Takuji Nakashima

Structure–activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)‐α secretion from RAW264.7 cells in a structure‐depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end‐structure of alginate oligomers was important for the TNF‐α‐inducing activity. Among the unsaturated guluronate (G3–G9) and mannuronate (M3–M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio‐Plex assay revealed that interleukin (IL)‐1α, IL‐1β, and IL‐6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure–activity relationship profiles as seen in TNF‐α, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll‐like receptor (TLR)2 and TLR4 effectively inhibited the G8‐ and M7‐induced production of TNF‐α, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.

Characterization of bacterium isolated from the sediment at coastal area of omura bay in Japan and several biological activities of pigment produced by this isolate

Recently we discovered a bacterial strain (MS‐02–063) that produces large amounts of red pigment from coastal area of Nagasaki Prefecture, Japan. Comparative 16S rDNA gene sequencing analysis revealed that strain MS‐02–063 was phylogenetically closely related to γ‐proteobacterium Hahella sp. MBIC 3957 that produces prodigiosin. However, some physiological and biochemical differences between strain MS‐02–063 and Hahella sp. MBIC 3957 were observed. The red pigment (RP‐063) produced by this isolate was highly purified from the culture supernatant. It was speculated that RP‐063 might be prodigiosin‐like pigment in physical properties and biological activities such as antibacterial and cytotoxic activity. Antibacterial activity of RP‐063 was examined by an agar dilution method. The results indicated that RP‐063 showed antibacterial activity for specific for pathogenic gram‐positive bacteria such as Staphylococcus aureus. The potency of antibacterial activity against S. aureus was nearly equal to those of tetracycline. Moreover, RP‐063 showed inhibition of the superoxide generation by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐stimulated mouse macrophage RAW 264.7 cell line. Prodigiosin members have a wide variety of biological properties, including anticancer and antimalarial, etc. Especially, potent immunosuppressive properties have been reported for prodigiosin members with the mechanism of action different from that of the other well known immunosuppressors in atopic dermatitis therapy such as cyclosporin A, FK506 and rapamycin. It is suggested that RP‐063 may be able to arrest the inflammation caused by superantigens secreted from S. aureus, which colonized skin on atopic dermatitis as well as suppression of activated lymphocyte proliferation and superoxide generation from leucocytes.

Trehangelins A, B and C, novel photo-oxidative hemolysis inhibitors produced by an endophytic actinomycete, Polymorphospora rubra K07-0510

Three new natural products, designated trehangelins A, B and C, were isolated by solvent extraction, silica gel and octadecylsilyl silica gel column chromatographies and subsequent preparative HPLC from the cultured broth of an endophytic actinomycete strain, Polymorphospora rubra K07-0510. The trehangelins consisted of a trehalose moiety and two angelic acid moieties. Trehangelins A (IC50 value, 0.1 mg ml−1) and C (IC50 value, 0.4 mg ml−1), with symmetric structures, showed potent inhibitory activity against hemolysis of red blood cells induced by light-activated pheophorbide a. However, trehangelin B, with an asymmetric structure, displayed only a slight inhibition (IC50 value, 1.0 mg ml−1).

Mangromicins A and B: structure and antitrypanosomal activity of two new cyclopentadecane compounds from Lechevalieria aerocolonigenes K10-0216.

Two new cyclopentadecane antibiotics, named mangromicins A and B, were separated out from the culture broth of Lechevalieria aerocolonigenes K10-0216 by Diaion HP-20, silica gel and ODS column chromatography, and were finally purified by HPLC. The chemical structures of the two novel compounds were elucidated by instrumental analyses, including various NMR, MS and X-ray crystallography. Mangromicins A and B consist of cyclopentadecane skeletons with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. Mangromicins A and B showed in vitro antitrypanosomal activity with IC50 values of 2.4 and 43.4 μg ml−1, respectively. The IC50 values of both compounds were lower than those of cytotoxicity against MRC-5 human fetal lung fibroblast cells.

A Novel Indole-diterpenoid, JBIR-03 with Anti-MRSA Activity from Dichotomomyces cejpii var. cejpii NBRC 103559

A new indole-diterpene, JBIR-03 (1), was isolated from the fungus Dichotomomyces cejpii var. cejpii NBRC 103559 and its structure was determined based on the spectroscopic data. 1 exhibited anti-MRSA (methicillin-resistant Staphylococcus aureus) activity and antifungal activity against apple Valsa canker-causing fungus, Valsa ceratosperma, while it exhibited no toxicity towards human cancer cells.

