Changes in glycogen concentration and gene expression levels of glycogen-metabolizing enzymes in muscle and liver of developing masu salmon.

Glycogen, as an intracellular deposit of polysaccharide, takes important roles in energy balance of many animals. In fish, however, the role of glycogen during development is poorly understood. In the present study, we assessed changes in glycogen concentration and gene expression patterns of glycogen-metabolizing enzymes in developing masu salmon (Oncorhynchus masou masou), a salmonid species inhabiting west side of North Pacific Ocean. As we measured glycogen levels in the bodies and yolk sacs containing the liver separately, the glycogen concentration increased in both parts as the fish developed, whereas it transiently decreased in the yolk sac after hatching, implying glycogen synthesis and breakdown in these tissues. Immunofluorescence staining using anti-glycogen monoclonal antibody revealed localization of glycogen in the liver, muscle and yolk syncytial layer of the pre-hatching embryos and hatched larvae. In order to estimate glycogen metabolism in the fish, the genes encoding homologs of glycogen synthase (gys1 and gys2) and glycogen phosphorylase (pygma, pygmb and pygl) were cloned, and their expression patterns were assessed by quantitative PCR and in situ hybridization. In the fish, gys1 and gys2 were robustly expressed in the muscle and liver, respectively. Also, expression of pyg isoforms was found in muscle, liver and yolk syncytial layer during hatching. With changes in glycogen concentration and expression patterns of relevant genes, our results suggest, for the first time, possible involvement of glycogen in energy balance of fish embryos, especially during hatching.

Assessment of the timing and degree of smolt development in southern populations of masu salmon Oncorhynchus masou.

The present study assessed whether non-anadromous masu salmon Oncorhynchus masou in Miyazaki, southern Japan, smoltify, and if so, at what time of the year. Yearling O. masou of Miyazaki and an anadromous population from Hokkaido, northern Japan, were reared in hatcheries in their respective regions and sampled monthly from February to June to examine the spring smoltification period. The Hokkaido population showed a peak of gill Na+ -K+ -ATPase (NKA) activity in May, which was accompanied with an increase in mRNA levels of the seawater (SW)-type NKA alpha subunit, nkaα1b. Increases in gill NKA activity and nkaa1b levels were not seen in Miyazaki populations. Transferring yearling Miyazaki population to 70% SW (salinity of 23) in mid-April resulted in an increased serum osmolality over 4 days. These results suggest that they do not smoltify in their second spring. Next, profiles of gill NKA activity and its subunit mRNA levels in under-yearling Miyazaki population in the autumn were examined. Two phenotypes differing in body color during this period were categorized as parr and smolt-like fish. Smolt-like fish had higher gill NKA activity than parr in December while there was no significant difference in gill nkaα1b levels. Smolt-like fish acclimated to 70% SW better than parr as judged by lower serum osmolality. However, serum osmolality in smolt-like fish did not return to the basal level 7 days after transfer to 70% SW, suggesting that their hypo-osmoregulatory ability was not fully developed to a level comparable to anadromous populations of this species. The present study suggests that, if O. masou in Miyazaki go though a smoltification process, it occurs in its first autumn instead of the second spring and is less pronounced compared with anadromous populations.

Inquiring about insomnia may facilitate diagnosis of depression in the primary care setting.

