Simple aromatics identified with a NFAT-lacZ transcription assay for the detection of immunosuppressants.

Determination of the mechanism of action of FK506 and cyclosporin A has yielded new molecular targets involved in signal transduction during T cell activation. A common target of FK506 and cyclosporin A is inhibition of activation of the NFAT transcription factor, for which a specific binding region is present in the promoter of the IL-2 gene. A reporter gene assay has been used to screen for agents that interfere with this early step in T cell activation. Simple aromatic compounds that block NFAT-dependent transcription and show in vitro immunosuppressive activity were isolated from the broth and mycelia of two Streptomyces sp. fermentations. The compounds were active at concentrations that were not directly cytotoxic.

MLR-52, (4'-demethylamino-4',5'-dihydroxystaurosporine), a new inhibitor of protein kinase C with immunosuppressive activity.

In the course of screening with the mixed lymphocyte reaction, a new inhibitor of protein kinase C with immunosuppressive activity was isolated from the fermentation broth and mycelia of Streptomyces sp. AB 1869R-359. Although certain similarities exist, this strain is morphologically and physiologically distinct from other reported producers of staurosporine-related compounds. We have found that this strain produces relatively high levels of staurosporine and the new minor compound MLR-52, which possesses the indolo[2,3-a]carbazole chromophore of staurosporine, but differs in the substitution pattern of the sugar moiety. Their structures have been elucidated by mass and NMR spectra. MLR-52 has been shown to inhibit the enzymatic activity of protein kinase C and the murine mixed lymphocyte reaction.

Dunaimycins, a new complex of spiroketal 24-membered macrolides with immunosuppressive activity. III. Immunosuppressive activities of dunaimycins.

The immunosuppressive effects of the dunaimycins, a new complex of spiroketal 24-membered macrolides, were compared to cyclosporin A, ascomycin, and rapamycin. Each dunaimycin was a potent inhibitor of the mitogenic response observed in mixed murine splenocyte or human leukocyte cultures, and like immunosuppressive drugs these compounds were relatively less potent inhibitors of the constitutive proliferation of murine EL4 thymoma cells. Dunaimycin D4S showed no selectivity in inhibiting the mitogenic response of spleen cells to concanavalin A, pokeweed mitogen, lipopolysaccharide, or phytohemagglutinin. Cyclosporin A and ascomycin did not inhibit interleukin 2 dependent proliferation, whereas the dunaimycins and rapamycin blocked the uptake of [3H]thymidine in mixed cultures supplemented with exogenous interleukin 2. In addition, dunaimycin D4S had no apparent affinity for cyclosporin A or FK-506 immunophilins. Although the dunaimycins inhibited the activity of Na+, K(+)-ATPase, inhibition of this enzyme appeared insufficient to explain the biological activity of these new macrolides. Over a narrow concentration range, dunaimycin D4S showed in vivo immunosuppressive activity in the murine popliteal lymph node hyperplasia model.

Macquarimicins, microbial metabolites from Micromonospora. I. Discovery, taxonomy, fermentation and biological properties.

A novel series of microbial metabolites were discovered in fermentation broths of two soil isolates. Both cultures were identified as strains of Micromonospora chalcea. Production of the metabolites, named macquarimicins, was monitored by an HPLC assay. A seven-day fermentation yielded 27 mg/liter of macquarimicin A. With MICs of 50 to 100 micrograms/ml, macquarimicin A has only very low activity against strains of Bacteroides and other anaerobes. Macquarimicin B has inhibitory activity against the leukemia cell line P-388.

Dibefurin, a novel fungal metabolite inhibiting calcineurin phosphatase activity.

The novel calcineurin inhibitor, dibefurin, has been isolated from the fungal culture AB 1650I-759. The isolation was bioactivity-directed fractionation using an assay which measures the phosphatase activity of calcineurin. The compound was purified by countercurrent, reverse phase and gel filtration chromatographies. Several studies, including crystallographic, NMR and MS, revealed that dibefurin is a novel dimeric compound of a unique structural type.

Phylogeny and taxonomy of mesophilic Methanococcus spp. and comparison of rRNA, DNA hybridization, and phenotypic methods.

The phylogeny and taxonomy of the mesophilic methane-producing archaea of the order Methanococcales were examined by DNA relatedness, 16S rRNA sequence analysis, cellular protein patterns, and phenotypic methods. The mesophilic species Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltaei, and "Methanococcus aeolicus" formed a deep group with 5 to 30% DNA relatedness and 92 to 96% 16S rRNA sequence similarity. Twenty-two additional isolates and Methanococcus deltae were similar to the type strain of either M. voltaei or M. maripaludis. Two isolates, strains A2 and A3, exhibited 37% DNA relatedness and 99.2% 16S rRNA sequence similarity to M. voltaei PS(T) (T = type strain). In the absence of phenotypic differences, these organisms were assigned to M. voltaei. Similarly, four autotrophic isolates, strains C5, C6, C7, and C8, exhibited 54 to 69% DNA relatedness and 99.2% 16S rRNA sequence similarity to M. maripaludis JJT and were assigned to M. Maripaludis. While these isolates were sufficiently genetically diverse to justify classification in novel species, few differences were apparent in the phenotypic properties available for measurement. Thus, the phenotypic properties of these lithotrophic archaea were highly conserved and poor indicators of genetic diversity. Partial sequencing of about 200 bases of both the 16S and 23S rRNAs of the isolates demonstrated allelic diversity within methanococcal species. This allelic diversity did not correlate with diversity measured by DNA relatedness, cellular protein pattern, and other methods. Similarly, antisera to whole cells of the type strains did not cross-react strongly to whole cells of strains that were genetically similar, and serological cross-reactivity was not a useful taxonomic method for methanococci. Lastly, on the basis of the results of 16S rRNA sequence analyses and biochemical data, the ancestor of the mesophilic methanococci may have been an autotrophic thermophile.