Cedarmycins A and B, new antimicrobial antibiotics from Streptomyces sp. TP-A0456.

Two novel butyrolactone antibiotics, cedarmycins A and B, were isolated from the cultured broth of the actinomycete Streptomyces sp. TP-A0456. The new compounds were purified by HP-20 resin, silica gel, ODS column chromatographies and preparative HPLC, consecutively. The structure of cedarmycin was determined by spectroscopic methods as an alpha,beta-unsaturated butyrolactone with a fatty acid side chain. These compounds showed antibiotic activity against Gram-positive and -negative bacteria and yeasts. Among the tested organisms, cedarmycins potently inhibited the growth of Candida glabrata IFO 0622 with the MIC of 0.4 microg/ml, comparable to that of amphotericin B.

TPU-0031-A and B, new antibiotics of the novobiocin group produced by Streptomyces sp. TP-A0556.

Two novel antibiotics, TPU-0031-A and B, were isolated from the culture broth of an actinomycete strain. The producing strain, TP-A0556, was identified as Streptomyces sp. based on the taxonomic study. The new antibiotics were obtained by solvent extraction and chromatographic purification. Spectroscopic analyses showed that TPU-0031-A and B were 7'-demethylnovobiocin and 5"-demethylnovobiocin, respectively. These compounds showed antibiotic activity against Gram-positive and -negative bacteria.

Association of induced disease resistance of rhododendron seedlings with inoculation of Streptomyces sp. R-5 and treatment with actinomycin D and amphotericin B to the tissue-culture medium.

Seedlings of rhododendron were treated by adding Streptomyces sp. strain R-5, actinomycin D and/or amphotericin B to the tissue-culture medium. HPLC analysis showed that all of the treated seedlings contained these antibiotics at concentrations higher than the suppressive levels to mycelial growth of Pestalotiopsis sydowiana, a major pathogen of rhododendron. Occurrence of disease caused by this fungus in the seedlings was suppressed by treatment of the medium surface with strain R-5, but not by treatment with these antibiotics, suggesting that growth of strain R-5, an antibiotic producer, could be essential for induction of disease resistance in tissue-cultured seedlings of rhododendron.

Goadsporin, a chemical substance which promotes secondary metabolism and Morphogenesis in streptomycetes. II. Structure determination.

The structure of goadsporin was determined by using spectroscopic techniques. NMR analysis revealed that goadsporin consists of 19 amino acids, two of which are dehydroalanines (Deala), and six of which are cyclized to oxazoles (Oxz) and thiazoles (Thz) by dehydrative cyclization and dehydrogenation from serine, threonine and cysteine. NMR analysis established seven partial structures, and their sequence was determined by CID-MS/MS. Negative mode FAB-MS/MS gave product ions arising from charge-remote fragmentation that allowed determination of the sequence of the amino acid components as AcNH-Ala-MeOxz-Val-Deala-MeOxz-Ile-Leu-Thz-Ser-Gly-Gly-MeOxz-Leu-Deala-Oxz-Ala-Gly-Thz-Val-OH. The chiral amino acids were determined by the advanced Marfey's method to have L-configurations.

Goadsporin, a chemical substance which promotes secondary metabolism and morphogenesis in streptomycetes. I. Purification and characterization.

Streptomycetes, which belong to the Gram-positive bacteria, produce secondary metabolites and sporulate. The timing of starting the secondary metabolite production and the sporulation depends on environmental conditions such as nitrogen and carbon sources. In order to obtain a tool for understanding the regulation mechanism, we carried out screening for chemical substances that induce secondary metabolism and sporulation in streptomycetes and found an active substance from the culture broth of Streptomyces sp. TP-A0584. This substance designated goadsporin promoted the formation of red pigment and sporulation at a concentration of 1 microM in Streptomyces lividans TK23 which does not produce the pigment under normal growth conditions. Goadsporin is an oligopeptide consisting of 19 amino acids with the molecular formula C72H97N19020S2. Sporulation and/or secondary metabolite production was induced in 36 streptomycetes strains among 42 strains tested. These results suggest that goadsporin acts on a common regulation pathway for sporulation and secondary metabolism in streptomycetes and can be a powerful tool to analyze the regulation mechanism.