AIM: Depression is often undiagnosed in primary care. Asking about sleep status is much easier than asking about mood. This study was conducted to examine the relation between insomnia and depression. METHODS: New patients aged 35-64 years were recruited from internal medicine clinics in Japan. Self-administered questionnaires were employed. Depression was evaluated by the Zung Self-Rating Depression Scale and the Profile of Mood States. Sleep status was investigated using the Pittsburgh Sleep Quality Index. Likelihood ratios of insomnia for depression were calculated. To assess the relation between insomnia and depression independent of confounding factors, adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated using multiple logistic regression analyses. RESULTS: Among 598 subjects, 153 (25.6%) were assessed as having depression. 'Very bad sleep quality, with difficulty falling asleep within 30 min ≥3 times/week' showed a positive likelihood ratio of 20.36 (95%CI, 2.53-164) while 'not very good sleep quality' had a negative likelihood ratio of 0.32 (95%CI, 0.14-0.72). Adjusted for sex, age, underlying diseases, major life events, lifestyle habits, and relationship problems, significant OR for depression were observed for 'difficulty falling asleep within 30 min ≥3 times/week' (OR, 2.53; 95%CI, 1.07-5.98), 'waking up in the middle of the night or early morning ≥3 times/week' (OR, 3.09; 95%CI, 1.58-6.05), and 'fairly bad sleep quality' (OR, 3.65; 95%CI, 1.34-9.96). CONCLUSION: Inquiring about the weekly frequency of difficulty 'falling asleep within 30 min,' 'waking up in the middle of the night or early morning,' and 'sleep quality' may help to diagnose depression.

Molecular cloning of cytochrome P450 side-chain cleavage and changes in its mRNA expression during gonadal development of brown hagfish, Paramyxine atami.

Since hagfishes are considered the most primitive vertebrate known, extant or extinct, studies on their reproduction are indispensable for understanding phylogenetic aspects of vertebrate reproduction. However, little information is available on the endocrine regulation of the gonadal function in the hagfish. Based on EST analysis of the testis of the brown hagfish (Paramyxine atami), P450 side chain cleavage (CYP11A), which is the first and essential enzyme for steroidogenesis in jawed vertebrates, was cloned. The deduced amino acid sequence of hagfish CYP11A shows high identity to other animal forms especially in two functional domains, adrenodoxin binding domain and heme-binding domain. In the phylogenetic analysis, hagfish CYP11A forms a clade with the vertebrate CYP11A. Following the real-time PCR analysis, CYP11A mRNA expression levels were clearly correlated to the developmental stages of gonads in both sexes of the brown hagfish. By in situ hybridization, CYP11A mRNA signals were found in the theca cells of the ovarian follicles and Leydig cells and the tubule-boundary cells of the testis. These molecular and histological evidences are suggesting that CYP11A plays functional roles as a steroidogenic enzyme in gonadal development. Moreover, native GTH purified from hagfish pituitary stimulated the transcriptional levels of CYP11A in the organ-cultured testis in vitro, clearly suggesting that the steroidogenic activity of the hagfish is under the control of the pituitary GTH. It is suggested that vertebrates, during their early evolution, have established the pituitary-gonadal reproductive system.

Gluconeogenesis in the extraembryonic yolk syncytial layer of the zebrafish embryo.

Yolk-consuming (lecithotrophic) embryos of oviparous animals, such as those of fish, need to make do with the maternally derived yolk. However, in many cases, yolk possesses little carbohydrates and sugars, including glucose, the essential monosaccharide. Interestingly, increases in the glucose content were found in embryos of some teleost fishes; however, the origin of this glucose has been unknown. Unveiling new metabolic strategies in fish embryos has a potential for better aquaculture technologies. In the present study, using zebrafish, we assessed how these embryos obtain the glucose. We employed stable isotope (13C)-labeled substrates and injected them to the zebrafish embryos. Our liquid chromatography-mass spectrometry-based isotope tracking revealed that among all tested substrate, glutamate was most actively metabolized to produce glucose in the zebrafish embryos. Expression analysis for gluconeogenic genes found that many of these were expressed in the yolk syncytial layer (YSL), an extraembryonic tissue found in teleost fishes. Generation 0 (G0) knockout of pck2, a gene encoding the key enzyme for gluconeogenesis from Krebs cycle intermediates, reduced gluconeogenesis from glutamate, suggesting that this gene is responsible for gluconeogenesis from glutamate in the zebrafish embryos. These results showed that teleost YSL undergoes gluconeogenesis, likely contributing to the glucose supplementation to the embryos with limited glucose source. Since many other animal lineages lack YSL, further comparative analysis will be interesting.

