A new PCR approach for the identification of Fusarium graminearum / Um novo protocolo de PCR para a identificação de Fusarium graminearum

The main objective of this work was to develop a PCR protocol for the identification of Fusarium graminearum, based on a pair of primers targeted to a segment of the 3' coding region of the gaoA gene that codes for the enzyme galactose oxidase (GO). This region has low homology with the same region of GO genes from other fungi. Genomic DNA from 17 strains of Fusarium spp. isolated from diseased cereals, from several other Fusarium species, and from other fungi genera was analyzed in a PCR assay using this primer set. The 17 strains of Fusarium spp. were also analyzed for the GO enzyme production in submerse fermentation in a new formulated liquid medium. All strains that were morphologically and molecularly identified as F. graminearum were able to secrete the enzyme and had a positive result in the used PCR protocol. No DNA fragment was amplified using genomic DNA from other Fusarium species and species of other fungi genera. The results suggest that the proposed PCR protocol is specific and can be considered as a new molecular tool for the identification of F. graminearum. In addition, the new formulated medium is a cheap alternative for screening for GO screening production by F. graminearum.

O principal objetivo deste trabalho foi desenvolver um novo protocolo de PCR para identificação de isolados de Fusarium graminearum, baseado no uso de um par de iniciadores direcionado para um segmento da região 3' codificadora do gene gaoA que codifica a enzima galactose oxidase (GO). Esta região possui baixa homologia com a mesma região de genes da GO de outros fungos. O DNA genômico de 17 cepas de Fusarium spp. isoladas de cereais infectados com sintomas, de vários outras espécies de Fusarium e de outros gêneros de fungos foi analisado em um protocolo de PCR utilizando os iniciadores desenhados. Os 17 isolados de Fusarium spp. também foram analisados para a produção da enzima GO em fermentação submersa em um novo meio líquido. Todas as cepas que foram morfologicamente e molecularmente identificadas como F. graminearum foram capazes de secretar a enzima e tiveram um resultado positivo no protocolo de PCR, utilizando os iniciadores direcionados para o gene gaoA. Nenhum fragmento de DNA foi amplificado quando foi utilizado o DNA genômico de várias outras espécies de Fusarium e de espécies de outros gêneros de fungos. Os resultados sugerem que o protocolo de PCR gerado é específico e pode ser considerado como uma nova ferramenta molecular para a identificação de cepas de F. graminearum. Além disso, o meio líquido formulado é uma alternativa barata para a avaliação da produção de GO por F. graminearum.

Technique for detection of T-antigen, an early marker of prostatic carcinoma

The tumor marker, D-galactose-B[l-3]-N-acetyl-D-galactosamine [GaI-GalNAc, also known as T-antigen] can be identified by a very simple galactose oxidase-Schiff's GOS] reaction either on tissues from patients with adenocarcinoma neoplasms. Gal-GalNAc is expressed in the neoplastic mucosa as well as the remote non-neoplastic mucosa. It is, however, not expressed in mucosa of normal subjects. We have tested the usefulness of the tumor marker D-galactose-beta-[l-->3]-N-acetyl-D-galactosamine in differentiating the benign from the malignant and premalignant lesions of the prostate. A sequential galactose oxidase technique, followed by Schiff's reaction, was done on deparaffinized tissue sections of 35 carcinomas, 25 hyperplasias, 20 foci of adenosis, and 10 normal specimens. While none of the 35 benign prostates and 11 foci of adenosis expressed D-galactose-beta-[l-->3]-N-acetyl-D-galactosamine [100 percent specificity], 62 [95.4 percent sensitivity] of 65 adenocarcinomas variably expressed the marker. We therefore propose that this simple technique may have potential use in routine histopathological analysis of prostatic specimens. This marker may also serve as the basis of assays for early detection of prostatic malignancies

Production of mycotoxins by galactose oxidase producing "Fusarium" using different culture media

The original isolate of the galactose oxidase producing fungus "Dactylium dendroides, and other five galactose oxidase producing "Fusarium" isolates were cultivated in different media and conditions, in order to evaluate the production of 11 mycotoxins, wich are characteristic of the genus "Fusarium": moniliform, fisaric acid, deoxyvalenol, fusarenone-X, nivalenol, 3-acetyldeoxynivalenol, neosolaniol, zearakenol, zeralenone, acetyl T-2, and iso T-2. The toxicity of the culture extractes to "Artemia salina" larvae was tested

P53 expression and D-galactose-B- [1--3] -N-acetyl-D-galactosamine in colorectal bilharziasis, precancerous lesions and carcinoma

In an attempt to evaluate the role of bilharziasis in the pathogenesis of colorectal cancer, the specific molecular changes associated with specific morphologic changes in colorectal bilharzial lesions, precancerous lesions as well as selected cases of cancer were studied. A total of 36 tissue samples were selected and carefully investigated in this study. They included 20 cases of non-cancerous colonic bilharziasis [5 bilharzial colitis, 5 bilharzial granuloma and 10 bilharzial polyps], 3 cases of ulcerative colitis, 3 cases of juvenile polyps and 10 cases of colorectal adenocarcinoma [8 with bilharziasis and 2 without bilharziasis]. p53 and GO-Schiff reaction were done on these cases. It was found that cancer cases expressed both p53 and Gal-GalNAc markers. Moreover, involved mucosa remote from the tumor revealed Gal-GalNAc staining. All bilharzial cases, as well as juvenile polyps, from non-cancerous patients showed no positivity for both markers. However, one case of ulcerative colitis associated with moderate dysplasia exhibited p53 as well as Gal-GalNAC expression

Restriction enzyme fragment patterns of mtDNA from a galactose oxidase-producing mold

1. Mitochondrial DNAs from Dactylium dendroides, Hypomyces rosellus, Fusarium graminearum, Gibberella fujikuroi, Fusarium tricinctum strains and a galactose oxidase (GAO)-producing mold (original strain) presented distinctive restriction enzyme fragment patterns with the endonucleases Hind III and EcoRI. 2. A small number of comigrating bands was found when the GAO-producing mold was compared with the others. The molecular size of mtDNA from the GAO-producing mold, as judged by summation of fragment sizes produced by digestion with EcoRI, Hind III and Bgl II, is 61.3 +/- 2.16 kb. 3. The results suggest that the mtDNA from the GAO-producing mold strain is distinct from that of D. dendroides and all other ascomycetes analyzed

A Dactylium dendroides morphological mutant defective in D-galactose metabolism

1. A morphological mutant of the mold Dactylium dendroides was and the phenotype characterized as D-Gal-and L-Ara-. 2. The transport system for D-galactose seemed to be inducible in wild type and mutant and was altered in the mutant. 3. Galactose-1-P- uridylyl transferase activity was absent in the mutant. 4. The levels of intracellular galactose oxidase activity were similar in the wild type and in the mutant, theraby excluding a possible participation of this enzymes in glactose catabolism inthe mold. 5. The low level of galactose oxidase activity found in the extracellular medium indicates a defect in galactose oxidase secretion by the mutant