The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform
Piotr M. Skowron - One of the best experts on this subject based on the ideXlab platform.
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chemically induced affinity star restriction specificity a novel tspgwi Sinefungin endonuclease with theoretical 3 bp cleavage frequency
BioTechniques, 2011Co-Authors: Agnieszka żyliczstachula, Olga żolnierkiewicz, Joanna Jezewskafrąckowiak, Piotr M. SkowronAbstract:The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5′-ACGGA-3′, cleaving DNA 11/9 nucleotides downstream. Here we show that Sinefungin, a cofactor analog of S-adenosyl methion...
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Chemically-induced affinity star restriction specificity: a novel TspGWI/Sinefungin endonuclease with theoretical 3-bp cleavage frequency.
BioTechniques, 2011Co-Authors: Agnieszka Żylicz-stachula, Olga Żołnierkiewicz, Joanna Jeżewska-frąckowiak, Piotr M. SkowronAbstract:The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5′-ACGGA-3′, cleaving DNA 11/9 nucleotides downstream. Here we show that Sinefungin, a cofactor analog of S-adenosyl methion...
Kenji Inagaki - One of the best experts on this subject based on the ideXlab platform.
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Antiviral effect of Sinefungin on in vitro growth of feline herpesvirus type 1
The Journal of Antibiotics, 2019Co-Authors: Yudai Kuroda, Haruka Yamagata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura, Ken MaedaAbstract:Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, Sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that Sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.
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Draft Genome Sequence of Streptomyces incarnatus NRRL8089, which Produces the Nucleoside Antibiotic Sinefungin
Genome announcements, 2015Co-Authors: Kenshiro Oshima, Michiko Nemoto, Kenji Inagaki, Koji Fukuda, Masahira Hattori, Hitomi Shimizu, Takashi TamuraAbstract:A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic Sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.
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Production improvement of antifungal, antitrypanosomal nucleoside Sinefungin by rpoB mutation and optimization of resting cell system of Streptomyces incarnatus NRRL 8089
Journal of bioscience and bioengineering, 2009Co-Authors: Koji Fukuda, Takashi Tamura, Kozo Ochi, Hideyuki Ito, Sayaka Yamamoto, Kenji InagakiAbstract:Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the Sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the beta-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM L-Arg. D-Arg or L-ornithine did not enhance the Sinefungin production, and >50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of L-Arg and ATP.
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Improvement of Sinefungin-Producing Strain of Streptomyces incarnatus by Conferring Rifampicin-Resistance through Ultraviolet Light Irradiation and Protoplast Regeneration
2002Co-Authors: Takashi Tamura, Hidehiko Tanaka, Kenji InagakiAbstract:Secondary metabolite production by gram-positive bacteria is strictly regulated at the transcription of the biosynthetic genes to mRNA in response to certain stringent conditions. Therefore, some mutational disruption of regulatory domains of the bacterial RNA polymerase might increase the production of the antibiotics. In this study, we have attempted to improve the Sinefungin-producing strain of Streptomyces incarnatus NRRL 8057 by irradiating ultraviolet light on the protoplast, and selecting mutants that acquired the resistance to rifampicin, the antibiotic which specifically binds to the β-subunit of bacterial RNA polymerase. After three rounds of mutation, 10 strains were obtained with varied resistance to rifampicin. A mutant which showed the highest resistance was found to have the highest Sinefungin production, which was 2.4 times higher(0.45±0.11μg/ml)than the wild type strain (0.19±0.07μg/ml). The breeding approach by rifampicin-resistance may be advantageous over the classical random screening since it requires much smaller number of candidates to be examined.
Malka Robert-gero - One of the best experts on this subject based on the ideXlab platform.
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Sinefungin and Taxol Effects on Cell Cycle and Cytoskeleton ofLeishmania donovaniPromastigotes
Experimental cell research, 1996Co-Authors: Lamya Moulay, Malka Robert-gero, Spencer Brown, Marie-claude Gendron, Frédéric TournierAbstract:Abstract Sinefungin is an antibiotic possessing a strong anti-leishmanial activity. Among the most important effects of this molecule onLeishmania donovanipromastigotes are morphological modifications and a very rapid and effective inhibition of DNA synthesis. These cells contain a single DNA-rich mitochondrion whose division cycle is coordinated with the nuclear division cycle. We have developed a flow-cytometric procedure based upon mithramycin as fluorochrome that can perform quantitative cell cycle analysis on the nuclear DNA. Cell cycle progression was analyzed to establish that Sinefungin irreversibly blocks the promastigotes in early S phase. Sinefungin did not react with stationary cells as they were arrested in G1. Surprisingly, taxol, a microtubule-stabilizing drug, induced the same morphological modifications as Sinefungin although it interfered with the G2/M progression. According to immunofluorescence studies, the stable microtubular network is apparently affected neither by taxol nor by Sinefungin.
