Shigeji Aoki

Fluorescence microscopic studies on mitochondria and mitochondrial nucleoids in a wild-type strain and respiratory mutants of Candida albicans

A wild-type strain and two respiratory mutants of Candida albicans were examined for mitochondria and mitochondrial nucleoids (mt-nucleoids) using the fluorescent dyes, 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) and 4, 6-diamidino-2-phenylindole (DAPI). Rapidly growing cells of the wild type possessed one or a few giant branched mitochondria that were intensely stained with DASPMI. When a bud emerged, an end of the giant mitochondrion extended into the bud and the mitochondrion was divided and partitioned into mother and daughter cells by cytokinesis. Cell cycle-associated fragmentation or fusion of mitochondria were not demonstrated. The mutant KRD-8, that possesses cristate mitochondria but respires at a lower level, was shown to contain one or a few, less stainable giant mitochondria per cell. DASPMI failed to stain cells of the mutant KRD-19 which lacks cytochrome aa3 and cristate mitochondria. About eight and 10 mt-nucleoids were detected as discrete fluorescent spots in DAPI-stained cells of the wild type and KRD-8, respectively. KRD-19 cells also possessed mt-nucleoids, although the number of mt-nucleoids per cell seemed to be smaller than that of the wild type. In all the strains, mt-nucleoids existed discretely throughout the budding cycle, and the increase of their number per cell appeared to correlate with the cellular volume but not with nuclear division.

UV Survival of Human Mycoplasmas : Evidence of Dark Reactivation in Mycoplasma buccale

The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony‐forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony‐forming ability of UV‐irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post‐irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony‐forming units (CFU) of UV‐irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony‐forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV‐irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV‐induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.

Induction of experimental Candida arthritis in rats

Experimental arthritis, caused by intravenously (IV) introduced Candida albicans, has been induced for the first time in rats. Four-week-old Sprague-Dawley rats were inoculated IV with three different strains of C. albicans and observed for 4 weeks. Each of the three strains tested was able to produce arthritis. The incidence of Candida arthritis increased in a dose-dependent manner and was more than 90% at sublethal doses. Joints of the limbs were affected predominantly, and at higher doses arthritis was produced in multiple (four or five) joints in individual animals, showing it to be polyarthritis. C. albicans was recovered from all cultures of affected limb joints, which were excised 12, 19 and 28 days after inoculation and showed different stages and degrees of joint swelling. Results of histopathology and radiography showed that the Candida arthritis involved not only periarticular inflammation but also changes in joint bones. In particular, metaphyseal enlargement, punched-out lesions at the diaphysis and the appearance of osteoclasts were the most prominent changes in affected bones. These pathological features are compared with those of Candida arthritis in humans.

Diversity of laccase among Cryptococcus neoformans serotypes

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra‐serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper‐binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.

Radiographic features of experimentalCandida arthritis in rats

Sprague-Dawley rats were inoculated intravenously (i.v.) withCandida albicans, and limb joints showing signs ofCandida-induced arthritis were subjected to radiographic and histologic examination. New bone formation and bone resorption were morbidly enhanced in bones sampled from the arthritic joints. Sparsely distributed needle-shaped calcified deposits began to be formed on bony surfaces in parallel with the onset of joint swelling. The calcified deposits gradually became denser and then covered the bony surfaces almost entirely, giving rise to an exostosis-like profile. In addition to the new bone formation, bone resorption was also observed in regions adjacent to the sites of new bone formation, and punched-out bone lesions were produced. Eventually, severe deformation of joint bones due to new bone formation and bone resorption was evident. Reflecting these unusual radiographic changes, abundant osteoblasts and osteoclasts were demonstrated histologically in the bones. On the basis of these results, possible mechanisms for the induction of arthritis byCandida infection are discussed.

Analysis of bacterial flora in dohyo soil

ObjectivesSumo wrestling is one of the most popular sports in Japan. Injuries are not uncommon as this is a vigorous contact sport. Sumo wrestlers have little in the way of protective clothing; their main garb is the mawashi, making them prone to exposure to any microorganisms in the dohyo. The bacterial flora of the dohyo has received little attention. If the constituent flora is identified, then appropriate treatment or prevention of any bacterial lesions or infections incurred by the wrestlers is possible.MethodsThe Vitek AMS system used in this study was developed by McDonnell Douglas Corporation. In this system, the physiological and biochemical properties of Gram-positive and negative bacilli, Gram-positive and-negative cocci, and fungi isolated from clinical materials and environments are examined using test cards specifically for each microorganism group, and the results are automatically read by a computer and encoded. Obtained codes are compared with a built-in database, and bacterial species of test strains are identified.ResultsIn this study, using the automatic identification kit VITEK or ATB, we describe the aerobic bacterial flora found in the dohyo over the four seasons of the year. We also investigated the effect of salt on the bacterial flora as sumo wrestlers toss salt on the dohyo before each match. We show the relationship between salinity changes and variations in the flora observed upon the addition of salt. Without salt, at the beginning of a match, Gram-negative bacteria predominate. When salt is added, there is a transient decrease in the incidence of flora followed by an increase in the incidence Grampositive cocci.ConclusionsSixteen bacterial genera were identified using the bacterial identification systems in dohyo soil samples during the year. The number of identified bacterial species was 32. Even in the presence of salt, there is a measurable amount of bacterial flora in dohyo soil; salt does not act as an antibacterial agent.