The novel gluconeogenesis inhibitor FR225654 that originates from Phoma sp. no. 00144. I. Taxonomy, fermentation, isolation and physico-chemical properties.

FR225654, a novel gluconeogenesis inhibitor, was isolated from the culture broth of Phoma sp. No. 00144 and purified by adsorptive resin and reverse-phase column chromatography. This compound is a potent inhibitor of gluconeogenesis and is a promising candidate of anti-diabetic agent.

The novel gluconeogenesis inhibitor FR225654 that originates from Phoma sp. no. 00144. II. Biological activities.

The novel gluconeogenesis inhibitor FR225654, isolated from the culture broth of Phoma sp. No. 00144, has an unique structure that consists of a highly oxygenated trans-decalin ring and a beta-keto-enol, with a characteristic side chain. This compound selectively inhibited gluconeogenesis of rat primary hepatocytes and had hypoglycemic effects in several in vivo mouse models.

The novel gluconeogenesis inhibitor FR225654 that originates from Phoma sp. no. 00144. III. Structure determination.

A novel gluconeogenesis inhibitor, FR225654 was isolated from the culture broth of Phoma sp. No. 00144. Spectroscopic analysis concluded that FR225654 has a highly oxygenated trans-decalin ring and beta-keto-enol in its main part, and has a characteristic side chain possessing a conjugated carboxylic acid and tri-substituted olefin.

Selective ligand purification using high-performance affinity beads.

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.

FR225659-binding proteins: identification as serine/threonine protein phosphatase PP1 and PP2A using high-performance affinity beads.

FR225659 was originally isolated as a novel gluconeogenesis inhibitor produced by fungal strain Helicomyces sp. No. 19353. To identify the target protein of FR225659, we synthesized high-performance affinity latex beads that immobilized FR225659 derivative FR253761 or FR259383. Using these beads, we identified FR225659 binding proteins as serine/threonine protein phosphatase type1 (PP1) and type2A (PP2A) from rat hepatocyte crude extract. FR225659 and its synthetic derivatives were strongly inhibited the enzyme activities of purified catalytic subunits of PP1 and PP2A in vitro.

Characterization of double-stranded RNA elements in the violet root rot fungus Helicobasidium mompa.

Double-stranded (ds) RNA of various types was detected by electrophoresis in 23 of 25 isolates of Helicobasidium mompa. These dsRNAs varied in size from ca. 2 kbp to more than 10 kbp. dsRNAs from an isolate V1 had two distinct nucleotide sequences for putative RNA-dependent RNA polymerase (RDRP). Their complete sequences revealed that V1 dsRNA1 was 2247 bp in length, with a single ORF that encoded a 706-amino acid residue polypeptide with a predicted molecular mass of 82.6 kDa, and that V1 dsRNA3 was 1776 bp in length, with a single ORF that encoded a 538-amino acid residue polypeptide with a predicted molecular mass of 62.6 kDa. RDRP-conserved motifs were identified in both predicted amino acid sequences. Phylogenetic analysis indicated that V1 dsRNA1 was most closely related to Fusarium poae virus 1, while V1 dsRNA3 was most closely related to Helicobasidium mompa 70 virus. These results indicate coinfection of isolate V1 by two distinct partitiviruses.

FR171456, a novel cholesterol synthesis inhibitor produced by Sporormiella minima No. 15604. I. Taxonomy, fermentation, isolation, physico-chemical properties.

FR171456 and FR173945, novel and potent cholesterol synthesis inhibitors, have been isolated from the fermentation broth of a fungal strain No. 15604. This strain was identified Sporormiella minima from its mycological characteristics. FR171456 and FR173945 strongly inhibited cholesterol synthesis in human hepatoma cell line Hep G2. These compounds also have in vitro antifungal activity against Candida albicans and Aspergillus fumigatus.

The novel gluconeogenesis inhibitors FR225659 and related compounds that originate from Helicomyces sp. No. 19353. II. Biological profiles.

The novel gluconeogenesis inhibitor FR225659 and four related compounds were isolated from the cultured broth of a fungal strain No. 19353. These compounds inhibited the glucagon-stimulated gluconeogenesis of rat primary hepatocytes and had hypoglycemic effects in two different in vivo models.