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Relationship Between Chemical Structure and Antileishmanial Effect of Sinefungine Analogues
Nucleosides and Nucleotides, 1996Co-Authors: Pierre Blanchard, Françoise Lawrence, M. S. El Kortbi, Jean-louis Fourrey, Malka Robert-geroAbstract:Abstract The synthesis of various analogues of Sinefungin (l), having structures 2-5, has been developed by means of an original approach which uses radical chemistry. The study of their biological activities revealed that for the antileishmanial effect of Sinefungin, the presence of the amino group at C-6′ in the (S)-configuration and the presence of the carboxyl group at C-9′ are necessary.
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Distribution of Macromolecular Methylations in Promastigotes of Leishmania donovani and Impact of Sinefungin
The Journal of eukaryotic microbiology, 1993Co-Authors: Françoise Lawrence, Malka Robert-geroAbstract:. Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent both in vitro and in vivo. This molecule, structurally related to S-adenosylmethionine, is a good competitive inhibitor of methyltransferases in vitro. The aim of this report is to analyze the impact of Sinefungin on methylation pattern and the subcellular localisation of methyl groups and various methylases in promastigotes of Leishmania donovani. We have shown the presence of various methylated macromolecules in different subcellular fractions, with somewhat higher concentration in membrane fraction. In vitro, Sinefungin inhibits the three main protein methylases, but in cells cultured in its presence the protein carboxylmethylations are specifically inhibited.
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Leishmania donovani: Antagonistic effect of S-adenosyl methionine on ultrastructural changes and growth inhibition induced by Sinefungin
Experimental parasitology, 1992Co-Authors: Marie-anne Phelouzat, Lamya Moulay, Françoise Lawrence, Caroline Borot, Jean Schaeverbeke, Madeleine Schaeverbeke, Malka Robert-geroAbstract:Abstract Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent in vitro and in vivo (Bachrach et al. 1980, FEBS Letters121, 287–291; Neal et al. 1985, Transactions of the Royal Society of Tropical Medicine and Hygiene79, 85–122). It was previously shown that this molecule is a competitive inhibitor of AdoMet for transmethylases (Paolantonacci et al. 1986, Molecular and Biochemical Parasitology21, 47–54; Avila et al. 1987, Molecular and Biochemical Parasitology26, 69–76) and that it induces shape changes of Leishmania donovani promastigotes as observed by light microscopy (Lawrence and Robert-Gero 1990; Bulletin de la Societe Francaise de Parasitologie8, 13–18). In the present work the effect of the antibiotic on the ultrastructure was analyzed by electron microscopy. The main changes induced at sublethal concentrations (0.26 μM Sinefungin for 16 hr) were progressive rounding, decreased motility, enlargement of the flagellar pocket, and shortening and loss of the external part of the flagellum. The comparison with control cells showed shorter Golgi saccules and fragmentation of the trans-Golgi network into vesicles, indicating a stimulated Golgi apparatus activity. This result, associated with the enlarged flagellar pocket, suggests an unbalanced cytoplasmic exchange between exocytosis and endocytosis. These effects are quite different from those induced by tunicamycin (Dagger et al. 1984, Biology of the Cell50; 173–180) or paromomycin. In addition, other nucleoside and nonnucleoside growth inhibitors failed to induce similar changes. AdoMet antagonized the Sinefungin-induced shape changes and ultrastructural modifications but had no effect with respect to other growth inhibitors. This suggests that the Sinefungin activity at the cellular level is specifically related to competition with AdoMet. A comparative study of N-methylation and carboxylmethylation of proteins in Sinefungin-treated promastigotes showed that the antibiotic preferentially inhibits the latter, catalyzed by protein-O-methyltransferases. These enzymes are known to regulate the function of various proteins involved in secretion. Overall the results suggest that one of the main targets of Sinefungin in exponentially growing cells is the protein carboxylmethylation involved in membrane transport.
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Total synthesis of uracil analogues of Sinefungin.
