Toshio Kanbe

A ring structure around the dividing plane of theCyanidium caldarium chloroplast

The unicellular hot-spring alga Cyanidium caldarium RK-1 (RK-1) is remarkable in that it has only a single chloroplast, a single mitochondrion and a cell nucleus, and that it generates four cyst cells following two cell divisions (NAGASHIMA and FUKUDA 1984). These features make it convenient for the study of the behavior of these organelles during cell division. The fine structure of the RK-1 cell was investigated using the freeze-substitution technique of VAN HARREVEI~D and CROWEI~L (1964). We found a plastiddividing ring structure (PD ring) around the constricting isthmus of the dividing chloroplast of RK1, which appeared specifically during the first (Figs. 1 and 2) and second chloroplast divisions (Figs. 3-6). The figures show an RK-1 cell before (Figs. 1 and 2) and after the first cell division (Figs. 3-6). The upper cell of the two-cyst stage RK-1 cell exhibits the representative opposite cell organelle location of chloroplast and cell. nucleus, with the mitochondrion between them (Fig. 4). The chloroplasts were characterized by the presence of a large amount of the ribosomes in their central region and by the cord-type phycobilisomes on the thylakoid membrene (Figs. 1-6). At higher magnification in Fig. 6, an electron-dense

The role of the cytoskeleton in the polarized growth of the germ tube in Candida albicans.

Cells of the dimorphic yeast Candida albicans are easily induced to germinate in synchrony. Using germinating cells of strain FC18, we examined the effects of several drugs that are known to affect the cytoskeleton on growth and cytoskeletal organization. Cytochalasin A (CA), an inhibitor of actin function, inhibited the germination of the yeast cells and changed the cylindrical expansion of the apex of the germ tube to swelling growth. Effects of CA on the organization of actin were examined with rhodamine-phalloidin (Rh-Ph), which specifically stains F-actin. In CA-untreated cells, Rh-Ph staining resulted in condensed dot-like fluorescence at the growing tip, as well as filamentous fluorescence (actin cables) that ran from the apex to the basal region. In CA-treated cells, condensed dot-like fluorescence was still observed at the swelling tip, but actin cables had disappeared completely. This result indicates that CA does not affect the asymmetrical distribution of actin, and suggests that the actin cables are not required for maintenance of the polarized localization of actin. Benomyl, an anti-microtubule drug, inhibited the germination of yeast cells and the apical growth of germinated cells. Benomyl not only disrupted microtubules (MTs), but also affected the distribution of actin. In benomyl-treated cells, actin dots were randomly dispersed all over the cell. This result indicates that benomyl destroyed the mechanism that maintains the asymmetrical distribution of actin, and suggests that MTs are involved in such a mechanism. The polarized localization of organelles is one of the most important factors associated with dimorphism.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular Approaches in the Diagnosis of Dermatophytosis

Dermatophytosis is one of the most common infectious diseases in the world and can be caused by several dermatophyte species. These species are closely related in genetic structure in spite of different phenotypic and ecological features. The morphological similarity, variability, and polymorphism of dermatophytes have meant that species identification for dermatophytes is time consuming and requires a significant degree of knowledge and technological expertise. Molecular biology-based techniques have solved problems concerning the morphology-based identification of dermatophytes and have improved our knowledge on the epidemiology of dermatophytosis. Further development of molecular diagnosis of dermatophytosis requires the investigation of additional molecular markers for diagnostic tools targeting multiple loci as well as the improvement of techniques.

PCR-based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes

For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR‐based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCR‐based identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials. Copyright

Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments.

Four strains of aerobic, mesophilic, acidophilic bacteria that produced bacteriochlorophyll (BChl) a were isolated from acidic hot springs and mine drainage. The characteristics of the four isolates were almost identical. The isolates were strictly aerobic and chemo-organotrophic. They were gram-negative, non-motile cocci and coccobacilli, formed salmon-pink colonies on solidified media and produced BChl a and carotenoids only under aerobic growth conditions. The cells also produced small amounts of zinc-substituted BChl a when grown in the presence of 1 mM zinc sulfate. Anaerobic growth in the light was not found, but aerobic growth was stimulated by continuous incandescent illumination. The isolates grew in a pH range of 3.5-6.0, with pH optima of 4.5-5.0. A phylogenetic analysis based on 16S rDNA sequences showed that the isolates clustered in the major acidophilic group of the class Proteobacteria, which includes species of the genera Acidiphilium and Rhodopila. The anaerobic phototrophic bacterium Rhodopila globiformis was the closest relative to the new isolates (95% level of sequence similarity). The G+C content of the genomic DNA of the isolates was 69.1-69.8 mol%. On the basis of these results, it was concluded that the four isolates should be classified into a new genus and a new species, for which the name Acidisphaera rubrifaciens is proposed. The type strain is strain HS-AP3T (= JCM 10600T).

High frequency variation of colony morphology and chromosome reorganization in the pathogenic yeast Candida albicans

A clinical isolate of the pathogenic yeast Candida albicans varied in its colony morphology from smooth (o-smooth) to semi-rough type (SRT) and concomitantly lost its virulence for mice. In terms of DNA content, the smooth parent was near triploid when Saccharomyces cerevisiae strains of known ploidy were used as references. The SRT variant showed several features characteristic of polyploidy. From the SRT variant, revertant-like smooth (r-smooth) variants with recovered virulence were derived at a frequency of 5 x 10(-3). The results of pulsed-field gel electrophoresis on chromosomal DNA showed changes in patterns of chromosome-sized DNA bands in the SRT variant as well as in r-smooth variants, which correlated with these variations. Correlations between colony morphology, state of ploidy and virulence of this asporogenous yeast are considered.

Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes

BACKGROUND We have focused on the DNA topoisomerase II genes of pathogenic fungi and have previously applied polymerase chain reaction (PCR)-based identification of several species including the some of the major dermatophyte species. OBJECTIVE To identify the dermatophytes (18 species) to a species level by PCR and PCR-restriction fragment length polymorphism (RFLP) techniques, without determining the nucleotide sequence. METHODS The genomic DNAs of the dermatophytes (ten species of Trichophyton, seven species of Microsporum, and Epidermaphyton floccosum) were amplified by PCR using a common primer set (dPsD1) for the dermatophytes, followed by nested PCR using other primer sets (dPsD2, PsT and PsME) that contained primers specific for the DNA topoisomerase II genes of the dermatophytes. PCRs using PsT and PsME were used for the species-identification of Trichophyton, Microsporum and E. floccosum. The PCR products generated by dPsD2 were digested with restriction enzymes (Hinc II, Hinf, Afl II and PflM I), and the restriction profiles were analyzed. RESULTS Of the eighteen species of dermatophytes, five species (T. rubrum, T. violaceum, M. canis, M. gypseum and E. floccosum) were specifically identified by the PCR using PsT and PsME to the species level, and the remaining species were identified by the unique restriction profiles for each species in the PCR-RFLP analysis, except that the restriction profile of T. mentagrophytes var. interdigitale was identical to that of T. mentagrophytes var. quinckeanum. CONCLUSION PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene are simple and rapid, and quite useful as tools for the identification of dermatophytes to the species level.

Disruption of the human pathogenic yeast Candida albicans catalase gene decreases survival in mouse-model infection and elevates susceptibility to higher temperature and to detergents.

Catalase‐deficient strains of the human pathogenic yeast Candida albicans were constructed using the URA‐blaster method. The disruptant was viable and grew normally in an ordinary culture condition, but became extremely sensitive to treatment with hydrogen peroxide. No catalase activity was observed in a catalase (CCT)‐gene‐disrupted strain, 1F5‐4‐1, suggesting that there were no other catalase or catalase‐like enzymes in this yeast. The disruptant was shown to be sensitive to higher temperature and to low concentrations of SDS, NP‐40, or Triton X‐100. After a wild‐type CCT gene was reintroduced into the disruptant, catalase activity was restored and the strain became moderately sensitive to treatment with hydrogen peroxide. However, neither the temperature sensitivity nor the susceptibility to SDS observed in the disruptant was restored in the CCT‐reintroduced strain. A model infection experiment using wild‐type and dCCT strains showed that the disruptants disappeared more rapidly than the wild‐type strain in mouse liver, lung, and spleen. These results suggest that the catalase plays a significant role in survival in the host immune system and thus leads this organism to establish infection in the host.

PCR-based identification of common dermatophyte species using primer sets specific for the DNA topoisomerase II genes

BACKGROUND We have determined nucleotide sequences of the DNA topoisomerase II genes of the dermatophyte species, and conducted a PCR-based identification system using species-specific primers for the nucleotide sequences. OBJECTIVE To identify the major dermatophytes, Trichophyton rubrum, T. mentagrophytes, T. violaceum, M. gypseum, M. canis and E. floccosum, by PCR amplifications at the species level, without determining the nucleotide sequence. METHODS For PCR-based identification of the major dermatophyte species, a common primer set (dPsD1) for these species and species-specific primer sets (PsT and PsME) for each species were designed based on the genomic sequences of the DNA topoisomerase II genes of the dermatophytes, and tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by the common primer set, followed by a second PCR with the primer sets consisting of species-specific primers for each dermatophyte species. RESULTS Using dPsD1, a DNA fragment of 3390 bp was amplified from the genomic DNA of all the dermatophyte species. In the subsequent nested PCR using species-specific primer sets (PsT and PsME), both sets amplified unique sizes of PCR products, all of which corresponded to a species of the dermatophytes even in the presence of other fungal DNA. CONCLUSION We demonstrate that the PCR-based identification targeting the DNA topoisomerase II gene is rapid and simple, and is available as a tool for the identification of the major dermatophyte species.

Detection of IgE antibody against Candida albicans enolase and its crossreactivity to Saccharomyces cerevisiae enolase

Candida albicans 46 kDa protein, a glycolytic enolase enzyme, is an important allergen of the yeast. The purpose of the study was to detect circulating IgE and IgG antibodies against C. albicans enolase (CAE). We isolated CAE using sequential DEAE Sephacel and Pl 1 column chromatography from spheroptasts of C. albicans, and delected IgE and IgG antibody against CAE by immunoblotting. Crossreactivity of enolose of C. albicans and Saccharomyces cerevisiae was also examined by immunoblotting and immunoblot inhibition test. Among 54 sera with positive IgE RAST to C. albicans, IgE antibody against CAE was detected in 20 sera (37%) and IgG antibody in 27 sera (50%). The allergenic potency of CAE was confirmed using a skin‐prick test in three patients. Simultaneous IgE binding to S. cerevisiae enolase was only observed in four out of 20 sera reacting to CAE. Pre‐treatment of sera with CAE completely inhibited IgE binding to S. cerevisiae enolase. Whereas the latter only partially inhibited IgE binding to CAE. These results suggest that CAE shares some crossreacting epitopes with S. cerevisiae enolase, representing minor components of CAE but dominant segments of S. cerevisiae enolase.