Avaliação da mutagenicidade de Piper methysticum L. f. no sistema methG1 em Aspergillus nidulans / Mutagenic evaluation of Piper methysticum L. f. in Aspergillus nidulans methG1 system

O extrato seco da raiz de Piper methysticum L. f. Forster (PIPERACEAE), a kava-kava, é usado no tratamento de diversos problemas envolvendo ansiedade como um dos sintomas. Por não causar dependência, sedação e ter ação ansiolítica, muitas pessoas têm recorrido a kava-kava para auxiliá-las no emagrecimento. Isto pode levar ao consumo indiscriminado da planta e acarretar riscos, pois todo medicamento fitoterápico deve respeitar limites de doses. Um risco na utilização de plantas medicinais é a toxicidade e, dentro deste, a mutagenicidade. Como a mutagenicidade está relacionada com a carcinogenicidade torna-se importante testar este potencial na kava-kava. Assim, o objetivo deste trabalho foi avaliar o potencial mutagênico do extrato seco da raiz de P. methysticum no sistema methG1 em Aspergillus nidulans. A linhagem utilizada foi a biA1methG1, auxotrófica para biotina e metionina. Conídios dormentes de colônias crescidas por cinco dias foram tratados com soluções da kava-kava nas concentrações de 0,35 mg mL-1 e 3,5 mg mL-1, e depois de 24h, semeados em meio seletivo contendo metionina, para análise dos sobreviventes, e sem metionina, para a análise dos mutantes. Os números de sobreviventes e mutantes dos tratamentos foram comparados aos do controle. Os resultados indicaram que o extrato da raiz da kava-kava é mutagênica, pois a freqüência de mutação dos tratamentos foi maior que da mutação espontânea, porém não ocorrendo diferença significativa entre as doses.

The dry root extract of Piper methysticum L. f. Forster (Piperaceae), kava-kava, is used as to treat several health problems involving anxiety symptoms. As it causes no addiction, it can be applied as a sedative and anxiolytic. Many people have been relying on kava-kava as an auxiliary treatment. This can lead to an indiscriminate plant consumption and lead to risks, because all phytotherapic medications must observe dosage limits. One risk in the folk medicinal plant use is toxicity, and within it, mutagenicity. As mutagenicity is closely related to carcinogenicity, it is important to test the kava-kava mutagenicity potential. Thus, the purpose of this work was to test the mutagenicity of the dry root extract of P. methysticum in the methG1 system of Aspergillus nidulans. The bia1methG1 lineage, which is auxotrophic for biotine and methione, was used. Conidia from five-day-old colonies were collected and treated with kava-kava solutions at 0.35 mg mL-1 and 3.5 mg mL-1 concentrations and, after 24h, they were planted in selective growth medium with and without methione, in order to analyze the survivors and mutants, respectively. The number of survivors and mutants analyzed under effect of the treatments was compared with the control. The results indicated that the kava-kava dry root extract is mutagenic, since the mutation frequency of the treatments was higher than spontaneous mutation, however, there were no differences between the doses tested.

Cytological characterization of an Aspergillus nidulans mutant from a strain with chromosomic duplication

A development mutant, named V103, was obtained spontaneously from the A strain of A. nidulans. The A strain contains a duplicated segment of chromosome I that has undergone translocation to chromosome II (I ¨ II). It is mitotically unstable and generates phenotypically deteriorated types, some with enhanced stability. The deteriorated variants of A. nidulans show abnormal development, exhibiting slower colony growth, variations in colony pigmentation and changes in conidiophore structure. The alterations observed in the conidiophore include fewer metulae and phialides, further elongation and ramification of these structures, delayed nuclear migration and the presence of secondary conidiophores.

Isolation and characterisation of cycloheximide-sensitive mutants of Aspergillus nidulans.

