Aims:The characterization by molecular and physiological methods of wild apiculate strains, isolated from ÔAglianico del VultureÕ grape must. Methods and Results:The restriction analysis of 18S rDNA allowed the identification of strains at the species level, which were predominantly Hanseniaspora uvarum. The RAPD analysis and the evaluation of technological traits, such as the metabolic and enzymatic activities, were useful to evaluate the polymorphism of this species. Conclusions: The RAPD analysis clustered the wild H. uvarum strains in four main genetic groups and a very high phenotypic variability confirmed this genetic polymorphism. The technological variables, which determined the strain biodiversity differed significantly, demonstrating that these technological traits are strain dependent. A certain correlation was found between the strain behaviour and its isolation zone, indicating the influence of the environment on the genetic patrimony of the population. Significance and Impact of the Study: The genetic and technological biodiversity recorded among H. uvarum wild strains represents the basis for organizing a collection of apiculate strains exhibiting oenological characteristics at different levels, such as high/low production of secondary compounds, and, therefore, potentially useful for a selection programme.

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The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent-and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.The 90 kDa heat shock protein (Hsp90) 1 is a central part of a chaperone meshwork chaperoning a large number of substrate proteins (1-5). Besides being a partner of a large number of co-chaperones and substrates, Hsp90 binds to filamentous actin and tubulin (6 -8) and the involvement of the cytoskeleton in the traffic of Hsp90 substrates has also been demonstrated (5, 9). Together with other chaperones, like Hsp27 and Hsp70, Hsp90 is involved in cytoprotection (10 -12).Cell lysis is one of the most commonly used methods to test cellular integrity. Moreover, lysis rate anomalies (13,14) together with diffusional anomalies (15, 16) were used as important arguments for the organization of the cytoplasm. Since cellular integrity is preserved after a partial cell lysis to a large extent (13,14), partial lysis provides a highly sensitized, but still somewhat organized cellular system, where the contribution of various components to both the cytoplasmic organization and cellular stability can be studied.The original aim of the present study was to examine whether Hsp90 inhibition induces any change in the rate of cell lysis induced by mild detergent treatment or hypotonic shock. The rationale behind these experiments was to test, whether Hsp90, a cytoprotective chaperone, binding to "thousand-andone" substrates and other proteins is involved in the maintenance of cellular integrity (17), and whether its inhibition renders cells more "lysis-prone." The first experiments were very promising: geldan...

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We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were Candida norvegensis and one was C. inconspicua. Although C. norvegensis is considered a pseudohyphaproducing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C. inconspicua.The frequency of human infections caused by yeasts is increasing. Candida albicans is still the opportunistic pathogenic yeast most frequently isolated from humans with fungal diseases; however, several non-C. albicans Candida species are also cultured more and more frequently from different body sites (3,15). Preliminary identification of non-C. albicans Candida species has been incorporated into the majority of routine laboratory algorithms (18). In doubtful cases, additional techniques are required to obtain a precise identification of the isolated species, e.g., the determination of different biochemical patterns with the API 20C and API ID32C systems (API/ ATB package inserts, BioMerieux, 1996). Recently, molecular biology techniques have been introduced for the more precise identification of Candida isolates (21,22). This urgent need is based on at least two factors: the spread of Candida species resistant to antifungal agents (6) and the threat of nosocomial Candida outbreaks (4). An additional advantage of these techniques is the ability to differentiate between closely related species or previously misidentified yeasts.C. inconspicua and C. norvegensis are rarely isolated opportunistic pathogens (17,20). During the past 5 years, the number of yeast isolates identified in the Laboratory for Bacteriological Diagnostics, University of Debrecen, as C. inconspicua or C. norvegensis increased from 3 to 4 per year to 29 per year (in 2001), while the overall increase in the rate of yeast isolation was only threefold (9). We could not reliably distinguish the two species by traditional identification methods (with the API ID32C system and detection of pseudohypha production) because the species identities obtained by different methods were contradictory. To precisely identify our isolates we applied ribosomal DNA (rDNA) analysis.The genetic background of the method chosen can be summarized as follows. The tandemly repeated rDNA genes of yeasts contain highly conservative and less conservative regions. Sequences coding for the 18S, 5.8S, and 25S rRNAs evolved slowly during the phylogenesis of fungi; thus, they are suitable for use as tools for discrimination and identification at the interspecies or higher level. On the contrary, the internal transcribed spacer region 1 (ITS1) and ITS2 rDNA regions are more variable; thus, they can serve as a tool in the discrimination of closely related yeast species (21). Restriction enzyme analysis of PCR-amplified rDNA is a superior typing method suitable for correct identification, even with a large number of yeast isolates. In addition, this method can correct misidentification r...

The response of Schizosaccharomyces pombe towards the oxyanions selenate [Se(VI)] and dichromate [Cr(VI)] was investigated in order to establish the involvement of the yeast ATP sulfurylase in their reduction. An ATP sulfurylase-defective/selenate-resistant mutant of S. pombe (B-579 Se(R) -2) and an ATP sulfurylase-active/selenate-sensitive strain of S. pombe (B-579 Se(S)) were included in this study. The inhibitory effect of Se(VI) and Cr(VI) oxyanions on growth and bioaccumulation was measured. The sensitive strain showed natural sensitivity to selenate while the resistant mutant tolerated a 100-fold higher concentration of selenate. These results indicate that selenate toxicity to microorganisms is connected with the reduction of selenate to selenite. Both strains showed similar sensitivity to Cr(VI) and in this study there was no evidence that ATP sulfurylase participates in the reduction process of Cr(VI).

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