Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii.

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

Activation of beta-glucan synthases by wall-bound purple acid phosphatase in tobacco cells.

Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis.

When pDHA4, a vector carrying all five pfaA-pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA-pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.

Isolation and characterization of a novel thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid.

A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 degrees C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n-3) at 43% of the total fatty acids. It had a growth rate of 0.38 h(-1). The DHA production rate of 2.8 +/- 0.7 g l(-1) day(-1) is the highest value reported for any microorganism.

Vital growth factors of Malassezia species on modified CHROMagar Candida.

A comparison of several media, i.e., potato dextrose agar with olive oil (Oil-PDA), modified Dixon agar (mDIX) and variations of Leeming and Notman agar (LNA) for the isolation and growth of Malassezia and Candida species was examined. Since LNA supported the highest growth of Malassezia species its key components, i.e., ox bile, glycerol monostearate, glycerol and Tween 60, were added to CHROMagar Candida. All 7 species of Malassezia grew well on this modified medium (LN-CHROM) after incubation for 4 days at 30 degrees C and development was equal to that observed on LNA. Colonies on LN-CHROM were smooth and from pink to dark purple in color. Furthermore, the use of LN-CHROM did not alter the colony characteristics of Candida species as compared to that found on CHROMagar Candida. The results of the present investigation indicate that the use of LN-CHROM would make possible the simultaneous isolation and identification of Malassezia and Candida species.

Comparative evaluation of standard dilution method and commercial kit for frozen plate antifungal susceptibility testing of yeasts using 200 clinical isolates.

A comparative evaluation of standard microdilution methods and a commercial kit for frozen plate antifungal susceptibility testing of yeasts was performed using amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole on 200 yeast isolates. The isolates included 100 strains of Candida albicans, eight of C. tropicalis, twelve of C. parapsilosis, eight of C. glabrata, five of Cryptococcus neoformans, thirteen of Trichosporon asahii, and 54 other strains of seven other species of ascomycotic yeasts. Microdilution testing was performed according to the standard method for antifungal susceptibility testing published by the Japanese Society for Medical Mycology (JSMM), which are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) M27-P. The commercial kit was prepared according to the manufacturer's instructions. The degree of agreement within +/-1 dilution for 200 clinical isolates against five antifungal agents was excellent with values for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole of 100%, 99.0%, 97.5%, 97.0%, and 97.0%, respectively. Overall, the frozen plate antifungal susceptibility testing kit provided convenient and reproducible results comparable to those obtained with the JSMM standard method.

Cell shape regulation and co-translocation of actin and adenosyl homocysteinase in response to intermediate hypertonicity.

Hypertonic stimulation induced association of S-adenosyl-L-homocysteine hydrolase (SAHH) with the F-actin-rich cell cortex in Dictyostelium. At intermediate, but not higher, levels of hypertonicity, SAHH further translocated from the cortex to the cytosol in company with a fraction of actin and cofilin. At the same time the cells rounded up. Acidification of the cells stimulated both the cell rounding and the translocation of actin and SAHH, whereas alkalinization retarded these responses, suggesting that cellular pH is involved in their control. On the other hand, mutant analysis suggested that neither cGMP signaling nor conventional myosin is required.

The MADS-box transcription factor SrfA is required for actin cytoskeleton organization and spore coat stability during Dictyostelium sporulation.

The MADS-box transcription factor SrfA is involved in spore differentiation in Dictyostelium [Development 125 (1998) 3801]. Mutant spores show an altered morphology and loss of viability. A detailed structural analysis of mutant spores has been performed to gain insight into the specific aspects of spore differentiation in which SrfA is involved. Two main structural defects have been observed. One is the formation of high order actin structures, the so-called actin rods. SrfA mutant spores showed the initial stages of rod formation but no mature rods were found in older spores either in the nucleus or the cytoplasm. Moreover, phosphorylation of actin, that is believed to stabilize the actin rods, is strongly reduced in the mutant. The other defect observed was the formation of the spore coat. Young srfA- spores show basically normal trilaminar coat structures suggesting that release of prespore vesicles and basic assembly of the coat takes place in the absence of SrfA. However, the outer layer gets wavier as the spore ages and suffers a progressive degradation suggesting a late defect in the stability of the spore coat. Taken together, these results suggest that SrfA is involved in late events of spore maturation necessary for spore stability.

Hypertonic signal promotes stability of Dictyostelium spores via a PKA-independent pathway.

Differentiation of Dictyostelium spores initiates with rapid encapsulation of prespore cells under the control of cAMP-dependent protein kinase (PKA), followed by further maturation processes involving cytoskeletal reorganization. Constitutive activation of PKA induces precocious formation of viable spores in development and confers the ability to encapsulate under specific submerged conditions. In this study, we show that the stability of these spores depends upon conditions of high osmotic strength during spore differentiation, indicating that a hypertonic signal is required in addition to PKA to induce maturation to stable spores. The formation of stable spores under hypertonic conditions requires high cell density, suggesting the involvement of additional cellular signaling.

