Identification of phoslactomycin E as a metabolite inducing hyphal morphological abnormalities in Aspergillus fumigatus IFO 5840.

In our survey for antifungal compounds, a fermentation broth of Streptomyces sp. HA81-2 was found to inhibit the in vitro growth of Aspergillus fumigatus IFO 5840 accompanied by hyphal morphological abnormalities. One of the isolated antibiotics was identified as phoslactomycin E based on LC-MS and NMR spectral data. In a preliminary assay using the membrane fractions of A. fumigatus, phoslactomycin E was found to inhibit the activity of 1,3-beta glucan synthase.

Synthesis of the sugar moiety of TIME-EA4, a glycopeptide isolated from silkworm diapause eggs.

We describe the efficient synthesis of the tetrasaccharide, 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,4-di-O-acetyl-3-O-allyl-beta-D-mannopyranosyl-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl azide, which is the protected form of the sugar unit of TIME-EA4 that is isolated from the diapausing eggs of the silkworm, Bombyx mori. The beta-linked D-mannoside of the tetrasaccharide was obtained using the conventional oxidation-reduction method for inversion of the configuration at the C-2 hydroxyl group of beta-D-glucoside. The reduction was effected with NaBH(4) in a methanolic solution in a ratio of 98:2 in favor of the beta-D-mannoside that was obtained in 87% yield.

Remarkably simple sequence requirement of the M-factor pheromone of Schizosaccharomyces pombe.

The mating reaction is triggered by specific pheromones in a wide variety of organisms. Small peptides are used as mating pheromones in yeasts and fungi. In the fission yeast Schizosaccharomyces pombe, M-factor is a C terminally farnesylated nonapeptide secreted from M-cells, and its counterpart, P-factor, is a simple peptide composed of 23 amino acids. The primary structure requirements for the biological activity of pheromone peptides remain to be elucidated. Here, we conducted comprehensive substitution of each of the amino acids in M-factor peptide and inspected the mating ability of these missense mutants. Thirty-five sterile mutants were found among an array of 152 mutants with single amino acid substitutions. Mapping of the mutation sites clearly indicated that the sterile mutants were associated exclusively with four amino acid residues (VPYM) in the carboxyl-terminal half. In contrast, the substitution of four amino-terminal residues (YTPK) with any amino acid had no or only a slightly deleterious effect on mating. Furthermore, deletion of the three N-terminal residues caused no sterility, although truncation of a fourth residue had a marked effect. We conclude that a farnesylated hexapeptide (KVPYMC(Far)-OCH(3)) is the minimal M-factor that retains pheromone activity. At least 15 nonfunctional peptides were found to be secreted, suggesting that these mutant M-factor peptides are no longer recognized by the cognate receptor.

The protozoan toxin climacostol inhibits growth and induces apoptosis of human tumor cell lines.

Climacostol (5-(Z)-non-2-enyl-benzene-1,3-diol) is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids, compounds that show antimicrobial, antiparasitic and antitumor activities. We investigated the cytotoxic activity of the chemically synthesized toxin on: (1) human tumor squamous carcinoma A431 cells, (2) human promyelocytic leukaemia HL60 cells, and (3) human non-tumor endothelial EA.hy926 cells. The results showed that climacostol effectively inhibited the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death, with non-tumor cells proving significantly more resistant to the toxin.

Synthesis of fluorescent molecular probes specific for the receptor of blepharismone, a mating-inducing pheromone of the ciliate Blepharisma japonicum.

Blepharismone (gamone 2) is a mating-inducing pheromone of the ciliate Blepharisma japonicum. N-Pyrenylbutyryl-blepharismone and N-biphenylacetyl-blepharismone, which are fluorescent derivatives of blepharismone, were synthesized as molecular probes for the gamone 2 receptor. Further, we proved that they have inhibitory activities against the blepharismone-induced monotypic pairing of B. japonicum.

Structure-activity relationship studies on UK-2A, a novel antifungal antibiotic from Streptomyces sp. 517-02. Part 5: Roles of the 9-membered dilactone-ring moiety in respiratory inhibition.

Several open-chained analogues of UK-2A, a novel antifungal antibiotic isolated from Streptomyces sp. 517-02, were prepared for structure-activity studies. The in vitro antifungal activities of these compounds against Rhodotorula mucilaginosa IFO 0001 and the inhibition of uncoupler-stimulated respiration in bovine heart submitochondrial particles (SMP) were evaluated. Oxidative potentials were measured by cyclic voltammetry. An analogue prepared from dihexyl L-glutamate showed comparable inhibitory activity as UK-2A.

Structure-activity relationship studies on niphimycin, a guanidylpolyol macrolide antibiotic. Part 1: The role of the N-methyl-N''-alkylguanidinium moiety.

Several N-methyl-N''-alkylguanidinium derivatives were synthesized and used as simplified analogues of niphimycin (NM), a guanidylpolyol macrolide, in structure-activity relationship studies. The C16-alkylated derivative exerted fungicidal activity by directly damaging the fungal plasma membrane and inducing oxidative stress in a manner similar to niphimycin. These results indicate that the N-methyl-N''-alkylguanidinium moiety is required for antifungal activity by NM.

Developmentally and environmentally regulated expression of gamone 1: the trigger molecule for sexual reproduction in Blepharisma japonicum.

Sexual reproduction (conjugation) in protozoan ciliates is induced by specific cell-cell interactions between cells of complementary mating types. The ancestral ciliate Blepharisma japonicum has two mating types, I and II. The substances that act as signaling molecules in this extracellular interaction for conjugation are called gamones. The glycoprotein gamone 1, produced by mating type I cells, is a key factor that triggers this interaction. We have previously isolated gamone 1 and determined its complete amino acid sequence. To elucidate the mechanism of initiation of conjugation in ciliates, we investigated the transcription of the gamone 1 gene and found that it is controlled by various internal and external factors. The gamone 1 gene transcript appeared specifically when sexually mature mating type I cells were starved. It was not detected in immature cells, mating type II cells or proliferating cells. The level of transcription was markedly increased in type I cells when they were stimulated with gamone 2, which is secreted by type II cells. This is the first report that the transcription of gamone genes in ciliates is strictly regulated by developmental and environmental factors. This study suggests that the onset of transcription of gamone 1 is linked to the switching mechanism that converts mitotically proliferating cells to differentiated preconjugants, the mechanism of differentiation from immature to mature cells in clonal development, and the mechanism that ensures mating type-specific gene silencing.

Semi-synthesis and biological evaluation of analogues of UK-2A, a novel antifungal antibiotic from Streptomyces sp. 517-02.

Several analogues of UK-2A, a novel antifungal antibiotic isolated from Streptomyces sp. 517-02, were semi-synthesized for structure-activity studies. In vitro antifungal activities of these compounds against Saccharomyces cerevisiae IFO 0203 were evaluated by the conventional paper disk method. Several derivatives exhibited growth inhibitory activity similar to UK-2A.