M.Morita and O.Ohneda contributed equally to this workAn HLF (HIF-1a-like factor)/HIF-2a-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF kd/kd ) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF kd/kd mice expressing 80±20% reduction, depending on the tissue, in wildtype HLF mRNA were fertile and apparently normal. Hyperoxia±normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF kd/kd mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF kd/kd mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF kd/kd mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was signi®cantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF kd/kd mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.

Anactinomycete, strain C-38,383, was selected in a screening program for the isolation of novel antitumor agents. A yellow crystalline product, named rebeccamycin, was isolated from the mycelium and was found to have activity against P388 leukemia, L1210 leukemia and B16 melanoma implanted in mice. Rebeccamycin inhibits the growth of humanlung adenocarcinoma cells (A549) and produces single-strand breaks in the DNAof these cells. No DNA-protein cross-links were detected. A related antibiotic, staurosporine, is produced by Streptomyces staurosporeus and Streptomyces actuosus. Strain C-38,383 was found to resemble closely strains of Nocardia aerocolonigenes recently renamedSaccharothrix aerocolonigenes. A strain selection isolate without aerial mycelium, C-38,383-RK-l, failed to produce rebeccamycin while a strain with aerial mycelium, C-38,383-RK-2, was found to be a suitable strain for production. A description of the producing strain is presented and its taxonomic position is reviewed. A fermentor containing 37 liters of production medium gave a rebeccamycin yield of 663 mg/liter after 204 hours of incubation with strain C-38,383-RK-2.Strain C-38,383, an actinomycete isolated from a soil sample collected in Panama, was selected for further study since clarified culture fluid inhibited KB cell culture growth.10 A product, crystallizing as fine yellow needles, was isolated from the mycelium of strain C-38,383 grown in submerged culture. This compound was found to be soluble in dimethyl sulfoxide and tetrahydrofuran, poorly soluble in commonlyused organic solvents and insoluble in water. Significant in vivo antitumor activity was obtained with murine tumor tests. The active compound,a novel iV-glycoside containing a symmetric heterocyclic aglycone with two chlorine atoms, was named rebeccamycin (Fig.

An actinomycete strain No. Q996-17 produced a novel compound, epoxomicin, which exhibited in vivo antitumor activity against B16melanoma. Structural studies indicated that it is a newmember of the epoxy-jS-aminoketonegroup, and is closely related to eponemycin.In our continuous search for antitumor agents showing specific activity against B16 murine melanoma, an unidentified actinomycete strain No. Q996-17 was found to produce a new compound epoxomicin. It wasextracted by rc-butanol from the fermentation broth and purified by various chromatographies. Structural studies revealed that epoxomicin has an epoxy-^-aminoketone moiety in the structure and is a newmemberof the eponemycin^group. Epoxomicin exhibited strong in vitro cytotoxicities against various tumor cell lines, whereas it did not exhibit anti-bacterial and anti-fungal activities. It showed strong in vivo inhibitory activity against B16 melanoma but moderate activity against P388 mouse leukemia.

In the course of screening for new antibiotics active against fungi, an actinomycete strain No. PI57-2 that had been isolated from a soil sample collected in Fiji Island was found to produce novel antibiotics, pradimicins A and B1*^. Pradimicin A, the major component showed moderate in vitro activity against a wide variety of fungi and yeasts including clinically important pathogens. More interestingly, it exhibited marked in vivo therapeutic activity against systemic fungal infections caused by Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans strains in mice. Degradation studies revealed that pradimicin A has a unique structure containing the following moieties : D-Alanine, D-xylose, 4 ,6-dideoxy-4-methylamino-D-galactose and a substituted 5,6-dihydrobenzo[a]naphthacenequinone. This communicationpresents production, isolation, chemical and biological properties and structures of pradimicins A and B. Based on the taxonomic studies performed, strain PI57-2 is classified as a heretofore undescribed species of the genus Actinomaduraand named Actinomadurahibisca sp. nov. (ATCC 53557), and the details will be reported in a separate pap er. Pradimicin was produced in a 200-liter tank fermentor using a mediumconsisting of glucose 3%, soybean meal 3%, Pharmamedia 0.5%, yeast extract 0.1 % and CaCO3 0.3%. The tank was operated at 28°C with agitation of 250 rpm and aeration of 120 liters/minute. Antibiotic production was monitored by bioassay using C, albicans A9540 as a test organism and visible absorption at 500 nm in 0.01 n NaOH-MeOH (1 :1). After 5~6 days of fermentation, antibiotic potency reached the maximum of 500~700 /*g/ml.

Visual observation is a powerful approach for screening bioactive compounds that can facilitate the discovery of attractive druggable targets following their chemicobiological validation. So far, many high-content approaches, using sophisticated imaging technology and bioinformatics, have been developed. In our study, we aimed to develop a simpler method that focuses on intact cell images because we found that dynamic changes in morphology are informative, often reflecting the mechanism of action of a drug. Here, we constructed a chemical-genetic phenotype profiling system, based on the high-content cell morphology database Morphobase. This database compiles the phenotypes of cancer cell lines that are induced by hundreds of reference compounds, wherein those of well-characterized anticancer drugs are classified by mode of action. Furthermore, we demonstrate the applicability of this system in identifying NPD6689, NPD8617, and NPD8969 as tubulin inhibitors.

Cytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1 beta, tumour necrosis factor-alpha, and interferon-gamma) induced apoptotic cell death in the mouse pancreatic beta-cell line beta TC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line alpha TC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in beta TC1 cells. The abundance of endogenous Bcl-2 in beta TC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in beta TC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.

Streptomyces hygroscopicus No. P247-7 1 (ATCC53709) produced a novel antibiotic eponemycin which exhibited specific in vivo antitumor effect against B 16 melanoma.Structural studies assigned {AS)-1 ,2-epoxy-2-hydroxymethyl-4-(7V-isooctanoyl-L-serylamino)-6-methylhept-6-ene-3-one to eponemycinwhichis unrelated to the knownantitumor antibiotics.In our continuing search for new microbial metabolites with antitumor activity, Streptomyces hygroscopicus No. P247-71 , isolated from a soil sample from the Philippines, was found to produce a novel antibiotic with specific activity against B16 melanoma. The active principle, eponemycin, was recovered from the fermentation broth with ethyl acetate extraction and purified by chromatography. Structural studies revealed it to be a novel molecule composedof isooctanoic acid, L-serine and an aminoepoxyketone. 9-Methylstreptimidone wasisolated as a co-product from the crude extract.Eponemycin showed potent growth inhibition against various tumor cells, but no inhibitory activity against bacteria and fungi. It induced significant prolongation of survival time of mice transplanted with B16 melanoma, while no antitumor activity was observed against P388 leukemia.In this paper, we describe the production, isolation, physico-chemical characterization, structural elucidation and biological properties of eponemycin.Taxonomy of the Producing Strain Source of Organism Strain P247-71 was isolated from a soil sample collected near the root of a tamarind at Mt. Apo, Davao, Mindanao Island, Philippines. MorphologyBoth substrate and aerial mycelia are formed. They are long, well-branched and not fragmented into short filaments. Chains ofarthrospores are born on the aerial hyphae. The spore chain and spore morphology are as follows: 1) Spiral spore chains with 2 to 8 turns, 2) monopodially branched sporophores, 3) spores, oval or barrel-shaped (0.5 to 0.7 by 0.5 to 1.2/mi), and 4) spore ornamentation, rugose or smooth.Sporangium, motile spore and sclerotium are not observed. Cultural and Physiological CharacteristicsStrain P247-71 grows well in most descriptive media. Gray aerial mycelium with hygroscopic black patches is observed on International Streptomyces Project (ISP) agar media except for ISP No. 6 medium.Eponemycin was originally designated BMY-28647 or BU-3862T.

Maduropeptin, a complexof newmacromolecular antitumor antibiotics, is a metabolite of Actinomadura madurae H710-49. The active components maduropeptins A1? A2 and B are acidic chromopeptides with MWof around 22,500 and composed of 14 types of amino acids and an unstable chromophore. The antibiotics are active in vitro against Gram-positive bacteria and highly cytotoxic to tumor cells. They produced significant prolongation of survival time of mice implanted with P388 leukemia and B16 melanoma. 403 In the course of screening for novel metabolites active against murine P388 leukemia in mice, we found a complexof newmacromolecularantibiotics designated maduropeptin. The producing culture H710-49, isolated from a soil sample collected in Germany, was identified as Actinomadura madurae (ATCC39144). Maduropeptin is an acidic macromolecular substance from its extraction behavior; the active principle was recovered from the fermentation broth by use of a basic ion exchange resin. HPLC analysis indicated that the crude solid contained at least four active components (maduropeptins Al9 A2, B and D) and an inactive component (maduropeptin C) having similar physico-chemical properties. Upon UVirradiation in a cold room, components B and D were decomposed yielding a complex of components A1? A2 and C which were isolated as single entities by chromatography. The bioactive maduropeptin components showed MW'saround 22,500 and characteristic UVabsorption maximaat 210, 286 and 308 nm. They exhibited potent inhibitory activity against Gram-positive bacteria and tumor cells and strong in vivo antitumor effect against P388 leukemia and B16 melanomaimplanted in mice. In this paper, we report the producing organism, production, isolation, chemical properties and biological activities of maduropeptin. Producing OrganismAn actinomycete strain, No. H710-49 (ATCC39144), was isolated from a soil sample collected in Germany. Strain H710-49 forms both substrate and aerial mycelia. The substrate mycelium is long, branched and not fragmented into short filaments. Short spore-chains are born on the tip or monopodial branch of the aerial mycelium. The spore-chains contain 2~15 spores (mostly 4~8 spores), per chain, and are straight, hooked or spiral in shape. The spores are oval to elliptical (0.5~0.6 x 0.7~1.2 urn) with Maduropeptin was originally called as BBM-1644.