DNA base alignment and taxonomic study of genus Malassezia based upon partial sequences of mitochondrial large subunit ribosomal RNA gene.

The sequences of the large subunit of mitochondrial ribosomal RNA (LsmtrRNA) gene of Malassezia species were analysed. The sequences of the seven species of Malassezia are well separated in each species. Therefore the LsmtrRNA gene is thought to be one of the gene targets for species identification in the genus Malassezia. The dendrogram obtained from this gene supports the previous study of Malassezia species based upon the chromosomal genes. This is the first report of taxonomic analysis of Malassezia species based upon the mitochondrial gene.

Distribution and movement of Caenorhabditis elegans on a thermal gradient.

To analyze thermal responses of Caenorhabditis elegans in detail, distribution of a worm population and movement of individual worms were examined on a linear, reproducible and broad temperature gradient. Assay methods were improved compared with those reported previously to ensure good motility and dispersion of worms. Well-fed, wild-type worms distributed over a wide temperature range of up to 10 degrees C, and, within this range, worms migrated in both directions of the gradient at similar frequencies without any specific response to the growth temperature in most cases. By contrast, worms migrated down the gradient if put in a region warmer than the warm boundary of distribution. The distribution range changed depending on the growth temperature and starvation, but active avoidance of a starvation temperature was not detected. These findings contradict previous hypotheses of taxis or migration to the growth temperature in association with food and instead indicate avoidance of a warm temperature. Our results favor a model for thermal response of C. elegans that postulates a single drive based on warm sensation rather than downward and upward drives in the physiological temperature range. Mutants in ttx-3, tax-2, tax-4 or egl-4 genes showed abnormal thermal responses, suggesting that these genes are involved in warm avoidance. Laser ablation and gene expression studies suggest that AFD neurons are not important, and tax-4 expression in neurons other than AFD is required, for warm avoidance.

Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts.

Methods of rapidly extracting chromosomal DNA from human pathogenic yeasts were used in mitochondrial DNA (mtDNA) studies. This paper is concerned with rapid and reliable methods of extracting mtDNA for sequence analysis for species or strain identification, and epidemiological study of medically important fungi and fungal infections. To determine the optimal method of mtDNA extraction without isolating mitochondria, we examined three commonly used methods: 1). boiling, 2). glass bead disruption, and 3). a commercially available kit. We assessed the amount and quality of DNAs using a spectrophotometer and specific polymerase chain reaction (PCR). The DNA yield depended on the extraction method used and the yeast species. An adequate amount of mtDNA was obtained with both glass beads and a commercially available kit to amplify the mitochondrial gene using PCR without isolating the mitochondria. These techniques are convenient for extracting DNA from a variety of small-scale samples.

Programmed cell death of Pinus nucellus in response to pollen tube penetration.

In ovules of Pinus densiflora, pollen tubes elongate and branch into the nucellar tissue in the direction of the female gametophyte. After pollination, nucellar cells located around the pollen grain and tube die off. We showed here that the nuclei of the nucellar cells were stained by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling). The number of TUNEL-positive cells increased during pollen tube growth. The tips of pollen tube branches protruded into the nucellar cells to form a convex-concave junction. At this junction, the cell membrane of nucellar cells was separated from the cell wall and the protoplast shrank. Small vesicles and amorphous materials were released from the protoplast into the space between the cell membrane and wall. Vacuoles were collapsed, chromatin was condensed, and mitochondria and plastids were deteriorated in the shrunken protoplast. Agarose gel analysis of DNA isolated from the ovules showed a DNA ladder, suggesting that the nuclear DNA had undergone internucleosomal cleavage. These results suggest that nucellar cells undergo programmed cell death in response to pollen tube penetration with some features resembling apoptosis and other features peculiar to nucellar cells.

[Overview of lipophilic yeast Malassezia: the current status of the molecular diagnosis].

The lipophilic yeast, genus Malassezia is a part of the normal cutaneous microflora of human and warm-blooded vertebrates. Species of the genera were re-classified to seven species; M. pachydermatis, M. globosa, M. furfur, M. obtusa, M. restricta, M. slooffiae and M. sympodialis. However, the means of species identification in conventional clinical laboratories have not been reported and the clinical significance of each species is not clearly understood. Species identifications of genus Malassezia which depend on the morphological, physiological characters are difficult and time-consuming. Recently, many molecular techniques have been developed for identification or typing of Malassezia. PCR-mediated methods, PCR-direct sequencing and nested-PCR using specific primers, are useful to identify the spices. The basic information obtained from these approaches have been contributing to the understanding of these pathogenic yeasts and related diseases.