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.

Purification and characterization of a 15 kDa protein (p15) produced by Helicosporium that exhibits distinct effects on neurite outgrowth from cortical neurons and PC12 cells.

We have found that a 15 kDa protein (p15) produced by a fungus of Helicosporium genus induced characteristic morphological changes on neurites extending from rat cerebral cortex neurons in culture. In the presence of p15, neurite elongation from cortical neurons was markedly inhibited, and they extended short, slender, and less branched neurites. Laminin-induced promotion of neurite outgrowth was also dramatically suppressed. In contrast, p15 promoted nerve growth factor (NGF)-induced neurite outgrowth from PC12 cells, although treatment of cells with p15 alone had little effect. Partial amino acid sequence analysis of p15 revealed that it had limited homology to plant lectins. These results suggest that the mechanism of neurite outgrowth was considerably different between cortical neurons and PC12 cells and that p15 modulated neurite outgrowth in cell type-specific manners.

Cytotoxic benzyl dihydroflavonols from Cudrania tricuspidata.

Three new dihydroflavonols, gericudranins A-C were isolated from the stem bark of Cudrania tricuspidata. They were identified as 6,8-di-p-hydroxybenzyltaxifolin, 8-p-hydroxybenzyltaxifolin and 6-p-hydroxybenzyltaxifolin, respectively, by means of spectral studies. These compounds were cytotoxic to human tumor cell lines, such as CRL 1579 (skin), LOX-IMVI (skin), MOLT-4F (leukemia), KM12 (colon) and UO-31 (renal) in culture, with ED50 values of 2.7-31.3 micrograms ml-1.

Sparoxomycins A1 and A2, new inducers of the flat reversion of NRK cells transformed by temperature sensitive Rous sarcoma virus. II. Isolation, physico-chemical properties and structure elucidation.

Sparoxomycins A1 and A2, isolated from the fermentation broth and mycelium of Streptomyces sparsogenes SN2325, are new inducers of the flat reversion of NRK cells transformed by temperature sensitive Rous sarcoma virus. Sparoxomycins A1 and A2 were isolated by active carbon chromatography, centrifugal partition chromatography (CPC), ODS column chromatography and preparative HPLC. The structure of sparoxomycins were determined by spectroscopic evidences. Stereochemical assignments of these inducers were executed from the analyses of CD spectra.

Costunolide and dehydrocostus lactone as inhibitors of killing function of cytotoxic T lymphocytes.

Costunolide and dehydrocostus lactone were isolated from an extract of mokko (Saussurea lappa Clarke) as inhibitors of killing activity of cytotoxic T lymphocytes (CTL). Mokko lactone was also isolated as an inactive compound from the extract. The structure-activity relationship indicated that alpha-methylene-gamma-butyrolactone is required for the inhibitory effect. Costunolide markedly inhibited the granule exocytosis and the production of inositol phosphates in response to anti-CD3 monoclonal antibody (mAb) stimulation at a concentration that did not affect the binding of anti-CD3 mAb. Tyrosine phosphorylation induced by crosslinking of CD3 molecules was significantly inhibited by costunolide in a dose-dependent manner. These results suggest that costunolide inhibits the killing activity of CTL through preventing the increase in tyrosine phosphorylation in response to the crosslinking of T-cell receptors.

1'-Acetoxychavicol acetate as an inhibitor of phagocytosis of macrophages.

We screened extracts of edible plants for inhibitors of phagocytosis by peritoneal exudate macrophages. 1'-Acetoxychavicol acetate was isolated from the ethyl acetate extract of Languas galanga, and this compound strongly inhibited phagocytosis at an IC50 value of 1.2 microM with negligible effects on pinocytosis and cell viability. Target(s) of 1'-acetoxychavicol acetate was suggested to be downstream of the signal transduction pathway that is mediated by protein kinase C.

Anti-inflammatory effects and specificity of L-156,602: comparison of effects on concanavalin A and zymosan-induced footpad edema, and contact sensitivity response.

We investigated the in vivo selective anti-inflammatory effect of L-156,602, which was first identified as a preferential delayed-type hypersensitivity-suppressant in our screening program and first reported to be a C5a antagonist. The agent most profoundly suppressed footpad edema 4 h after elicitation by concanavalin A (con A) and also caused a significantly impaired response after a further 20 h. Footpad edema induced by either serotonin, carrageenan or zymosan was not much influenced by the agent. Although the dominant cell population that migrated in response to con A and zymosan 4 h after elicitation was neutrophils, L-156,602 specifically prevented the con-A-induced migration of neutrophils, suggesting a distinct mechanism of neutrophil recruitment between con A and zymosan-induced inflammation. The agent also reduced the contact-sensitivity response, especially in host mice sensitized with a moderate dose of picryl chloride and almost completely suppressed the infiltration of mononuclear leukocytes and neutrophils into the site of inflammation. These selective effects of L-156,602 on inflammatory reactions appeared to be not merely via C5a antagonism.

Screening for specific inhibitors of phagocytosis of thioglycollate-elicited macrophages.

Phagocytosis is one of the basic and characteristic properties of macrophages. We screened metabolites of Actinomyces for low molecular weight substances that selectively inhibited phagocytosis of dried yeast but not pinocytosis of neutral red by thioglycollate-elicited peritoneal macrophages. Inhibitors of actin filament organization, protein kinases, respiration, and lipid synthesis selectively inhibited phagocytosis, and blockers of proton gradients selectively inhibited pinocytosis. This suggests that these functions are differently regulated. We applied this system to screening of metabolites of Actinomyces, and identified mycotrienin, piericidin, and genistein as selective inhibitors of phagocytosis.

Effects of microbial products on glucose consumption and morphology of macrophages.

