Behaviour studies of the fungicide cymoxanil in two strains of the fungus Botrytis cinerea and in haemolymph of locust and lobster. I. In situ monitoring by internal surface reversed-phase high-performance liquid chromatography.

A method for following the metabolism of the fungicide cymoxanil in various biological media is described. By using a recently developed high-performance liquid chromatographic method, with an internal surface reversed-phase column, it is unnecessary to clean up the sample before analysis. Thus this technique makes monitoring in fungi as well as in arthropod haemolymph easier and faster.

Nuclear staining of Colletotrichum gloeosporioides f. sp. malvae conidia with fluorescent and nonfluorescent stains.

Six different staining techniques were evaluated for their suitability to stain nuclei of Colletotrichum gloeosporioides f. sp. malvae (C.g.m.) spores. Of the three fluorescent stains, DAPI (4',6-diamidino-2-phenylindole) and bisbenzimide (Hoechst 33258) stained spore nuclei well; mithramycin did not. To achieve consistent results with the bisbenzimide staining protocol, the spores had to be fixed prior to staining and the stain had to be supplemented with Triton X-100. Both safranin O and Giemsa were suitable nonfluorescent staining techniques; lomofungin was not. Safranin O staining was simple and rapid. However, reproducibility was better if the spore suspension and KOH droplets were rapidly mixed prior to adding the stain. There was no significant difference in the percentages of uninucleate and binucleate spores observed in spore preparations stained with DAPI, bisbenzimide, safranin O or Giemsa. Bisbenzimide and safranin O were found to be simple, rapid and reliable fluorescent and nonfluorescent techniques, respectively, for staining nuclei of C.g.m. spores.

CAF-603: a new antifungal carotane sesquiterpene. Isolation and structure elucidation.

A new antifungal compound, CAF-603, was isolated from the culture broth of Gliocladium virens IFO 9166. The structure of compound 1 has been elucidated by spectroscopic methods that also allowed the relative stereochemistry to be assigned. CAF-603 [1] was active against yeasts and dermatophytes.

Studies on a toxic metabolite from the mould Wallemia.

While monitoring the occurrence of toxigenic moulds in foods, using a bioassay screen, it was shown that an isolate of Wallemia sebi produced toxic effects in several of the bioassays. The toxic metabolite was isolated and purified using solvent extraction, TLC and HPLC coupled with the brine shrimp assay to monitor the toxic fractions. The purified toxin, which we propose to call walleminol A, has been partially characterized by mass spectroscopy, nuclear magnetic resonance, ultraviolet and infrared spectroscopy. It can be provisionally interpreted as a tricyclic dihydroxy compound, C15H24O2, with structural features characteristic of a sesquiterpene with an isolated double bond, but further work is required to characterize this compound unequivocally. The minimum inhibitory dose of walleminol A in the bioassays is approximately 50 micrograms/ml, which is comparable with a number of mycotoxins such as citrinin and penicillic acid.

Isolation, sequence, and conformation of seven trichorzianines B from Trichoderma harzianum.

From the antagonistic fungus Trichoderma harzianum, a group of acidic new peptides, trichorzianines B (TB), was isolated in addition to neutral trichorzianines A (TA) previously studied. TA and TB exhibit various biological activities related to their membrane properties and a different behaviour of the two groups was noticed. As observed for other peptaibols, TB consist in a microheterogeneous mixture which was resolved into pure peptides by reversed-phase C18 HPLC. The sequence of the seven main isolated TB, namely TB IIa, TB IIIc, TB IVb, TB Vb, TB VIa, TB VIb, TB VII, was determined by the combined use of positive ion FAB mass spectrometry and 2D 1H n.m.r. spectroscopy, including COSY and NOESY experiments. TB differ from the corresponding TA only by the replacement of Gln 18 in the TA sequence by a glutamic acid. The 1H n.m.r. data suggested that the TB are mainly organized in an alpha helix.

12,13-Deoxytrichoverrins from Myrothecium verrucaria.

The structures of two new diastereoisomeric 12,13-deoxytrichothecenes have been established by a combination of nmr and mass spectroscopy.

Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C.

Calphostin C (UCN-1028C), a newly isolated compound from Cladosporium cladosporioides, is a potent and specific inhibitor of protein kinase C, because it was 1000 times more inhibitory to protein kinase C (IC50, 0.05 microM) than other protein kinases such as cAMP-dependent protein kinase and tyrosine-specific protein kinase (IC50, greater than 50 microM). Calphostin C did not inhibit calcium activated neutral protease (calpain)-digested protein kinase C, indicating that it interacts with the regulatory domain of protein kinase C. In addition this compound showed inhibitory effects on the binding of [3H]PDBu to protein kinase C. The potent cytotoxic activity and antitumor activity of calphostin C might be due to the inhibition of protein kinase C, and thus it may be potentially useful for the therapeutic application.

Profiling of Alternaria mycotoxins in foodstuffs by high-performance liquid chromatography with diode-array ultraviolet detection.