The novel gluconeogenesis inhibitors FR225659 and related compounds that originate from Helicomyces sp. No. 19353. I. Taxonomy, fermentation, isolation and physico-chemical properties.

FR225659 and four related compounds are novel gluconeogenesis inhibitors that consist of a novel acyl-group and three abnormal amino acids. They were isolated from the culture broth of Helicomyces sp. No. 19353 and can be purified by absorptive resin and reverse-phase column chromatography. They are potent inhibitors of gluconeogenesis in primary cultured rat hepatocytes and thus may be useful as anti-diabetic agents.

The novel gluconeogenesis inhibitors FR225659 and FR225656 from Helicomyces sp. No. 19353. III. Structure determination.

During the course of screening for novel gluconeogenesis inhibitors, FR225659 and its related compounds were isolated from a fermentation broth of Helicomyces sp. No. 19353. Spectroscopic analysis concluded that FR225659 is an N-acyl tripeptide consisting of a novel acyl, a 3-chloro-4-hydroxyarginine, a 3-hydroxy-3-methylproline and a dehydrovaline. Degradation study allowed assignment of the absolute configuration of the 3-hydroxy-3-methylproline to be (2S,3R). FR225656 was shown to possess a dehydroisoleucine instead of the dehydrovaline of FR225659.

Molecular characterization of dsRNA segments 2 and 5 and electron microscopy of a novel reovirus from a hypovirulent isolate, W370, of the plant pathogen Rosellinia necatrix.

A hypovirulent isolate, W370, of the white root rot fungus Rosellinia necatrix has previously been shown to harbour 12 dsRNA segments. In this study, complete nucleotide sequences of segments 2 and 5 of W370 dsRNAs were determined. The nucleotide sequence of genome segment 2 was 3773 bases long with a single long open reading frame (ORF) encoding 1226 amino acid residues with a predicted molecular mass of approximately 138.5 kDa. The nucleotide sequence of segment 5 was 2089 bases long with a single long ORF, whose deduced polypeptide contained 646 amino acid residues with a predicted molecular mass of about 72 kDa. Comparative analysis showed that the deduced protein sequence of segment 2 had significant homology with the putative VP2 of Colorado tick fever virus (CTFV) and European Eyach virus (EYAV) in the genus Coltivirus, but the deduced protein sequence of segment 5 had no similarity with other virus proteins. Double-shelled spherical particles approximately 80 nm in diameter associated with W370 dsRNAs were observed in a preparation from the mycelial tissue of isolate W370. The results demonstrated that the virus associated with W370 dsRNAs is a novel reovirus of the family Reoviridae. The virus was named Rosellinia anti-rot virus (RArV).

Cloning and characterization of a totivirus double-stranded RNA from the plant pathogenic fungus, Helicobasidium mompa Tanaka.

Virus-like particles (VLPs, named HmTV1-17), about 40 nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5,207 and 2,096 bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5' located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the -1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.

Detection of a double-stranded RNA virus from a strain of the violet root rot fungus Helicobasidium mompa Tanaka.

Three double-stranded (ds) RNA species (ca. 1.30, 1.27 and 1.23 x 106) were isolated by CF-11 cellulose chromatography from a strain of the violet root rot fungus Helicobasidium mompa recovered from apple roots. Purified virion preparations contained isometric particles about 25 nm in diameter, and also the same three species of dsRNA isolated from total extracts by CF-11 cellulose chromatography. The molecular mass of the coat protein was about 67 K when estimated by SDS-PAGE. The largest dsRNA (referred to as dsRNA1) contains a single, long open reading frame of 1794 nucleotides that encodes a putative polypeptide containing 598 amino acid residues with a molecular mass of 69.9 K. This polypeptide contains amino acid sequence motifs conserved in putative RNA-dependent RNA polymerases of RNA viruses. Phylogenetic analysis revealed similarities to RNA-dependent RNA polymerases from Atkinsonella hypoxylon 2H virus, a member of the family Partitiviridae.

Nucleotide sequences of double-stranded RNA segments from a hypovirulent strain of the white root rot fungus Rosellinia necatrix: possibility of the first member of the Reoviridae from fungus.

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41 x 10(6) to 2.95 x 10(6). Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5'-end (5'-ACAAUUU-3') and at the 3'-end (5'-UGCAGAC-3') were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus.