Transformation of Trichoderma species with dominant selectable markers

Summary We have developed a transformation system for Trichoderma hamatum and Trichoderma harzianum Rifai, using dominant markers for selection based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the -tubulin gene (bml) from Neurospora crassa, respectively. Transformation frequencies and protoplast regeneration were low in both species. All the T. hamatum hygromycin-resistant transformants analysed were mitotically stable, in contrast to those of T. harzianum derived by benomyl resistance, in which only 50% of the transformants analysed were stable. Molecular analysis of transformants showed the integration of the transforming DNA into the genome and indicated that the number and sites of integration varied among the transformants.     

Cuticle-induced endo/exoacting chitinase CHIT30 from Metarhizium anisopliae is encoded by an ortholog of the chi3 gene

The characterization of chitinase genes and enzymes is an important step toward global understanding of the chitinolytic system in entomopathogenic fungi. Chitinase CHIT30 from Metarhizium anisopliae var. anisopliae (strain E6) has both endo- and exochitinase activities and is a potential determinant of pathogenicity. Serum anti-CHIT30 specifically detected this chitinase amongst five isoenzymes shown in glycol-chitin activity gels. Chitinase CHIT30 secretion is upregulated by chitin, tick cuticle and low concentrations of N-acetylglucosamine (0.25%) and is downregulated by both high N-acetylglucosamine (1%) and glucose (1%) concentrations. Chitinase CHIT30 was produced at tick cuticle during fungal infection. The chi3 gene was assigned to code chitinase CHIT30 in M. anisopliae var. anisopliae.     

Clinical and polysomnographic characteristics of 34 patients with Kleine-Levin syndrome

There is only scant information on sleep characteristics and long-term follow-up in patients with Kleine-Levin syndrome (KLS). This study describes the clinical course, results of polysomnography and long-term follow-up in a relatively large group of patients with KLS. During the years 1982-97, we encountered 34 patients (26 males and eight females) with KLS. We were able to obtain the original polysomnographs from 28 males and four females. In 25 patients, data regarding their present state of health were obtained. Fourteen agreed to be present at a detailed interview and examination while 11 gave the information by phone. The mean age at onset was 15.8 +/- 2.8 years and the mean diagnostic delay, 3.8 +/- 4.2 years. The mean duration of a single hypersomnolent attack was 11.5 +/- 6.6 days. The main abnormal findings extracted out of 35 polysomnographs obtained from 32 patients during and/or in-between attacks included: decreased sleep efficiency, and frequent awakenings from sleep stage 2. All 25 patients reported present perfect health, with no evidence of behavioral or endocrine dysfunction. In adolescents with periodic hypersomnia, the diagnosis of KLS should be explored. Sleep recordings during a hypersomnolent period will often show frequent awakenings from sleep stage 2. The long-term prognosis is excellent.     

Endochitinase CHI2 of the biocontrol fungus Metarhizium anisopliae affects its virulence toward the cotton stainer bug Dysdercus peruvianus

Chitinases have been implicated in fungal cell wall remodeling and play a role in exogenous chitin degradation for nutrition and competition. Due to the diversity of these enzymes, assigning particular functions to each chitinase is still ongoing. The entomopathogenic fungus Metarhizium anisopliae produces several chitinases, and here, we evaluate whether endochitinase CHI2 is involved in the pathogenicity of this fungus. We constructed strains either overexpressing or lacking the CHI2 chitinase. These constructs were validated by Southern, Northern and Western blot analysis, and chitinase production. To access the effects of CHI2 chitinase in virulence, the cotton stainer bug Dysdercus peruvianus was used as a host. CHI2 overexpression constructs showed higher efficiency in host killing suggesting that the production of this chitinase by a constitutive promoter reduces the time necessary to kill the insect. More significantly, the knock out constructs showed decreased virulence to the insects as compared to the wild type strain. The lack of this single CHI2 chitinase diminished fungal infection efficiency, but not any other detectable trait, showing that the M. anisopliae family 18, subgroup B endochitinase CHI2 plays a role in insect infection.     

Purification and ultrastructural localization of a copper-zinc superoxide dismutase (CuZnSOD) from the entomopathogenic and acaricide fungus Metarhizium anisopliae

The entomopathogenic fungus Metarhizium anisopliae contains three superoxide dismutases. One of these enzymes was purified and partially characterized as a CuZnSOD. The enzyme has an estimated molecular mass of 30690 Da and a specific activity of 3838.89 Umg(-1). SDS-PAGE and 2D gels show a single band of protein in the fractions eluted from the gel filtration column with a molecular mass of 20000 and approximately 15000 Da, respectively, and a pI of 6.0. These results suggest that the native enzyme is a dimer consisting of two subunits. Polyclonal antiserum were raised against purified CuZnSOD and used to determine its subcellular localization by immunoelectron microscopy. M. anisopliae CuZnSOD is present in the cell wall.     

Typing and patterns of cellular morphological alterations in Cryptococcus neoformans and Cryptococcus gattii isolates exposed to a panel of killer yeasts.

Cryptococcus neoformans and Cryptococcus gattii are encapsulated basidiomycetous yeasts that cause meningoencephalitis. The action of killer yeasts on the growth of one hundred genotypically characterized C. neoformans var. neoformans, C. neoformans var. grubii, and C. gattii clinical and environmental isolates was evaluated. Killer studies were performed on yeast malt-methylene blue (YM-MB) agar Petri dishes, and a dendrogram was obtained based on a quantitative data matrix using the diameter of the inhibition halo. The cellular morphological characteristics of dead cells within the halo were observed by means of optical and scanning electron microscopy. There was no formation of pores on the cell surface of the sensitive cells in contact with the toxins, at least for C. neoformans. The sensitivity patterns of clinical and environmental isolates to the killer toxins demonstrated that there is correlation between killer sensitivity of Cryptococcus species or varieties and some of the killer strains. In this case, the isolates were discriminated using the killer sensitivity patterns, and this could be used as a complementary tool to PCR-fingerprinting in epidemiological studies.     

Can police car colored flash light induce electroencephalographic discharges and seizures?

AIM: To explore the epileptogenic potential of a newly introduced police car flash light device (930 Heliobe Lightbar). METHODS: A 930 Heliobe Lightbar was installed in the EEG laboratory. Thirty patients with known epilepsy, 30 subjects with chronic headache who were otherwise healthy and 15 healthy volunteers were examined. RESULTS: All the subjects signed an informed consent and underwent an EEG during which photostimulation was performed with the standard stroboscope and later with the Police lightbar. In all 75 examined the lightbar did not induce clinical or electrographic seizures. In a single patient with epilepsy the lightbar enhanced epileptiform activity induced by standard photic stimulation. CONCLUSION: In this study the new Police lightbar was found to be non-epileptogenic.