We studied the effects of microbial products on glucose consumption and morphology of macrophages which were elicited with thioglycollate medium. Macromolecules such as lipopolysaccharide (LPS), tumor promoters, and respiratory inhibitors increased macrophage glucose consumption without inducing evident morphological changes. The assay system was used to screen for active substances in culture broth extracts from actinomycetes. Among them, aureothin increased glucose consumption of macrophages and inhibited respiration of a rat mitochondrial fraction. Concanamycin A induced morphological changes of macrophages into needle-like shapes but not of cloned cells including the macrophage-like cells J774.1. This compound changed fibrosarcoma L929 cells into round shapes without affecting the shape of a nontransformed fibroblast, BALB/3T3 cells. Antimycin and concanamycin A increased tumor-killing activity of macrophages when added during the effector phase. These results suggest that this assay system is simple and sufficiently reproducible and thus usable for screening for modulators of macrophage function among natural products.

Melastin, a novel product of Streptomyces that selectively inhibits leukemia cell growth.

In the course of our screening for new immunomodulators, a novel compound, melastin, was purified from the culture broth of Streptomyces. Melastin was purified through absorption to Diaion HP-20, ethanol precipitation, and anion exchange column as a brown powder. The molecular weight was estimated as 5000 +/- 3000 by gel filtration HPLC. Melastin suppressed lipopolysaccharide (LPS)-induced blastogenesis of B cells more profoundly than concanavalin A (con A)- or phytohemagglutinin (PHA)-induced blastogenesis of T cells. Moreover, it selectively inhibited the growth of several leukemia cells as compared with interleukin-dependent nontransformed leukocytes. No selectivity was observed between nontransformed fibroblasts and their oncogene-transformed variants. Melastin did not selectively inhibit macromolecule synthesis of leukemia cells.

New pyrrolobenzodiazepine antibiotics, RK-1441A and B. II. Isolation and structure.

Antibacteriophage antibiotics, RK-1441A and B, related to neothramycin were isolated from the culture broth of Streptomyces sp. and their structures were deduced from spectroscopic analyses. The structure of RK-1441A was 8,11-dihydroxy-3,7-dimethoxy-5-oxo-1H-pyrrolo[2,1-c:1,4]benzodiazepine. RK-1441B is a tautomeric mixture at C-3 of the structure, 3,8,11-trihydroxy-7-methoxy-5-oxo-1H-pyrrolo[2,1-c:1,4]benzodiazepine.

Epiderstatin, a new inhibitor of the mitogenic activity induced by epidermal growth factor. II. Structure elucidation.

The structure of a novel antibiotic, epiderstatin, was determined as 4-[3-((Z)-3,5-dimethyl-2-oxopiperidin-6-ylidene)-2-oxopropyl]-2, 6-piperidinedione by spectroscopic analyses of 1H NMR, 13C NMR, 1H-1H correlation spectroscopy (COSY), 13C-1H COSY, heteronuclear multiple bond correlation spectroscopy, UV and IR spectra. The antibiotic belongs to the glutarimide antibiotics, however, the characteristic feature of epiderstatin is that it has a piperidone ring instead of a cyclohexanone ring.

A new antifungal antibiotic, cystargin: fermentation, isolation, and characterization.

A new sulfur-containing peptide antifungal antibiotic, cystargin, was isolated from the fermentation broth of a new species of genus Kitasatosporia, designated as Kitasatosporia cystarginea. On acid hydrolysis, cystargin (C60H77N19O17S6) gave equimolar glycine, proline, aspartic acid and arginine. By performic acid oxidation, cysteic acid was detected after hydrolysis. It showed a growth inhibitory activity against various phytopathogenic fungi and inhibition of beta-1,3-glucan synthetase from Saccharomyces cerevisiae.

Studies on binding of toromycin, an antitumor antibiotic, to DNA.

Toromycin, an antitumor, bactericidal and antiviral compound, was found to bind to DNA in such a way as to interfere with the dissociation of double helix at an elevated temperature. The antibiotic did not introduce strand scission into DNA. Single-strand-specific nuclease S1-susceptibility of negatively supercoiled DNA was not influenced by its binding. The antibiotic was shown to bind to both of the alternating purine-pyrimidine copolymers, poly(dG-dC):poly(dG-dC) and poly(dA-dT):poly(dA-dT). The unique C-glycoside molecule of toromycin interacted with single-stranded DNA, but was found to have no affinity for RNA.

Structure and biological activity of ascamycin, a new nucleoside antibiotic.

A newly isolated strain of Streptomyces sp. produces a new nucleoside antibiotic, ascamycin and the corresponding dealanyl derivative. The structure of ascamycin was determined to be 2-chloro-9-beta-[5-O-(N-L-alanyl)sulfamoyl-D-ribofuranosyl]-adenine. Remarkable selective toxicity of ascamycin compared to the dealanyl derivative was accounted for on the basis of a dealanylating enzyme present in the envelope of sensitive bacteria. After dealanylation, it becomes permeable to cell membrane.

Specific binding of chartreusin, an antitumor antibiotic, to DNA.

Chartreusin, an antitumor and antibacterial antibiotic, was found to inhibit negatively superhelical DNA-relaxation catalyzed by prokaryotic topoisomerase I and conversion of the superhelical DNA into unit length linear form catalyzed by single-strand-specific S1 nuclease. The inhibitory effect of the agent was due to the binding to DNA causing the alteration of tertiary structure. To characterize the binding specificity, we investigated the protection of DNA against cleavages by various restriction endonucleases. It was evidenced that the binding of the agent is not at random and correlates to the sequence 5'CGC 3' 3'GCG 5' on DNA stretch.