6-Methyl-1,2,4-benzenetriol, a new intermediate in penicillic acid biosynthesis in Penicillium cyclopium.

Penicillic acid-negative mutants were obtained from a color mutant derived from Penicillium cyclopium NRRL 1888 through N-methyl-N'-nitro-N-nitrosoguanidine treatment. One mutant (SK2N6) accumulated 6-methyl-1,2,4-benzenetriol, which was not previously known to be a metabolite of P. cyclopium, in addition to orsellinic acid and orcinol. The radioactivity of [1-14C]acetic acid was rapidly incorporated into 6-methyl-1,2,4-benzenetriol in a culture of P. cyclopium SK2N6. Moreover, the radioactivity of [14C]6-methyl-1,2,4-benzenetriol was efficiently incorporated into penicillic acid in a culture of P. cyclopium NRRL 1888. These data indicate that 6-methyl-1,2,4-benzenetriol is a precursor for penicillic acid biosynthesis. The results on the addition of 1,4-dihydroxy-6-methoxy-2-methylbenzene, 6-methoxy-2-methylbenzoquinone(1,4), and 1-O-methylorcinol to a culture of P. cyclopium SK2N6 indicated that only the former two compounds are converted to penicillic acid. Thus, a new portion of the penicillic acid biosynthetic pathway is proposed.

Characterization of Flavobacterium species by analysis of volatile fatty acid production.

Seventy-four Flavobacterium strains were characterized by gas-liquid chromatographic analysis of volatile fatty acids produced in the culture medium. Principal components analysis permitted the graphic representation of the relative positions of the different strains, and aggregation according to the variance enabled a hierarchical classification to be established. The study revealed three subgroups each for F. meningosepticum and F. odoratum. Our F. breve, Flavobacterium sp. group IIb and F. multivorum strains appeared to be homogeneous. These results tallied with those of previous studies on DNA base composition and reassociation, electrophoretic protein profiles and cellular fatty acid composition.

An early steroidogenic defect in hormone-induced Leydig cell desensitization.

To define the nature of the lesion of the early steroidogenic pathway (prior to pregnenolone formation) in gonadotropin-induced desensitization of rat testicular Leydig cells, we evaluated cholesterol side-chain cleavage activity in isolated mitochondria by measurement of pregnenolone synthesis and [14C]isocaproic acid formation from [26-14C]cholesterol. The enzyme activity was shown to be reduced after in vivo treatment with 10 micrograms hCG when compared to that of mitochondria from control animals only when measured in the presence of limiting NADPH concentrations (100 microM). Sonication of mitochondria from control and hCG-treated rats caused complete loss of cholesterol side-chain cleavage activity. When acetone-powdered adrenal cell mitochondria were employed as the source of the enzyme, the addition of sonicated Leydig cell mitochondria from control and hCG-treated animals caused the same differences as those observed with intact Leydig cell mitochondria in the presence of low concentration of NADPH. The Km value of the adrenal enzyme for NADPH incubated with Leydig cell mitochondria increased from 0.111 mM in control to 0.37 mM after hCG, with no changes in Vmax. Moreover, cholesterol side-chain cleavage activity of adrenal mitochondria assayed in the presence of 100 microM cholesterol was progressively inhibited by increasing amounts of acetone powder from Leydig cell mitochondria of control and hCG-treated rats, with ID50 of 500 and 280 micrograms protein, respectively. The inhibiting factor was not a lipid or steroid but a heat-labile protein, with an approximate Stokes radius of 4.8 nm and an isoelectric point of 5.05 +/- 0.23 SD (n = 8). The inhibitory effect was confined to the Leydig cell mitochondrial membrane, and was not related to changes in oxidative phosphorylation. NADPH was not directly oxidized or immobilized by the mitochondrial factor, and this inhibiting substance was not adsorbed on 2',5' ADP-Sepharose 4B. These results have demonstrated that a heat-labile inhibiting protein factor is present in mitochondria from normal Leydig cells and is markedly activated or increased by hCG treatment. This substance that competitively modulates cholesterol side-chain cleavage activity could contribute to the early steroidogenic lesion, and also serve as an endogenous modulator of steroid hormone biosynthesis.