Mangromicins, six new anti-oxidative agents isolated from a culture broth of the actinomycete, Lechevalieria aerocolonigenes K10-0216

We have been continually searching for novel chemical compounds from culture broths of various actinomycetes using a physicochemical screening system. During the course of this program, we have previously reported the discovery of two new natural products, designated mangromicins A and B, discovered in a broth of a rare actinomycete strain, Lechevalieria aerocolonigenes K10-0216. Mangromicins have a unique and rare structure, a cyclopentadecane skeleton with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. New mangromicin analogs were isolated by using an improved production medium. As a consequence, six analogs, together with mangromicins A and B, were isolated from a cultured broth of L. aerocolonigenes K10-0216. We named them mangromicins D, E, F, G, H and I. All mangromicins showed radical scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and nitric oxide generated from LPS-stimulated RAW264.7 cells, a murine macrophage cell line. Among the analogs, mangromicins A and I showed the most potent DPPH radical scavenging activity and nitric oxide scavenging activity, respectively.

New Aureothin Derivative, Alloaureothin, from Streptomyces sp. MM23

A new polypropionate alloaureothin (1) possessing a nitro group, together with a known polypropionate aureothin (2), was isolated from mycelium of Streptomyces sp. MM23. The structure was determined on the basis of spectroscopic data. 1 exhibited growth inhibitory effect against human fibrosarcoma HT1080 cells with an IC50 value of 30 μM.

Mangromicin C, a new analog of mangromicin.

Mangromicin analogs were discovered in a cultured broth of a rare actinomycete, Lechevalieria aerocolonigenes K10-0216.1,2 The mangromicin analogs showed antitrypanosomal and reactive oxygen species (ROS) scavenging bioactivities. Mangromicin analogs have unique structures, including common partial structures, notably a cyclopentadecane skeleton with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. In a previous report, we described eight new mangromicin analogs, which possessed anti-oxidative properties. At that time, we discovered mangromicin C (1) but were unable to determine its structure, which was clearly different from all the other analogs. We have now found that 1 has a structure which includes a tetrahydropyran ring (Figure 1). In this paper, we report the fermentation, isolation, structural elucidation and biological activity of 1.

Development of a New Medium Useful for the Recovery of Dermatophytes from Clinical Specimens by Minimizing the Carryover Effect of Antifungal Agents

Two surface‐active compounds, egg lecithin and polysorbate 80, usually used as the deactivators of various preservatives were tested whether they also counteract either or all of the three major topical antifungal drugs, bifonazole (BFZ), lanoconazole (LCZ) and terbinafine (TBF). Both egg lecithin and polysorbate 80, when added to culture media up to final concentrations of 1.0 and 0.7%, respectively, antagonized the anti‐dermatophytic activity of the three drugs in a concentration‐dependent manner. A greater extent of antagonistic action was exerted when the two deactivators combined at their maximal levels tested were added; MICs of BFZ were increased more than 30‐fold and those of LCZ and TBF more than 200‐fold compared with the values obtained in the absence of the deactivators. Using the agar medium supplemented with the combined deactivators, culture studies were carried out with skin tissues specimens taken from guinea pigs whose feet were infected with dermatophytes and subsequently treated with 1% topical preparations of the three antifungal drugs. The experimental data from this animal study demonstrated that the combined deactivators‐supplemented medium yielded increased numbers of fungi compared with the basal medium. It looks, therefore, likely that the fungal recovery on the former medium more correctly reflects to actual fungal burden in the infected lesions than the latter. All these results suggest that the combined deactivators‐supplemented medium is more useful for mycological evaluation of therapeutic efficacy of imidazole and allylamine drugs against dermatophytoses in both preclinical and clinical studies.

Discovery of a pimaricin analog JBIR-13, from Streptomyces bicolor NBRC 12746 as predicted by sequence analysis of type I polyketide synthase gene

Sequence analysis of ketosynthase domain amplicons from Streptomyces bicolor NBRC 12746T revealed the presence of previously unreported type I polyketide synthases (PKS-I) genes. The clustering of these genes with the reference PKS-1 sequences suggested the possibility to produce a polyene compound similar to pimaricin. Thus, the cultured sample from NBRC 12746T was analyzed for the production of polyene compounds. The strain produced an antifungal compound which displayed the UV absorption spectrum of tetraene macrolides. The structure determination based on the spectroscopic analysis of the purified compound resulted in the identification of a novel pimaricin analog JBIR-13 (1). This study therefore strongly suggested that a careful analysis of PKS-I genes can provide valuable information in the search of novel bioactive compounds within a class predicted from phylogenetic analysis.