Xanthoepocin, a new antibiotic from Penicillium simplicissimum IFO5762.

A new antifungal antibiotic xanthoepocin was isolated from the culture broth of Penicillium simplicissimum IFO5762. Xanthoepocin was obtained from the culture fluid by solvent extraction and chromatographic purification. It showed antibiotic activity against Gram-positive bacteria and yeasts.

New Cdc25B tyrosine phosphatase inhibitors, nocardiones A and B, produced by Nocardia sp. TP-A0248: taxonomy, fermentation, isolation, structural elucidation and biological properties.

Strain TP-A0248 which produces two new Cdc25B tyrosine phosphatase inhibitors and also possessing antifungal activity, designated nocardiones A (1) and B (2), was considered to belong to the genus Nocardia on the basis of literature comparison of chemotaxonomic properties. The nocardiones were isolated by solvent extraction of fermentation broth of Nocardia sp. TP-A0248 and purified by the conventional column chromatography. Spectroscopic studies led to determination that 1 and 2 belong to a class compound of naphtho[1,2-b]furan-4,5-diones. Compound 1 inhibited the activity of Cdc25B, PTP1B and FAP-1 protein tyrosine phosphatases at a concentration of 10 microM. It also showed moderate in vitro antifungal and cytotoxic activity.

Arisostatins A and B, new members of tetrocarcin class of antibiotics from Micromonospora sp. TP-A0316. I. Taxonomy, fermentation, isolation and biological properties.

Arisostatins A and B, new members of tetrocarcin class of antibiotics were isolated from the culture broth of an actinomycete strain. The producing strain, TP-A0316, was identified as Micromonospora sp. Arisostatins were obtained from the culture fluid by solvent extraction and chromatographic purification. They showed antibiotic activity against Gram-positive bacteria and antitumor activity.

Arisostatins A and B, new members of tetrocarcin class of antibiotics from Micromonospora sp. TP-A0316. II. Structure determination.

Structures of arisostatins A and B were determined by spectroscopic analyses. Arisostatins were found to be new analogs of tetrocarcin A and possess an iso-butanoyldigitoxose unit instead of the acetyldigitoxose one. NMR analyses of arisostatins and tetrocarcin A led to the revision of the anomeric configurations in the tetrasaccharide moiety of tetrocarcin A.

Fistupyrone, a novel inhibitor of the infection of Chinese cabbage by Alternaria brassicicola, from Streptomyces sp. TP-A0569.

A new microbial metabolite, designated fistupyrone, was isolated from the culture broth of a plant-associated Streptomyces sp. TP-A0569. Fistupyrone inhibited the in vivo infection of the seedlings of Chinese cabbage by Alternaria brassicicola TP-F0423, the cause of Alternaria leaf spot, without any in vitro fungicidal activity.

High-mannose type oligosaccharide-dependent apoptosis in U937 cells induced by pradimicin, a mannose-binding antibiotic.

Cell surface oligosaccharides play a role in a variety of biological events such as cell adhesion and signal transduction. We have shown that BMY-28864, a semi-synthetic analog of pradimicin, induced apoptosis of U937 cells which had been incubated with 1-deoxymannojirimycin, an inhibitor of mannosidase I. BMY-28864 was not cytotoxic to the cells which had been cultivated with other glycosidase inhibitors such as castanospermine and swainsonine. We thus propose that BMY-28864 induces apoptosis by acting on a specific mannose-rich oligosaccharide, presumably (Man)9(GlcNAc)2+.

Involvement of Ca2+ ion and reactive oxygen species as a mediator in pradimicin-induced apoptosis.