Norleucine-tyrosine broth for rapid identification of Clostridium difficile by gas-liquid chromatography.

A new medium, norleucine-tyrosine (NT) broth, was developed for rapid identification of Clostridium difficile on the basis of caproic acid and p-cresol production. The NT broth consists of 0.5% Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 0.5% yeast extract (Difco Laboratories, Detroit), 0.2% L-norleucine and 0.2% L-tyrosine (wt/vol; final concentrations), and a mixture of salts. The procedure for demonstrating caproic acid and p-cresol production involves extracting NT broth cultures with ether or chloroform and analyzing the extracts with a gas-liquid chromatograph equipped with a thermal conductivity detector, as is customary for analysis of volatile fatty acids. A total of 120 strains of C. difficile from diverse geographic locations were tested by this procedure, and they all produced caproic acid and p-cresol in NT broth. No other Clostridium species or other microorganisms tested have been found to produce both products in NT broth.

[Enhancement factors of penicillic acid production by Penicillium verrucosum var. cyclopium in foodstuffs (author's transl)]. / Facteurs favorisant la production d'acide pénicillique par Penicillium verrucosum var. cyclopium dans les denrées alimentaires.

Main parameters of penicillic acid (P.A.) production by P. verrucosum var. cyclopium were investigated: substrate nature, water content (H: 22-70%), temperature (5-30% degrees C), confined air and incubation delay. 6100, 2820, 1832, 39 and 0.8 mg/kg were the maximal yields obtained in corn, barley, straw, colza and soya. Toxinogenesis rate was greatly enhanced in crushed corn. Maximal yields in corn increased exponentially with H; production and disappearance rates of the free toxin were an increasing function of H and temperature; so, low temperatures may lead to a high accumulation of P.A. within several months. In a confined environment, P.A. concentration was limited for H greater than or equal to 40%; but accession of air induced an accelerated P.A. biogenesis. Maximal yield of P.A. (8000 mg/kg) was obtained in corn with 60% water content, within one week at 25 degrees C.

In vitro C-20, 22 desmolase regulation of pregnenolone supply in first trimester human placenta.

Homogenates of first trimester human placentae were incubated with [26-14C]chloresterol. Using reverse-isotope dilution analysis [14C]isocaproic acid was isolated and characterized. A progressive increase in [14C]isocaproic acid formation with increasing gestation suggestive of a regulatory mechanism at the C-20, 22 desmolase site in the supply of placental pregnenolone was noted.

Production of penicillic acid and patulin by an atypical Penicillium roqueforti isolate.

Simultaneous production of penicillic acid and patulin by an atypical strain of Penicillium roqueforti isolated from cheddar cheese is reported. Mycotoxin production was confirmed by thin-layer and gas-liquid chromatography and by ultraviolet, infrared, and mass spectral analyses. Culture extracts were toxic to Bacillus megaterium and chicken embryos. Commercial strains of P. roqueoforti used in production of blue-veined cheeses were shown not to produce penicillic acid and patulin.

Penicillic acid production in submerged culture.

Twenty known penicillic acid (PA)-producing Aspergillus and Penicillium cultures were grown under various conditions in shaken flasks to determine the highest yielding strains and their requirements for maximum toxin production. Abilities of the cultures to utilize eight different carbon sources in Raulin-Thom medium for mycotoxin synthesis were determined at four different incubation temperatures: 15, 20, 25, and 28 degrees C. Of the 20 cultures, P. cyclopium NRRL 1888 was superior, yielding up to 4 mg of PG per ml, with mannitol as the carbon source and 25 degrees C as the incubation temperature. Fifteen of the cultures elaborated lesser amounts of PA, whereas four strains yielded none under the test conditions. Whey from the manufacture of cottage cheese by the cultured process was also a satisfactory medium for PA production. In whey medium, yields up to 3 mg/ml were obtained with P. cyclopium NRRL 1888.

