The Berkeley Pit, an acid mine waste lake, is a source of extremophilic microorganisms that produce interesting bioactive compounds. We have previously reported the isolation of berkeleydione 1, berkeleytrione 2, the berkeleyacetals and the berkeleyamides from the Pit Lake fungus Penicillium rubrum. In this paper we report the isolation and characterization of berkeleyones A-C (4, 5 and 7) as well as previously described preaustinoid A (3) and A1(6) from this same fungus. These compounds were evaluated as inhibitors of the signaling enzyme caspase-1 and as potential inhibitors of interleukin 1-β production by inflammasomes in induced THP-1 cell line assays.

We used six candidal strains (two Candida albicans and one each of four other species) to study the effects of test conditions on the activity of SCH 39304 compared with that of fluconazole in broth macro-and microdilution assays. Increasing the inoculum from 102 to 105 yeasts per ml raised the MICs for all isolates up to >512-fold. In contrast, results with a 50% turbidimetric endpoint (50% inhibitory concentration; IC,,2) varied no more than twofold. Similar effects were seen with fluconazole, and both drugs were found to have an associated delay in onset of action. Acidity was found to increase both MICs and IC1,2s. Other effects were observed among four synthetic media, but a consistent pattern was not identified. (1-6, 8, 10, 12, 15, 16, 20 [7,18,23]) and one each of C. glabrata (R87), C. tropicalis (F26), C. parapsilosis (3288), and C. lusitaniae * Corresponding author.(ATCC 64125). In addition, 39 isolates of C. albicans were used. These included 32 strains from a panel selected for their variable susceptibilities to an unrelated antifungal agent, flucytosine (18, 21), and 7 strains selected from among clinical isolates from our collection. Another strain was isolated from a patient whose ketoconazole treatment failed (strain K3, also identified by others as KB [9,10,18]). A final C. albicans isolate, strain F662, used previously with strain C17 to evaluate the effect of inoculum size on results obtained with fluconazole (18), was also used for inoculum studies. Locally isolated clinical isolates of C. glabrata (six strains), C. tropicalis (three strains), and C. parapsilosis (three strains) were tested with a single set of conditions. Isolates which had been stored in yeast nitrogen broth (YNB; Difco Laboratories, Detroit, Mich.) with 10% glycerol at -70°C were thawed and maintained on Sabouraud dextrose 3% agar plates (Sab-Dex broth, BBL Microbiology Systems, Cockeysville, Md.; Bacto-Agar, Difco). For studies, subcultures were inoculated into. 20 ml of YNB and incubated at 37°C overnight.Media and buffers. A 2 x concentration of unbuffered SAAMF was prepared as previously described (11), except that cystine was replaced with equimolar amounts of cysteine (14); filter sterilized (cellulose nitrate membrane; pore size, 0.20 pum; Nalge Co., Rochester, N.Y.); and stored at 4°C. This was the medium used for all of the experiments, except when specifically noted otherwise. YNB was prepared as recommended by the manufacturer to a lOx concentration, filter sterilized, and stored at 4°C. Minimal essential medium with Hanks balanced salt solution and RPMI 1640 medium (both from Sigma) were prepared on the day of use as recommended by the manufacturer. YNB and SAAMF were diluted to lx with deionized water, and all media were buffered, adjusted to the appropriate pH, and sterilized by filtration.

Two new drimane sesquiterpene lactones and one new tricarboxylic acid derivative were isolated from the Berkeley Pit extremophilic fungus Penicillium solitum. The structures of these compounds were deduced by spectroscopic analysis. Berkedrimanes A and B inhibited the signal transduction enzymes caspase-1 and caspase-3 and mitigated the production of interleukin 1-β in the induced THP-1 (promonocytic leukemia cell line) assay.

We examined the effects of temperature and inoculum on the agreement of macro-and microdilution broth Drugs. SCH 39304 (Schering-Plough Research Corp., Bloomfield, N.J.), fluconazole (Pfizer Research, Groton, Conn.), amphotericin B (E. R. Squibb & Sons, Princeton, N.J.), and ketoconazole (Janssen Pharmaceutical, Inc., Piscataway, N.J.) were used to prepare 1-mg/ml stock solutions in 2% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) for the triazoles and 10% dimethyl sulfoxide for the other drugs. Stock solutions of flucytosine (Hoffmann-La Roche Inc., Nutley, N.J.) were prepared at a concentration of 10 mg/ml in 10% dimethyl sulfoxide. All drugs were stored at -700C.Media and buffers. Unbuffered synthetic amino acid media for fungi (SAAMF) were prepared as previously described (2, 6), except that solutions of amino acids (Irvine Scientific Sales Co., Inc., Santa Ana, Calif.) were used instead of powdered amino acids and cystine was replaced with equimolar amounts of cysteine (7). Yeast nitrogen broth (Difco), RPMI 1640 (Sigma), and high-resolution broth (kindly supplied by Peter Troke, Pfizer Inc., Sandwich, England) were prepared as specified by the manufacturers, sterilized by filtration, and stored at 4°C. On the day of use, morpholinepropanesulfonic acid (MOPS) buffer (Sigma) was added to each medium to a final concentration of 0. 165 M, the pH was adjusted to 7.0 with 10 M sodium hydroxide or 6 M hydrochloric acid, and the medium was resterilized by filtration.Susceptibility testing. Macrodilution broth assays were performed as previously described (8,9). Briefly, twofold dilutions of the antifungal agents were prepared by standard methods (10) in media to 10 times the final concentrations, as listed below. Yeast inocula were prepared by suspending 1542

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