Pradimicin (PRM) induces apoptosis in mammalian cells which had been incubated with 1-deoxymannojirimycin (DMJ). Flow cytometric analysis revealed that PRM preferentially induced apoptosis to the cells of the G1 phase. Two possible mediators in this apoptotic cascade were identified. Exposure of DMJ-treated cells to PRM resulted in a rapid (approximately 5 seconds) and slow (approximately 30 minutes) elevation of the intracellular calcium level. Reactive oxygen species (ROS) were proved to be involved in this system by the fact that the apoptosis was completely inhibited by treating the cells with a ROS scavenger, N-acetylcysteine in prior to the PRM stimulation.

Development of a self-cloning system for Actinomadura verrucosospora and identification of polyketide synthase genes essential for production of the angucyclic antibiotic pradimicin.

A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 10(5) CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.

Signal transduction inhibitors, hibarimicins, A, B, C, D and G produced by Microbispora. I. Taxonomy, fermentation, isolation and physico-chemical and biological properties.

Strain TP-AO121 which produces a complex of novel tyrosine kinase inhibitors designated hibarimicins A, B, C, D and G was considered to be a new subspecies of Microbispora rosea, and the name, Microbispora rosea subsp. hibaria, was proposed. Hibarimicins A, B, C and D specifically inhibited the src tyrosine kinase activity without affecting protein kinase A or protein kinase C. They also showed in vitro anti-Gram-positive bacterial and antitumor activities. The molecular formulae of hibarimicins A, B, C, D and G were assigned to be C85H112O37, C85H112O37, C83H110O36, C85H112O38, and C85H112O39 respectively.

Signal transduction inhibitors, hibarimicins A, B, C, D and G produced by Microbispora. II. Structural studies.

The structure of hibarimicins A, B, C, D and G which are inhibitors for tyrosine specific protein kinase are determined using spectroscopic techniques. Hibarimicins described in this report consist of a common aglycon and six deoxyhexoses. The aglycon contains a highly oxidized naphtylnaphthoquinone as a chromophore. Among them, hibarimicin B was identical with angelmicin B.

Glucosylquestiomycin, a novel antibiotic from Microbispora sp. TP-A0184: fermentation, isolation, structure determination, synthesis and biological activities.

Glucosylquestiomycin, a novel N-glucopyranoside of questiomycin A, was isolated from the culture broth of Microbispora sp. TP-A0184. The absolute configuration of the sugar was determined as D-configuration by chemical synthesis. The new antibiotic showed antibacterial activity against gram-positive and -negative bacteria and yeasts and cytotoxic activity against U937 cells.

Cloning and nucleotide sequence of the putative polyketide synthase genes for pradimicin biosynthesis from Actinomadura hibisca.

We cloned the putative polyketide synthase genes (pms genes) for pradimicin A biosynthesis from Actinomadura hibisca using an oligonucleotide probe designed on the basis of conserved amino acid sequences of other polyketide synthases (PKSs). By DNA sequencing of an 8.2-kb SacI fragment that hybridized with the oligonucleotide probe, 11 open reading frames (ORFs) were found. All of the ORFs except for ORF10 were predicted to be translated in the same direction. Each of the deduced ORFs has significant sequence similarity to the protein responsible for polyketide biosynthesis or spore pigmentation. In particular, ORF1, ORF2, and ORF3 were 50-70% identical with genes coding for PKSs for actinorhodin biosynthesis. Specific DNA regions similar in sequence to pms genes were found with genomic Southern hybridization in all of the pradimicin producers examined, but were not found in pradimicin nonproducers, suggesting that the genes cloned in this study encode polyketide synthase for pradimicin biosynthesis.

Pradimicin, a mannose-binding antibiotic, induced carbohydrate-mediated apoptosis in U937 cells.

Pradimicin (PRM), a mannose-binding antifungal antibiotic, recognizes a D-mannoside in the presence of calcium. We demonstrated that BMY-28864, a semi-synthetic analog of PRM, induced apoptosis in U937 cells which had been incubated with 1-deoxymannojirimycin (DMJ). Characteristic morphological changes such as formation of apoptotic bodies and DNA fragmentation were observed in apoptotic cells.