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)

Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids. In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity. maCER1 consists of 287 amino acids, and it has a 28 and 32% identity to the Saccharomyces alkaline ceramidases (YPC1p and YDC1p) and the human alkaline phytoceramidase, respectively. Reverse transcriptase-PCR analysis demonstrated that maCER1 was predominantly expressed in skin. maCER1 was localized to the endoplasmic reticulum as revealed by immunocytochemistry. In vitro biochemical characterization determined that maCER1 hydrolyzed D-erythro-ceramide exclusively but not D-erythro-dihydroceramide or D-ribo-phytoceramide. Similar to other alkaline ceramidases, maCER1 had an alkaline pH optimum of 8.0, and it was activated by Ca2+ but inhibited by Zn2+,Cu2+, and Mn2+. maCER1 was also inhibited by sphingosine, one of its products. Metabolic labeling studies showed that overexpression of maCER1 caused a decrease in the incorporation of radiolabeled dihydrosphingosine into ceramide and complex sphingolipids but led to a concomitant increase in sphingosine-1-P (S1P) in HeLa cells. Mass measurement showed that overexpression of maCER1 selectively lowered the cellular levels of D-erythro-C24:1-ceramide, but not other ceramide species and caused an increase in the levels of S1P. Taken together, these data suggest that maCER1 is a novel alkaline ceramidase with a stringent substrate specificity and that maCER1 is selectively expressed in skin and may have a role in regulating the levels of bioactive lipids ceramide and S1P, as well as complex sphingolipids.
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PMID:Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase: an enzyme that preferentially regulates metabolism of very long chain ceramides. 1278 75

Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses. It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X. Wu, H. Liu, H. Xiao, J. A. Conway, E. Hehl, G. V. Kalpana, V. R. Prasad, and J. C. Kappes, J. Virol. 73:2126-2135, 1999). We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function. Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN. An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction. Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT. Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain. The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment. This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction. The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3' processing and joining reactions in the presence of both proteins. Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold. Thus, a functional interaction between these two viral enzymes may occur during viral replication.
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PMID:Interaction between human immunodeficiency virus type 1 reverse transcriptase and integrase proteins. 1511 87

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.
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PMID:Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. 1514 Sep 59

Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.
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PMID:Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta. 1576 66

Histological investigations have demonstrated that root canal sealers can induce mild to severe inflammatory alternations. However, there is little information on the precise mechanisms about root canal sealers-induced inflammatory reaction. Dysregulated cytokine productions at local disease sites have been considered to be major contributors to the development of inflammatory diseases. Interleukin (IL)-6 and IL-8 released have been reported to play an important role in the pathogenesis of inflammation. The aim of this study was to investigate the effects of root canal sealers N2 (zinc-oxide eugenol based) and AH Plus (epoxy resin based) on the expression of IL-6 and IL-8 mRNA gene in human osteoblastic cell line U2OS cells. The levels of mRNAs were measured by the semi-quantitative reverse-transcriptase polymerase chain reaction analysis. The exposure of quiescent U2OS cells to N2 and AH Plus resulted in the induction of IL-6 and IL-8 mRNA gene expression (p < 0.05). The intensity of IL-8 mRNA gene was found to be significant higher than IL-6 mRNA gene (p < 0.05). Taken together, the activation of IL-6 and IL-8 mRNA gene expression may be one of the pathogenesis of zinc oxide-eugenol based and epoxy resin based root canal sealers-induced periapical inflammation.
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PMID:Induction of interleukin-6 and interleukin-8 gene expression by root canal sealers in human osteoblastic cells. 1612 6

Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-A resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.
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PMID:Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1.85-angstrom resolution. 1730 Nov 46

A chloride current, I(Cl,H), slowly activating on hyperpolarisation was investigated in Drosophila melanogaster larval muscles using the two-electrode voltage clamp. Sizeable currents were observed after the intracellular chloride concentration ([Cl-]i) had been elevated by diffusion of Cl- from the electrodes. The time course of I(Cl,H) was rather variable and required two exponentials to be accurately described. The reversal potential, -40 to -20 mV in Cl- -loaded fires, shifted on lowering external [Cl-] in the positive direction. Steady-state activation of I(Cl,H) was characterised by V0.5 of approximately -120 mV and a slope factor, k, of approximately 10 mV at a [Cl-]i approximately 35 mmol l(-1). Raising [Cl-]i to approximately 50 mmol l(-1) caused a negative shift of V0.5 equivalent to the change of E(Cl) and led to a nearly threefold increase in maximal steady-state conductance. I(Cl,H) was resistant to 10 mmol l(-1) Zn2+ and 1 mmol l(-1) Cd2+ but was greatly reduced by 1 mmol l(-1) 9-anthracenecarboxylic acid (9-AC). I(Cl,H) was affected by changes of extracellular pH and increased on lowering extracellular osmolality. 9-AC also decreased muscle fibre resting conductance by approximately 20% and increased muscle contractions. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed the expression of all three ClC genes in muscle, and immunohistochemistry indicated location of Drosophila melanogaster chloride channel-2 (DmClC-2) at the Z-lines. We conclude that DmClC-2 accounts for the channels underlying I(Cl,H), and in part for the resting chloride conductance. DmClC-2 may serve general homeostatic mechanisms such as pH- and osmo-regulation or may support muscle function on high motor activity or during a particular neurohormonal state of the animal.
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PMID:Properties and possible function of a hyperpolarisation-activated chloride current in Drosophila. 1760 53