Journal of medicinal chemistry, 1992Co-Authors: Derek H.r. Barton, Malka Robert-gero, Françoise Lawrence, Stephane D. Gero, Béatrice Quiclet-sire, M. SamadiAbstract:Analogues of Sinefungin derivatives 18a and 18b have been prepared from uridine and L-aspartic acid. The key step in the synthesis was the coupling of the radical derived from 14 with the unsaturated amide 13. The latter was produced from the known N-hydroxy-2-thiopyridone ester of L-aspartic acid 12 with the olefin 11. Thus, the essential carbon skeleton was constructed by way of two radical coupling reactions. These analogues as well as 1a and 1b synthesized previously were tested for their antileishmanial effect in vivo and for their inhibitory activity of protein carboxymethylase (protein methylase II). The replacement of the adenine moiety by uracil or dihydrouracil considerably decreases the antiparasitic activity and the affinity for protein methylase II. The synthetic (S)-Sinefungin was as active as the natural one. Interestingly, the C-6' epimer 1b was 50% less active in vitro than the natural Sinefungin, but both had identical affinities for the target enzyme.
Koji Fukuda - One of the best experts on this subject based on the ideXlab platform.
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Multiple mutations in RNA polymerase β-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic Sinefungin: modeling rif cluster region by density functional theory.
Bioscience biotechnology and biochemistry, 2021Co-Authors: Saori Ogawa, Hitomi Shimidzu, Koji Fukuda, Naoki Tsunekawa, Toshiyuki Hirano, Fumitoshi Sato, Kei Yura, Tomohisa Hasunuma, Kozo Ochi, Michio YamamotoAbstract:Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic Sinefungin. To enhance Sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the Sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on Sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.
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Draft Genome Sequence of Streptomyces incarnatus NRRL8089, which Produces the Nucleoside Antibiotic Sinefungin
Genome announcements, 2015Co-Authors: Kenshiro Oshima, Michiko Nemoto, Kenji Inagaki, Koji Fukuda, Masahira Hattori, Hitomi Shimizu, Takashi TamuraAbstract:A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic Sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.
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Production improvement of antifungal, antitrypanosomal nucleoside Sinefungin by rpoB mutation and optimization of resting cell system of Streptomyces incarnatus NRRL 8089
Journal of bioscience and bioengineering, 2009Co-Authors: Koji Fukuda, Takashi Tamura, Kozo Ochi, Hideyuki Ito, Sayaka Yamamoto, Kenji InagakiAbstract:Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the Sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the beta-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM L-Arg. D-Arg or L-ornithine did not enhance the Sinefungin production, and >50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of L-Arg and ATP.
Takashi Tamura - One of the best experts on this subject based on the ideXlab platform.
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Antiviral effect of Sinefungin on in vitro growth of feline herpesvirus type 1
The Journal of Antibiotics, 2019Co-Authors: Yudai Kuroda, Haruka Yamagata, Michiko Nemoto, Kenji Inagaki, Takashi Tamura, Ken MaedaAbstract:Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, Sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that Sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.
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Draft Genome Sequence of Streptomyces incarnatus NRRL8089, which Produces the Nucleoside Antibiotic Sinefungin
Genome announcements, 2015Co-Authors: Kenshiro Oshima, Michiko Nemoto, Kenji Inagaki, Koji Fukuda, Masahira Hattori, Hitomi Shimizu, Takashi TamuraAbstract:A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic Sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.
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Production improvement of antifungal, antitrypanosomal nucleoside Sinefungin by rpoB mutation and optimization of resting cell system of Streptomyces incarnatus NRRL 8089
Journal of bioscience and bioengineering, 2009Co-Authors: Koji Fukuda, Takashi Tamura, Kozo Ochi, Hideyuki Ito, Sayaka Yamamoto, Kenji InagakiAbstract:Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the Sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the beta-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM L-Arg. D-Arg or L-ornithine did not enhance the Sinefungin production, and >50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of L-Arg and ATP.
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Improvement of Sinefungin-Producing Strain of Streptomyces incarnatus by Conferring Rifampicin-Resistance through Ultraviolet Light Irradiation and Protoplast Regeneration
2002Co-Authors: Takashi Tamura, Hidehiko Tanaka, Kenji InagakiAbstract:Secondary metabolite production by gram-positive bacteria is strictly regulated at the transcription of the biosynthetic genes to mRNA in response to certain stringent conditions. Therefore, some mutational disruption of regulatory domains of the bacterial RNA polymerase might increase the production of the antibiotics. In this study, we have attempted to improve the Sinefungin-producing strain of Streptomyces incarnatus NRRL 8057 by irradiating ultraviolet light on the protoplast, and selecting mutants that acquired the resistance to rifampicin, the antibiotic which specifically binds to the β-subunit of bacterial RNA polymerase. After three rounds of mutation, 10 strains were obtained with varied resistance to rifampicin. A mutant which showed the highest resistance was found to have the highest Sinefungin production, which was 2.4 times higher(0.45±0.11μg/ml)than the wild type strain (0.19±0.07μg/ml). The breeding approach by rifampicin-resistance may be advantageous over the classical random screening since it requires much smaller number of candidates to be examined.