Aspergillus nidulans is a non-pathogenic fungus with well-developed genetics which provides an excellent model system for studying different aspects of drug resistance in filamentous fungi. As a preliminary step to characterizing genes that confer pleiotropic drug resistance in Aspergillus, we isolated cycloheximide-sensitive mutants of A. nidulans, which is normally resistant to this drug. The rationale for this approach is to identify genes whose products are important for drug resistance by analysing mutations that alter the resistance/sensitivity status of the cell. Fifteen cycloheximide-sensitive (named scy for sensitive to cycloheximide) mutants of A. nidulans were isolated and genetically characterised. Each scy mutant was crossed with the wild-type strain and five of the crosses gave 50% cycloheximide-sensitive progeny suggesting that they carry a single mutation required for cycloheximide sensitivity. We examined ten scy mutants for resistance/sensitivity to other drugs or stress agents with different and/or the same mechanism of action. Six of these mutants exhibited other altered resistance/sensitivity phenotypes which were linked to the cycloheximide sensitivity. These six mutants were analyzed by pairwise crosses and found to represent six linkage groups, named scyA-F. One of the mutants showed fragmentation of its vacuolar system and, in addition, its growth was osmotic, low-pH, and oxidative-stress sensitive.

COVASIAM: an image analysis method that allows detection of confluent microbial colonies and colonies of various sizes for automated counting.

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties: Saccharomyces cerevisiae, Aspergillus nidulans, Escherichia coli, Azotobacter vinelandii, Pseudomonas aeruginosa, and Rhizobium etli. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (sigma = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (sigma = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.

Los alergenos del ambiente tabacalero / Allergens of tobacco environment

Se halló 16 a 60 esporos fúngicos/litro aire y de 100 a 100.000 esporos de actinomicetos termófilos/g polvo del piso en los secaderos de tabaco. Los hongos eran Alternaria sp (21 por ciento), Aspergillus sp (15 por ciento), Asp. fumigatus (7 por ciento), Asp. glaucus (5,5 por ciento), Asp. nidulans (13,3 por ciento), Chaetomiun sp (2,3 por ciento), Chrysosporium spp (5,5 por ciento), Cladosporium sp (3,1 por ciento), Curvularia sp (0,8 por ciento), Dactylaria sp (3,1 por ciento), Nogrospora sp (5,5 por ciento), Penicillium spp (11,7 por ciento), Talaromyces sp (2,3 por ciento) y no identificado (3,9 por ciento). Los Thermoactinomyces eran Tha. thalpophilus (66,6 por ciento), Tha. sacchari (6,7 por ciento), Tha. vulgaris (6,7 por ciento) y otros Tha. spp (20 por ciento). También el humo de cigarrillos contenía esporos viables de Thermoactinomyces. El 7 ñ 3,3 por ciento de los trabajadores tenían anticuerpos anti-Tha. thalpophilus demostrados por doble inmunodifusión (AU)

Los alergenos del ambiente tabacalero / Allergens of tobacco environment

Se halló 16 a 60 esporos fúngicos/litro aire y de 100 a 100.000 esporos de actinomicetos termófilos/g polvo del piso en los secaderos de tabaco. Los hongos eran Alternaria sp (21 por ciento), Aspergillus sp (15 por ciento), Asp. fumigatus (7 por ciento), Asp. glaucus (5,5 por ciento), Asp. nidulans (13,3 por ciento), Chaetomiun sp (2,3 por ciento), Chrysosporium spp (5,5 por ciento), Cladosporium sp (3,1 por ciento), Curvularia sp (0,8 por ciento), Dactylaria sp (3,1 por ciento), Nogrospora sp (5,5 por ciento), Penicillium spp (11,7 por ciento), Talaromyces sp (2,3 por ciento) y no identificado (3,9 por ciento). Los Thermoactinomyces eran Tha. thalpophilus (66,6 por ciento), Tha. sacchari (6,7 por ciento), Tha. vulgaris (6,7 por ciento) y otros Tha. spp (20 por ciento). También el humo de cigarrillos contenía esporos viables de Thermoactinomyces. El 7 ñ 3,3 por ciento de los trabajadores tenían anticuerpos anti-Tha. thalpophilus demostrados por doble inmunodifusión