombinant proteins had different preferences for octopamine and noradrenaline. The fourth cDNA, tomTHT1-4, directed synthesis of a truncated enzymatically inactive protein due to the presence of a premature stop codon.
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PMID:p-Coumaroylnoradrenaline, a novel plant metabolite implicated in tomato defense against pathogens. 1290 Apr 12

Neutrophil adhesion to extracellular matrix is necessary for an effective inflammatory response. Adhesion may accelerate neutrophil activation by affecting intracellular signaling pathways. The nuclear transcription factor kappaB (NF-kappaB) controls several cellular functions, including inflammation, proliferation, and cell survival. We explored the role of adhesion in NF-kappaB activation in human neutrophils. Cells were stimulated with tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin-8 (IL-8), and formyl-methionyl-leucyl-phenylalanine (fMLP). All four initiated neutrophil adherence to and spreading on fibronectin. GM-CSF and IL-8 did not activate NF-kappaB in suspended neutrophils but rapidly activated NF-kappaB under adherent conditions on matrix, as shown by IkappaB kinase activity assay, IkappaBalpha degradation, electromobility shift assay, and quantitative reverse transcriptase-PCR. In contrast, TNF-alpha activated NF-kappaB both in suspended cells and adherent cells. fMLP did not activate NF-kappaB in either suspended or adherent cells. Specific beta(2) integrin blockade prevented NF-kappaB activation by GM-CSF and IL-8 on fibronectin. Co-stimulating CD18 and CD11b with activating antibodies resulted in NF-kappaB activation by GM-CSF and IL-8 in suspended cells. We inhibited actin polymerization with cytochalasin and blocked the non-receptor kinase Syk with piceatannol. Both maneuvers prevented the co-stimulatory NF-kappaB-activating signal by beta(2) integrins. Thus, in addition to beta(2) integrin ligand binding, NF-kappaB activation depended on the formation of the receptor-associated intracellular focal adhesion complex. We conclude that beta(2) integrins may provide co-stimulatory signals allowing some soluble mediators to activate the NF-kappaB pathway even when they are not capable of doing so in suspension. This effect may become important when human neutrophils leave the circulating blood and migrate through extracellular matrix during inflammation.
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PMID:Integrins and cytokines activate nuclear transcription factor-kappaB in human neutrophils. 1461 35

There is increasing evidence for a role of 5-hydroxytrypta-mine-6 (5-HT6) receptors in cognitive function. In the rat and human brain, 5-HT6 receptors are widely expressed and highly enriched in the basal ganglia. However, in the mouse brain, only very low levels of 5-HT6 receptor mRNA and receptor protein, measured by TaqMan reverse transcriptase-polymerase chain reaction and selective radioligand binding, could be detected, with no evidence of enrichment in the basal ganglia. The mouse receptor was cloned and transiently expressed in human embryonic kidney 293 cells to characterize its pharmacological profile. Despite significant sequence homology between human, rat, and mouse 5-HT6 receptors, the pharmacological profile of the mouse receptor was significantly different from the rat and human receptors. Four amino acid residues, conserved in rat and human and divergent in mouse receptors, were identified, and various mutant receptors were generated and their pharmacologies studied. Residues 188 (tyrosine in mouse, phenylalanine in rat and human) in transmembrane region 5 and 290 (serine in mouse, asparagine in rat and human) in transmembrane region 6 were identified as key amino acids responsible for the different pharmacological profiles. Molecular modeling of the receptor and docking of selective and nonselective compounds was undertaken to elucidate the ligand receptor interactions. The binding pocket was predicted to be different in the mouse compared with rat and human 5-HT6 receptors, and the models were in excellent agreement with the observed mutation results and have been used extensively in the design of further selective 5-HT6 antagonists.
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PMID:Differences in the central nervous system distribution and pharmacology of the mouse 5-hydroxytryptamine-6 receptor compared with rat and human receptors investigated by radioligand binding, site-directed mutagenesis, and molecular modeling. 1464 56

The antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen Cryptococcus neoformans. To gain a broader understanding of the cellular response of C. neoformans to auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 micro M) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example, RHO1 and an open reading frame (ORF) encoding a protein with 91% similarity to the Schizophyllum commune 14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in Pleurotus sajor-caju was up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for C. neoformans. This is the first report of drug action using DD in C. neoformans.
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PMID:Differential gene expression in auristatin PHE-treated Cryptococcus neoformans. 1474 10

The tissue distribution, course of secretion, and sex differences of morphine were delineated in Ascaris suum. Nitric oxide (NO) release in various tissues in response to morphine and its metabolite morphine-6-glucuronide (M6G) were also examined. Ascaris suum of both sexes along with their incubation fluid were analyzed for morphine concentrations by high-performance liquid chromatography (HPLC) over a 5-day period. Various tissues were also dissected for HPLC and NO analysis. Morphine was found to be most prevalent in the muscle tissue, and there is significantly more morphine in females than males, probably because of the large amounts present in the female uterus. Morphine (10(-9) M) and M6G (10(-9) M) stimulated the release of NO from muscles. Naloxone (10(-7) M) and N-nitro-L-arginine methyl ester (10(-6) M) blocked (P < 0.005) morphine-stimulated NO release from A. suum muscle tissue. D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH2 (CTOP) (10(-7) M) did not block morphine's NO release. However, naloxone could not block M6G-stimulated NO release by muscles, whereas CTOP (10(-7) M) blocked its release. These findings were in seeming contradiction to our earlier inability to isolate a mu opiate receptor messenger RNA by reverse transcriptase-polymerase chain reaction using a human mu primer. This suggests that a novel mu opiate receptor was possibly present and selective toward M6G.
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PMID:Opiate alkaloids and nitric oxide production in the nematode Ascaris suum. 1504 Jun 62


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