Aspectos micromorfológicos do Penicillium marneffei em vida saprofítica. Produçäo de clamidoconídos com septaçöes transversas / Micromorphologic aspects of Penicillium marneffei in saprophytic life. Chlamydoconidia production with transversal septations

Examinando, ao microscópico óptico comum, Penicillium marneffei Segretain, 1959, amostra isolada de um paciente francês com AIDS, apó seu retorno da Indonésia (Ancelle e dols), cultivada em ágar-Sabouraud a 37-C, após 14 dias de incubaçäo, verificamos ao lado de formas de frutificaçäo caracteristicas do gênero Penicillium, clamidoconídios de parede espessa, com septaçöes transversais formando dois a três elementos fúngicos. Estes foram semelhantes aos observados por Drouhet e cols. quando estudaram à microscopia eletrônica as formas parasitárias desse microrganismo no hamster dourado inoculado com a referida amostra. Justifica-se este trabalho para mostrar que, tanto em vida parasitária como em vida saprofítica os clamidoconídios com parede espessa e septos transversais ocorrem com freqüência, näo havendo brotamento como em casos de histoplasmose clássica, em sua forma parasitária. Graças a este tipo de reproduçäo, podemos separar a histoplasmose da peniciliose pelo Penicillium marneffei com a qual as duas infecçöes podem se confundir ao exame histopatológico. Na histoplasmose, os fungos näo se dividem por fissäo binária

Restricticin, a novel glycine-containing antifungal agent.

Restricticin (1) is a naturally-occurring antifungal agent which contains triene, pyran and glycine ester functionalities and is unrelated to any previously known family of natural products. This unstable compound, as well as its corresponding N,N-dimethyl derivative (2), have been produced and isolated from both solid and liquid fermentations of Penicillium restrictum. The desglycyl hydrolysis product, restrictinol (3), was produced via the hydrolysis of pure restricticin and as an artifact of the isolation of restricticin.

Antigenicity of Penicillium notatum in animals and in atopic patients.

A mycelial and metabolic extract of Penicillium notatum (PN) was passed through Sephadex G-50 and DEAE-cellulose columns in order to separate soluble fractions that revealed a complex composition. Their proteins and hexoses were recorded with a LKB Uvicord spectrophotometer and quantified by the Lowry and the indol techniques. On the other hand an animal model was developed in rabbits and an IgG precipitin and hemagglutinating antibodies were obtained. The PN extract as well as those fractions with the higher protein content appeared positive when they were checked against the antiserum by means of Ouchterlony, Boyden and immunoelectrophoresis. The molecular weight of PN was established in 52,000 daltons approximately in comparison with well known marker proteins. Adult human beings suffering perennial rhinitis and bronchial asthma showed positive type I skin tests with PN and its most conspicuous fractions (glycoproteins). A RAST-IgE-anti-PN was prepared following Ceska's procedure and challenged against all human sera. Only 40% of the patients revealed a positive RAST IgE-anti-PN which correlated significatively with the protein fractions (35%) and with the skin tests (43% and 39%, respectively). These results reinforced the idea that PN is a potent antigenic mold both in animals and in humans in whom we detected an IgE specific antibody presumably related to their atopic clinical condition.

Neutral, alkaline and difference ultraviolet spectra of secondary metabolites from Penicillium and other fungi, and comparisons to published maxima from gradient high-performance liquid chromatography with diode-array detection.

The ultraviolet spectra of 6 predominantly secondary metabolites from filamentous fungi which, inter alia, are useful in the identification of the compounds after chromatography, were obtained in neutral (methanol) and alkaline solvents. Difference spectra were obtained by subtracting the neutral from the alkaline spectrum for each metabolite, using the spectrophotometer software. The data and method are of use in differentiating metabolites with similar chromophores. A database of the maxima was stored on a microcomputer for flexible storage, retrieval and updating of information. These data are compared to those published previously, obtained by diode-array detection using gradient high-performance liquid chromatography, which indicated that changes in solvent concentrations of the gradient affect the spectra of some metabolites. This could cause misidentification of chemosyndromes and metabolites which have been claimed to be of use in fungal chemotaxonomy.

Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi.

Improved methods are described for the isolation of pure, high molecular weight DNA from small and large scale cultures of filamentous fungi. The methods depend on the extraction of DNA under conditions which prevent nuclease activity and contamination by carbohydrate. The small scale method depends on enzymatic digestion of the wall whereas the large scale method uses partial damage followed by autolysis. High yields of DNA are obtained by both methods and the DNA is suitable for restriction analysis. Southern Blotting, RFLP analysis, dot blotting and the production of gene libraries. The small scale method can be used for the simultaneous analysis of multiple cultures.

Monoclonal antibody-based enzyme linked immunosorbent assay of aflatoxin B1, T-2 toxin, and ochratoxin A in barley.

Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1, 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile-0.5% KCl-6% H2SO4 (89 + 10 + 1) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonitrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCl buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1, 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were less than 12% for B1 and OA but as high as 17% for T2.