[Investigations on the production of mycotoxins and their quantitative evaluation. I. The production of penicillic acid by Penicillium cyclopium (author's transl)]. / Untersuchungen zur Bildung von Mykotoxinen und deren quantitative Bestimmung. I. Die Bildung von Penicillinsäure durch Penicillium cyclopium

A convenient thin-layer chromatographic screening procedure for the analysis of mycotoxins produced by Penicillium cyclopium is described. The production of penicillic acid is followed during a growth period of 44 days at two different temperatures. Quantitative evaluation is performed by UV densitometry at 234 nm. A biological profile is recorded showing the different ratios of four metabolites produced by Penicillium cyclopium and the definite effect of the growth temperature on the formation of penicillic acid in relation to the other metabolites.

Mycotoxin-producing potential of mold flora of dried beans.

To evaluate the potential for mycotoxin production by molds in dried beans, the mold flora of 114 samples was determined both before and after surface disinfection of the beans with 5% NaOCl. Surface disinfection substantially reduced mold incidence, indicating that contamination was mainly on the surface. The flora, both before and after disinfection, was dominated by species of the Aspergillus glaucus group, the toxicogenic species A ochracues, Penicillium cyclopium, and P. viridicatum, and species of Alternaria, Cladosporium, and Fusarium. The toxicogenic species Aspergillus flavis, A. versicolor, Penicillium Citrinum, P. expansum, P. islandicum, and P. urticae were encountered less frequently. Of 209 species of Aspergillus and Penicillium screened for mycotoxin production on sterile rice substrate, 114 produced one or more of the following mycotoxins: A. flavus, aflatoxins; A. ochraceus, ochratoxins; A. nidulans, A. unguis, and A. versicolor, sterigmatocystin; P. cyclopium, penicillic acid; P. citrinum and P. viridicatum, citrinin; P. urticae, patulin and griseofulvin. Sterigmatocystin production by A. unguis is reported for the first time.

Metabolism of glutamic acid in Aspergillus ochraceus during the biosynthesis of ochratoxin A.

The uptake and utilization of glutamic acid in the biosynthesis of ochratoxin A by Aspergillus ochraceus were studied. Uniformly labeled L[14C]glutamic acid was incorporated into both the phenylalanine and isocoumarin moieties of ochratoxin A. Penicillic acid was also labeled. During the early stages of development, the amino acid was used mainly for the synthesis of ribonucleic acid and protein. A portion of glutamic acid was oxidized and was recovered as metabolic 14CO-2. The initial uptake velocity of glutamic acid decreased with age and was pH and temperature dependent. No relationship was found between the initial uptake velocities and ochratoxin A biosynthesis.

Intermediates in the penicillic acid biosynthesis in Penicillium cyclopium.

Feeding experiments using 14C-labelled precurosrs demonstrate the following sequence of the biological formation of penicillic acid in Penicillium cyclopium: orsellinic acid leads to 2-O-methylorsellinic acid leads to 1,4-dihydroxy-6-methoxy-2-methylbenzene leads 6-methoxy-2-methyl-benzoquinone(1,4)leads to penicillic acid.

Mycotoxin-producing strains of Penicillium viridicatum: classification into subgroups.

Fifty-two isolates of Penicillium viridicatum Westling were divided into three groups based on ability to produce ochratoxin and/or citrinin, color, growth rate, type of growth, odor, and isolation source. Members of group I resemble one of the representative strains of P. viridicatum described in the literature; those belonging to group II differ from group I strains in several characteristics; group III is a heterogeneous series of highly variable isolates. Although three subgroupings can be recognized, retention of all isolates in the species P. viridicatum is deemed most appropriate at this time. Spore macerates of all isolates were examined for virus-like particles but none were detected.

Identification of the volatile compounds produced in sterile fish muscle (Sebastes melanops) by Pseudomonas fragi.

Volatile compounds produced by Pseudomonas fragi strain 18 in sterile fish muscle (Sebastes melanops) were identified by combined gas-liquid chromatography and mass spectrometry. Compounds positively identified included dimethyl sulfide, acetaldehyde, ethyl acetate, ethyl alcohol, and dimethyl disulfide. Methyl mercaptan, ethyl butyrate, ethyl hexanoate, and butanone were tentatively identified by relative retention times of the authentic compounds. The fruity odor that developed in fish muscle during incipient spoilage was attributed to a synergistic flavor interaction involving the ethyl esters of acetate, butyrate, and hexanoate.