Biomedical subjects
Publications and source records attributed to V Betina.
A new metabolite was isolated from the culture of Penicillium rugulosum and its structure was determined from physico-chemical data. Accompanying metabolites skyrin and rugulosin were characterized by UV, IR, CD, mass and NMR spectra.
Eurotium repens mycelium cultivated under static conditions was used to isolate and identify metabolities--echinulin, physcion, erythroglaucin, flavoglaucin and asperentin; the filtrate of the culture yielded asperentin 8-methylether. The broadest biological activity spectrum was displayed by asperentin which had antibacterial and antifungal effects and, at a concentration of 86 microgram/ml, caused 50% mor7 tality in Artemia saline larvae. The highest cytotoxicity towards HeLa cells was found in physcion which caused 50% growth inhibition at a concentration of 0.1 microgram/ml.
After a short irradiation at 366 nm with 200 lx, the intensity of conidiation of Trichoderma viride colonies grown in the dark increased for the first 10 s proportionally with time. The increase slowed down after 10 s-5 min of exposure and after 10-60 min of irradiation the conidiation intensity began to decrease. When photo-induced by daylight, the conidiation started at a high rate after 25 h and persisted even after 48 h. The conidiation had no circadian character and its periodicity depended on the periodicity of photo-induction. Its intensity was also influenced by the carbon sources used, a maximum being reached with glucose (1-2%). Higher glucose concentrations inhibited conidiation but had no influence on growth of colonies.
The photo-induced conidiation of Trichoderma viride is suppressed by ethidium bromide, acriflavin, lomofungin and 8-quinolinol at concentrations which do not inhibit the colony growth of this deuteromycete.
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We investigated the toxicity of 32 different mycotoxins, 7 macrolides, not 3 other fungal metabolites to Artemia saline (Brine Shrimp) larvae. We modified the known procedures of testing mycotoxins to A. salina larvae and found the latter to be a convenient model for comparing the toxicity of mycotoxins and other secondary metabolities. Results obtained were evaluated by the probit analysis.
The cytotoxic effect of 7 macrocyclic fungal metabolites on HeLa cells has been determined. The authors noted great differences in the cytotoxic activity of the substances investigated. Least effective had proved the metabolites curvularin and zearalenone which kept exhibiting ED50 at a concentration of 2 X 10(-4)M and at a concentration of 7.5 X 10(-5)M respectively, whereas the most effective cyanein and monorden gave rise to 50% inhibition of multiplication of HeLa cells at a thousandfold lower concentration than zearalenone. Cytochalasins A, B and D brought about 50% inhibition of proliferation of HeLa cells at concentrations of the same order, with ED50 ranging between 1.2-2.1 X 10(-6)M. The different biological activity of the studied macrocyclic fungal metabolites is discussed in connection with their chemical structure.
The analysis of 37 mycotoxins and other fungal metabolites by thin-layer chromatography is described. Their R(F) values in eight solvent systems, colour reactions after chemical detection and detection under UV light are presented. From the R(F) values, "chromatographic spectra" which can be used as one of the characteristics for the characterization of unknown mycotoxins were constructed.
A method for the bioautographic detection of mycotoxins on thin-layer chromatograms by using Artemia salina larvae is described. The method was tested on standard samples of mycotoxins (aflatoxin B1 kojic acid and sterigmatocystin) and on the extracts from toxicogenic fungi isolated from different sources.
Colonial growth of Neurospora sitophila phenotypically induced by ramihyphin A is accompanied by marked changes in the contents of DNA, RNA and proteins in the mycelium, and in the relative proportion of hexoses in cell wall hydrolysates. The glucosamine/glucose ratio is also characteristic for colonial growth. X-ray analysis of cell walls showed that ramilhyphin A suppresses the crystalline arrangement of chitin in cell walls. A combination of microbiological, biochemical and physico-chemical methods yielded a general picture of the changes accompanying the colonial growth of Neurospora sitophila.
The effect of antibiotics and metabolic inhibitors on mycelial growth of Botrytis cinera was followed. Inhibitors of protein synthesis, chloramphenicol, erythromycin and tetracycline inhibit growth or sporulation of Botrytis cinera. Ethidium bromide, 5-fluorouracil, phenylethylalcohol and K 20 cause granulation, vacuolization and undulation of hyphase. 2,4-Dinitrophenol, boromycin, macrotetrolides, monensin, scopathricin and TX2 at subfungistatic concentrations induce intensive branching of hyphal tips i.e. at the site of synthesis of the cell wall. In older hyphase grown in the absence of the antibiotics the branching begins after their addition, particularly in the septum region. When comparing the results referred to here with those obtained previously and on the basis of literature data it may be assumed that the changes in polarity of growth of Botrytis cinerea might be caused primarily or secondarily by impairing membrane functions and formation of cell walls.
The mycelium of Trichoderma viride grown in the dark under submerged conditions and transferred to membrane filters sporulated only after photoinduction. The optimum photoinduction of sporulation was reached when applying daylight for 3 min and near ultraviolet radiation (355 nm) for 10 to 30 sec. After the photoinduction probounced synthesis of DNA, RNA and protein was observed. The photoinduced sporulation was partially or fully inhibited in the presence of phenethyl alcohol, actinomycin D, 5-fluorouracil, cycloheximide and ethidium bromide. The same inhibitors blocked also the photoinduced sporulation of surface growing colonies of Trichoderma viride. Various inhibitiors of synthesis of nucleic acids and protein, inhibitors impairing the function of membranes and certain other compounds were also effective.
Ramihyphin A at subfungistatic concentrations stimulates ramification of hyphae of filamentous fungi. Stimulation of terminal ramification of hyphae that can be observed particularly in phytopathogenic fungi is most frequent. Hyphae of Microsporon canis, Trichophyton mentagrophytes, Blastomyces dermatitidis, Coccidioides immitis and Histoplasma capsulatum ramify intensively laterally. Stimulation of the lateral ramification was observed in Monilia fructigena, Penicillium marneffei and Penicillium chrysogenum. The antibiotic induces also formation of vesicular structures in phytopathogens. Due to the substantial ramification of hyphae, both terminal and lateral, the growth of colonies is interrupted. The addition of the antibiotic to a growing colony of Botrytis cinerea induces dichotomic ramification of terminal hyphae after 3 h of growth. Lateral hyphae begin to grow later and further ramify dichotomically. Dense bundles of ramified hyphae are formed after 24 h due to the unbalanced ramification and the colony no longer increases its size.
Some antibiotics and synthetic inhibitors affect, in several ways, the life cycle of Neurospora crassa (germination of conidia leads to myceliar growth leads to formation of conidia). Bikaverin, cyanein, scopathricin and phenethyl alcohol retard the germination of conidia, without inhibiting it completely. 5-Fluorouracil, ramihyphin A and zygosporin A (cytochalasin D) do not inhibit the germination. Bikaverin brings about a thickening of the hyphae of growing mycelium. Ramihyphin A, cyanein, scopathricin and zygosporin A stimulate the ramification of hyphae while 5-fluorouracil and phenethyl alcohol do not affect the myceliar morphology apart from their inhibitory effect on growth. Actinomycin D, 5-fluorouracil, cycloheximide, ramihyphin A and partially also sodium iodoacetate inhibit to a different degree the photoinduced formation of conidia. The inhibition by 5-fluorouracil is very conspicuous when the agent is present during the photoinduction but considerably weaker when it is applied 2 h after the photoinduction.
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