The potential of high-performance liquid chromatography (HPLC) with diode-array detection for the profiling of mycotoxins in food samples has been demonstrated. A gradient elution reversed-phase chromatographic method coupled with a suitable extraction procedure was devised for the separation and detection of major Alternaria mycotoxins in foodstuffs. Altenuene, alternariol, alternariol methyl ether (dibenzo-alpha-pyrone derivatives), altertoxin-I and altertoxin-II (perylene derivatives) were profiled in extracts of artificially infected maize, rice and tomato samples and naturally contaminated sunflower seeds. First evidence of the occurrence of a new dibenzo-alpha-pyrone derivative in Alternaria cultures is also reported.

Stemphyltoxin III from Alternaria alternata.

Stemphyltoxin III, (6aR*, 6bS*, 7R*, 8R*)-3,6a, 10-trihydroxy-4,9-dioxo-4,6a,6b,7,8,9-hexahydro-7,8-epoxyperylene, a known metabolite of Stemphylium botryosum var. lactucum, has been identified as a mutagenic metabolite of Alternaria alternata by spectroscopic studies. The 13C-nmr spectral data, which were not reported previously, are presented.

Ultrastructural study of galacturonic acid distribution in some pathogenic fungi using gold-complexed Aplysia depilans gonad lectin.

Aplysia gonad lectin, isolated from the mollusc Aplysia depilans, was successfully conjugated to colloidal gold and used for ultrastructural detection of galacturonic acids in some pathogenic fungi. These sugar residues were found to occur in the fibrillar sheath surrounding hyphal cells of Ascocalyx abietina and in intravacuolar dense inclusions of this fungus spores. In hyphae and spores of Ophiostoma ulmi, galacturonic acids were detected mainly in the outermost wall layers. In contrast, these saccharides appeared associated with the innermost wall layers and especially the plasma membrane of Verticillium albo-atrum cells. Galacturonic acids were found to be absent in cells of Fusarium oxysporum f.sp. radicis-lycopersici and Candida albicans. These cytochemical data indicate therefore that a heterogeneity in wall composition exists between ascomycete fungi. The significance of the presence of galacturonic acids in the cell walls of certain fungi is still open to question.

Application of flow cytometry to differentiating Exophiala dermatitidis, E. moniliae and E. jeanselmei from each other.

We applied a flow cytometry apparatus (FCM) to differentiating Exophiala dermatitidis, E. moniliae and E. jeanselmei from each other. The wavelength of the argon laser emitted from the FCM was 488 nm and the aperture of nozzle from which the stream of fluid containing single cells was blown out was 100 micron. By irradiating the stream with laser by either the forward light scatter (FLS) or by the perpendicular light scatter (PLS), we were able to get two pieces of informations. Histograms displayed by the FLS indicate the cell size, while dot displays by the PLS reflect the cell structure. As a result, E. dermatitidis was clearly differentiated from either E. moniliae or E. jeanselmei by their histograms by FLS. In addition, dot displays by the PLS differentiated E. moniliae from E. jeanselmei. In conclusion, flow cytometry is available for differentiating E. dermatitidis, E. moniliae and E. jeanselmei from each other.

Lipid composition and effect of amphotericin B on yeast cells of Paracoccidioides brasiliensis.

Yeast cells of Paracoccidioides brasiliensis strain SN, were obtained for analysis of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. Such analysis were made on cells cultured in the presence or absence of amphotericin B and on non proliferating cell suspensions exposed to the antibiotic. Marked alterations in lipid contents were observed in this different conditions. The major alterations were the reduction of total lipids, sterols, and palmitoleic acid in both, proliferating and non proliferating antibiotic exposed cells. The effect of amphotericin B was evaluated also in terms of viability and release of intracellular substances, at different times of exposure. The minimal inhibitory concentration (MIC) determined for that strain of this fungus was 0.2 microgram/mL.

Element localisation and distribution in the fungi Aureobasidium pullulans and Aspergillus niger, measured using the Oxford scanning proton microprobe.

The distribution and relative concentrations of essential elements were determined in germ tubes of Aureobasidium pullulans and hyphae of Aspergillus niger using the Oxford scanning proton microprobe. In both fungi, K, P and S were the major constituents, with Ca, Na and trace metals like Cu, Mn, Fe and Zn present at lower levels. In A. pullulans, elements were not distributed uniformly throughout the cells and, in general, the highest elemental concentrations occurred at the tip and in the older parts of the germ tube, particularly where there was yeast-like cell or branch development. In A. niger, elemental distribution was more uniform and there was a general gradient of increasing concentration away from the hyphal tip followed by a drop in levels in older regions. The scanning proton microprobe appears to have considerable potential for the investigation of fungal differentiation and morphogenesis.

Antitumor activity of polygalactosamine isolated from Paecilomyces sp. I-1 strain.