Dietary fat accelerates the ageing process and causes a greater mortality by accumulating lipid hydroperoxide (LPO) in Drosophila melanogaster. The present study found that the life span of D. melanogaster was shortened from 54 to 6 days in a dose-dependent manner when fat in diet increased from 0% to 25%. The results showed that supplementation of both green tea catechins (GTC) and broccoli extract (BE) reversed partially the fat-induced mortality. The maximum life span was 44 days for the control group fed with a 5% fat, whereas it increased to 50 and 59 days in the GTC- and BE-supplemented groups, respectively. The 50% survival time for the control flies fed with a 5% fat diet was 30 days. In contrast, it increased to 32 and 48 days when GTC and BE were supplemented in the diet. This was consistent with a significant reduction in total body LPO level in D. melanogaster maintained on the GTC- and BE-supplemented diet. Accordingly, catalase and superoxide dismutase (SOD) activities increased significantly in the flies fed with a GTC or a BE diet compared with those fed with a control 5% fat diet. Reverse transcriptase-polymerase chain reaction analysis indicated that the increase in enzymatic activities of catalase and SOD was accompanied by up-regulation of genes for catalase, copper-zinc containing SOD and manganese-containing SOD. It was concluded that GTC and BE reversed the fat-induced mortality in D. melanogaster, most likely but necessarily solely, by up-regulation of endogenous antioxidant enzymes.
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PMID:Green tea catechins and broccoli reduce fat-induced mortality in Drosophila melanogaster. 1770 29

Plants respond to environmental mechanical stimulation, such as wind, by modifying their growth and development. To study the molecular effects of stem bending on 3-week-old walnut trees, a cDNA-AFLP approach was developed. This study allowed the identification of a cDNA, known as Jr-ZFP2, encoding a Cys2/His2-type two-zinc-fingered transcription factor. Reverse transcriptase-polymerase chain reaction analysis confirmed that Jr-ZFP2 mRNA accumulation is rapidly and transiently induced after mechanical stimulation. After bending, Jr-ZFP2 transcript increase was restricted to the stem, the organ where the mechanical solicitation was applied. Furthermore, other abiotic factors, such as cold or salt, did not modify Jr-ZFP2 mRNA accumulation in walnut stems under our experimental conditions, whereas growth studies demonstrated that salt stress was actually perceived by the plants. These results suggest that the regulation of Jr-ZFP2 expression is more sensitive to mechanical stimulus. This gene will be a good marker for studying the early stages of mechanical perception in woody plants.
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PMID:Jr-ZFP2, encoding a Cys2/His2-type transcription factor, is involved in the early stages of the mechano-perception pathway and specifically expressed in mechanically stimulated tissues in woody plants. 1820 13

Flaviviruses are emerging pathogens of increasingly important public health concern in the world. For most flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) neither vaccine nor antiviral treatment is available. The viral RNA-dependent RNA polymerase (RdRp) non-structural protein 5 (NS5) has no equivalent in the host cell and is essential for viral replication. Here, we give an overview of the current knowledge regarding Flavivirus RdRp function and structure as it represents an attractive target for drug design. Flavivirus RdRp exhibits primer-independent activity, thus initiating RNA synthesis de novo. Following initiation, a conformational change must occur to allow the elongation process. Structure-function studies of Flavivirus RdRp are now facilitated by the crystal structures of DENV (serotype 3) and WNV RdRp domains. Both adopt a classic viral RdRp fold and present a closed pre-initiation conformation. The so-called priming loop is thought to provide the initiation platform stabilizing the de novo initiation complex. A zinc-ion binding site at the hinge between two subdomains might be involved in opening up the RdRp structure towards a conformation for elongation. Using two different programs we predicted common potential allosteric inhibitor binding sites on both structures. We also review ongoing approaches of in vitro and cell-based screening programs aiming at the discovery of nucleosidic and non-nucleosidic inhibitors targeting Flavivirus RdRps.
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PMID:The flavivirus polymerase as a target for drug discovery. 1861 13


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