Gradient high-performance liquid chromatography using alkylphenone retention indices of insecticidal extracts of Penicillium strains.

Purified extracts of four Penicillium strains which were active against the insect pest Spodoptera littoralis were analysed by gradient high-performance liquid chromatography (HPLC) for secondary metabolites using alkylphenone retention indices. HPLC of pure secondary metabolite standards detected previously in the extracts by thin-layer chromatography (TLC) was undertaken in order to obtain bracketed retention indices. More metabolites were detected by HPLC than by TLC, although some compounds detected by TLC in some strains were not detected by this HPLC method. A minority of metabolites were exclusive to each strain, and most were produced by more than one strain. The profiles were more characteristic of each strain when only the larger peaks were considered. This emphasizes the importance of detection limits in secondary metabolite analysis. Some of the implications of these analyses to fungus toxicity and systematic mycology are discussed.

[Cytophysiology of Penicillium solitum: a producer of lipase]. / Tsitofiziologiia Penicillium solitum--produtsenta lipazy.

The cell population of Penicillin solitum was studied during maximum accumulation of lipase in the medium with electron microscopic and immunofluorescence methods. The data provided a conclusion that 2 types of lypolytic enzymes with various substrate and antigenic characteristics formed in the cells of P. solitum. It is likely that there is a specific inductor for exolipase synthesis as well as relationship endoenzymatic systems.

FR65814, a novel immunosuppressant isolated from a Penicillium strain. Taxonomy, fermentation, isolation, physico-chemical and biological characteristics and structure assignment.

FR65814, a novel immunosuppressant, was isolated from the cultured broth of Penicillium jensenii F-2883. The structure was assigned to be 5,6-dihydroxy-4-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-1-oxaspiro++ +[2,5]octane by spectroscopic analyses. The compound suppressed the immune response at low concentration. In addition, a structually related component fumagillol, a known carcinolytic agent, was also isolated and found to show immunosuppressive activity.

Citreoviridin levels in Eupenicillium ochrosalmoneum-infested maize kernels at harvest.

Citreoviridin contents were measured in eight bulk samples of maize kernels collected from eight fields immediately following harvest in southern Georgia. Citreoviridin contamination in six of the bulk samples ranged from 19 to 2,790 micrograms/kg. In hand-picked samples the toxin was concentrated in a few kernels (pick-outs), the contents of which were stained a bright lemon yellow (range, 53,800 to 759,900 micrograms/kg). The citreoviridin-producing fungus Eupenicillium ochrosalmoneum Scott & Stolk was isolated from each of these pick-out kernels. Citreoviridin was not detected in bulk samples from two of the fields. Aflatoxins were also present in all of the bulk samples (total aflatoxin B1 and B2; range, 7 to 360 micrograms/kg), including those not containing citreoviridin. In Biotron-grown maize ears that were inoculated with E. ochrosalmoneum through a wound made with a toothpick, citreoviridin was concentrated primarily in the wounded and fungus-rotted kernels (range, 142,000 to 2,780,000 micrograms/kg). Samples of uninjured kernels immediately adjacent to the wounded kernel (first circle) had less than 4,000 micrograms of citreoviridin per kg, while the mean concentration of toxin in kernel samples representing the next row removed (second circle) and all remaining kernels from the ear was less than 45 micrograms/kg. Animal toxicosis has not been linked to citreoviridin-contaminated maize.

Absolute and relative configuration of erythroskyrin.

We have reisolated erythroskyrin from Penicillium islandicum and have determined the relative stereochemistry of the compound through extensive 1H- and 13C-nmr studies. The absolute stereochemistry was determined by nmr studies of the O-methylmandelate esters.

High-performance liquid chromatography of siderophores from fungi.

A reversed-phase HPLC separation of iron(III) chelates of 16 representative fungal siderophores including ferrichromes, coprogens and triacetylfusarinine C was established in order to investigate siderophore production of fungi. For comparison purposes, the widely used bacterial siderophore ferrioxamine B was included. Culture filtrates of the fungi Penicillium resticulosum, Fusarium dimerum, Aspergillus fumigatus and Neurospora crassa were quantitatively analyzed for the presence of known and unknown siderophores after growth in low-iron culture media and adsorption on XAD-2 columns using this HPLC separation system. Photodiode array detection allowed the distinction between siderophores and non-siderophores. According to their ultraviolet/visible spectra, a further classification of the siderophores into four types due to the number of anhydromevalonic acid residues per molecule (0-3) was possible.

Standardized high-performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone retention indices and UV-VIS spectra (diode array detection).