The inhibitory effect of polygalactosamine (PF102), which was isolated from Paecilomyces sp. I-1 strain, on a syngeneic murine solid tumor and its antitumor mechanism were studied. After an intravenous injection of PF102, 1 microgram/kg, an increase in cell mediated and humoral immunities in mice was observed and the growth inhibition of MM46 solid tumor in vivo was also evident. Macrophages induced by PF102 into the peritoneal cavity inhibited deoxyribonucleic acid synthesis of target cells. Moreover, PF102 caused a significant increase in the incorporation of 3H-thymidine into the thymic cells and the culture supernatant of T lymphocytes, stimulated with PF102, exhibited a marked activation of the cytostatic effect of the peritoneal macrophages. Furthermore, this culture supernatant fluid was found to contain interferon (IFN). Therefore, the antitumor activity of PF102 might be due in part to the activation of the macrophage lineage cells by macrophage activating factor and/or IFN produced from T lymphocytes stimulated by PF102.

Sporobolomyces yuccicola, a new species of ballistosporous yeast equipped with ubiquinone-9.

A hitherto undescribed ballistosporous yeast was isolated from a dead leaf of Yucca sp. in Canada. This yeast produces apiculate or short-ellipsoidal ballistospores, produces pale colored colonies, has Q-9 as the major ubiquinone, and does not contain xylose in the cells. This new yeast is described as Sporobolomyces yuccicola Nakase et Suzuki. Sporobolomyces yuccicola is the sixth species of the intermedius group, a group of atypical species of the genus Sporobolomyces equipped with Q-9.

Growth and macromolecular content of the dimorphic fungus Aureobasidium pullulans and the effect of hydroxyurea and other inhibitors.

The growth kinetics and the macromolecular content of the yeast and ethanol-induced hyphal forms of Aureobasidium pullulans were studied. During the morphological transition from yeasts to hyphae, both the protein and RNA content decreased significantly, the mycelial form containing only 76% of the amount of protein in the yeasts, and 38% of the RNA. The DNA was the only component tested whose level increased during the transition. Among several compounds inhibiting macromolecular synthesis, only hydroxyurea showed a remarkable effect on the morphology of A. pullulans, inducing the mycelial morphology. The macromolecular composition of hydroxyurea-treated cultures changed with time in a way similar to that of the ethanol-Tween 80-ammonia medium, and to that of carbon-starved cultures, without ethanol or glucose.

Vinigrol, a novel antihypertensive and platelet aggregation inhibitory agent produced by a fungus, Virgaria nigra. II. Pharmacological characteristics.

Vinigrol, a novel diterpene produced by Virgaria nigra, was tested orally in conscious spontaneously hypertensive rats (SHR) to confirm its antihypertensive activity. Vinigrol (2 mg/kg, po) decreased the mean arterial blood pressure of SHR by approximately 15% for at least 6 hours. Vinigrol induced contraction of rat aortic smooth muscle preparation at 1.5 X 10(-7) M, the contraction was blocked by nilvadipine, a Ca2+ entry blocker, but was not inhibited by prazosin or yohimbine. Radio-receptor binding assay of alpha adrenoceptors of rat brain membrane revealed that vinigrol had no affinity for these receptors.

Comparison of thermally-assisted fast atom bombardment (TA-FAB) with conventional FAB and EI mass spectrometry for the analysis of the Helminthosporium carbonum mycotoxins.

A new technique known as thermally-assisted fast atom bombardment (TA-FAB) has been applied to the analysis of a series of cyclic tetrapeptide mycotoxins in order to demonstrate the usefulness of the method for structural elucidation. TA-FAB uses saturated aqueous solutions of highly hydroxylated compounds, such as fructose, as alternatives to the usual viscous liquid matrices employed in conventional FAB. During the TA-FAB analysis, the probe tip is resistively heated causing differences to occur in the desorption profiles for the analyte and the matrix ions enabling an optimization for analyte desorption as a function of temperature. In this study, direct comparisons are made between TA-FAB, conventional FAB, and electron impact ionization for the analysis of the Helminthosporium carbonum mycotoxins at the 1.5 micrograms level. The results demonstrate the superior capacity of TA-FAB to provide both molecular weight confirmation and significant fragmentation to aid in the structural elucidation of these important biomolecules.

[IR spectrophotometric study of a mixed culture of Streptococcus lactis, strain MSU, and Rhodotorula colostri with agitation of the medium]. / IK-spektrofotometricheskoe issledovanie sovmestnoi kul'tury Streptococcus lactis shtamm MGU i Rhodotorula colostri v usloviiakh peremeshivaniia sredy.

Physiological aspects of Streptococcus lactis, strain MSU development in mixed culture with Rhodotorula colostri were studied in connection with nisin biosynthesis. By the IR spectra the average content of the main components in the monoculture cells of S. lactis and its association with the yeast in the stationary growth phase was the following by dry weight: 54-58 per cent of protein, 14-16 per cent of nucleic acids, 26-28 per cent of carbohydrates and 3-5 per cent of lipids. Glucose and nitrogen in the fermentation broth were consumed completely. A significant quantity of KH2PO4 remained in the fermentation broth. Lactic acid excretion was observed. S. lactis, strain MSU was the leading component of the association under the studied cultivation conditions because just its IR spectra dominated in the spectra of the mixed culture.