A general standardized method for the analysis of mycotoxins and other fungal secondary metabolites has been developed, based on high-performance liquid chromatography (HPLC) with an alkylphenone retention index and photodiode-array detection combined with thin-layer chromatography (TLC) in two different eluents. Each fungal secondary metabolite is characterized by its bracketed alkylphenone retention time index, its UV-VIS absorption maxima and its retardation factors relative to griseofulvin in two TLC eluents. This system is effective for the comparison of chemotaxonomic data in different laboratories and for a precise identification of fungi based on organic solvent extracts of fungal cultures. All important groups of mycotoxins and other fungal secondary metabolites could be detected in the HPLC system described and data are listed for 182 metabolites. The fungal secondary metabolites separated and characterized include aflatoxin B1, B2, G1 and G2, ochratoxin A, citrinin, penicillin acid, viomellein, penitrem A, patulin, sterigmatocystin, alternariol, tenuazonic acid, trichothecenes, roquefortines, fusarin C, zearalenone, PR-toxin, citreoviridin, viridicatumtoxin, verruculogen, rugulosin, cyclopiazonic acid, penicillin G and many other alkaloids, polyketides and terpenes.

High-performance liquid chromatographic determination of profiles of mycotoxins and other secondary metabolites.

A reversed-phase high-performance liquid chromatographic determination of profiles of mycotoxins and other fungal secondary metabolites has been developed. Penicillium, Aspergillus and Fusarium polyketides, terpenes and alkaloids have been emphasized. In a gradient elution, using water-acetonitrile containing 0.05% trifluoroacetic acid, 134 secondary metabolites were eluted evenly with retention times from 1.08 to 34.48 min. Metabolites with the same retention time were usually not produced by the same species. As UV detection at 254 nm was used, some mycotoxins (type A trichothecenes, viridicatumtoxin, peptide-like compounds and xanthomegnin) could not be detected. The method appears to be valuable for chemotaxonomic studies of fungi. Unpurified concentrated chloroform-methanol extracts of petri dish cultures analysed by the proposed method presented gave species-specific characteristic profiles of known and unknown secondary metabolites and mycotoxins.

New macrolides from Penicillium urticae mutant S11R59.

Two new macrolides, patulolide B and patulolide C, were isolated from a culture filtrate of Penicillium urticae S11R59 mutant. The structures of these macrolides were determined and their biological activities were investigated. These structures and biological activities were also compared with those of patulolide A which was produced by the same organism.

Separation of mycotoxin-containing sources in grain dust and determination of their mycotoxin potential.

Two distinct reservoirs of mycotoxins exist in fungal-infected cereal grains--the fungal spores and the spore-free mycelium-substrate matrix. Many fungal spores are of respirable size and the mycelium-substrate matrix can be pulverized to form particles of respirable size during routine handling of grain. In order to determine the contribution of each source to the level of mycotoxin contamination of dust, we developed techniques to harvest and separate mycelium-substrate matrices from spores of fungi. Conventional quantitative chromatographic analyses of separated materials indicated that aflatoxin from Aspergillus parasiticus, norsolorinic acid from a mutant of A. parasiticus, and secalonic acid D from Penicillium oxalicum were concentrated in the mycelium-substrate matrices and not in the spores. In contrast, spores of Aspergillus niger and Aspergillus fumigatus contained significant concentrations of aurasperone C and fumigaclavine C, respectively; only negligible amounts of the toxins were detected in the mycelium-substrate matrices of these two fungi.

[Effect of the exogenous lipids of complex media on the fatty acid composition of mycelial microorganisms]. / Vliianie ékzogennykh lipidov kompleksnykh sred na sostav zhirnykh kislot u mitselial' nykh mikroorganizmov.

The effect of exogenous lipid sources on the composition of fatty acids was studied in actinomycetes of the Streptomyces genus and in fungi belonging to the genera Blakeslea, Cunninghamella and Penicillium. The following sources of exogenous lipids were used: soybean and maize flour, sunflower by-products, chicken droppings, maize extract, yeast extract, peptone, sperm whale fat, sunflower and palm oil. The composition of fatty acids in total extracted lipids of the studied mycelial microorganisms was shown to reflect two processes: lipid synthesis de novo and assimilation of exogenous fatty acids. This fact ought to be taken into account both in the chemotaxonomic interpretation of fatty acid composition and in practical recommendations for the utilization of microbial lipids. It is of particular interest to study the physiological role of exogenous lipid metabolism in the cells of microorganisms.

Occurrence of mycotoxins in cereals and animal feedstuffs in Natal, South Africa.

During the period 1982-1983, just under 800 samples of agricultural commodities, comprising cereals, compound feeds, hay, and silage, were examined for molds and mycotoxins. Aflatoxin B1 showed the highest incidence rate; it occurred in over 27% of all samples analyzed, the highest levels being found in peanut meal at 1500 ppb. Other mycotoxins detected were patulin and a number of trichothecene toxins at incidence rates in all commodities of 5.6 and 3.1%, respectively. The commodities at highest risk were oil seeds, excluding soya bean; the latter was found to be fairly free from contamination with mycotoxins. The most prevalent fungi were Aspergillus flavus and parasiticus, which were found in over 22% of all samples, whereas Penicillium spp. showed the lowest incidence of genera, specifically identified in 8.3% of all samples examined. This latter finding explains in part the low incidence of Penicillium mycotoxins.