Relationships between plasma concentrations of sex steroid hormones and gonadal development in the brown hagfish, Paramyxine atami.

The relationship between sex steroid hormone profiles in plasma and gonadal function in hagfish is poorly understood. In the present study, plasma concentrations of estradiol, testosterone, and progesterone were examined with respect to gonadal development, sexual differences, and possible function of atretic follicles in the brown hagfish, Paramyxine atami, using a time-resolved fluoroimmunoassay. Plasma concentrations of these three hormones were low in juveniles of both sexes. In females, plasma estradiol showed a significant correlation with ovarian development, with the highest concentrations in late vitellogenic adults. Plasma testosterone and progesterone also increased significantly in non-vitellogenic adult females; however, plasma testosterone showed no significant differences among adult females at different ovarian developments, while plasma progesterone was significantly lower in late vitellogenic adults than it was in non-vitellogenic adults. Vitellogenic females that possessed atretic follicles showed significantly lower concentrations of all three hormones than females that only possessed normal follicles. In males, no significant differences were found in plasma estradiol or testosterone levels among groups of different developmental stages of the testis, while plasma progesterone showed a clear inverse relationship with testicular development. Thus, differences were found in plasma sex steroid hormone profiles between male and female P. atami. Moreover, plasma estradiol showed a significant correlation with ovarian development, which suggests that estradiol is involved in the regulation of ovarian development. The present study also revealed that steroid hormone production was strongly suppressed in females that possessed atretic follicles in their ovaries.

Effects of estradiol or testosterone treatment on expression of gonadotropin subunit mRNAs and proteins in the pituitary of juvenile brown hagfish, Paramyxine atami.

A single functional gonadotropin (GTH) comprising two subunits, α and ß, was recently identified in the pituitary of brown hagfish (Paramyxine atami). Little is known about the feedback mechanisms that regulate these GTH subunits by sex steroids in the hagfish. The present study was designed to examine feedback effects of estradiol and testosterone on mRNA expression and protein expression of both GTHα and GTHß subunits in the pituitary of the juvenile P. atami. Intraperitoneal administration of estradiol over the course of 27days resulted in substantial accumulation of immunoreactive (ir)-GTHα and ir-GTHß in the adenohypophysis, but testosterone treatments over 27days had no effects on ir-GTHα or ir-GTHß. Estradiol treatment for 1, 2, 4 or 14days had no effect on GTHα mRNA levels. In contrast, after 2days of estradiol treatment, GTHß mRNA levels had increased significantly from baseline, while these levels were not affected after treatment over 1, 4, or 14days. After 14days of testosterone treatment, both GTHα and GTHß mRNA levels had decreased significantly from baseline levels. These results indicate that estradiol acted primarily to suppress the secretion of GTH, and hence resulted in the accumulations of ir-GTHα and ir-GTHß in the pituitary. On the other hand, testosterone appeared to suppress both the synthesis and the secretion of GTH. Thus, estradiol and testosterone probably differ in their effects on the regulation of pituitary GTH synthesis and secretion in juvenile hagfish.

How histopathology can contribute to an understanding of defense mechanisms against cryptococci.

Invasive fungal infections, particularly those considered opportunistic, have become a common and significant complication of procedures performed in advanced contemporary medicine. Among such infections, cryptococcosis, which is usually caused by infection with Cryptococcus neoformans and Cryptococcus gattii, is particularly problematic because this fungal infection occurs in immunocompromised and apparently immunocompetent individuals. It has been largely accepted that Cryptococcus species are recognized by cellular receptors and that Th1-type immune responses play an important role in defense mechanisms against the yeast. However, the interaction between the yeast and host tissue varies depending on the characteristics of the yeast and the immune status of the host. To gain a better understanding of the pathophysiology of cryptococcosis, we wish to emphasize the usefulness of histopathological examinations, because it allowed more detailed information of an extremely complex interaction between the causative yeasts and tissue response. In the present review, we describe the pathophysiology of cryptococcosis as largely revealed in our previous histopathological investigations of the experimental infection.

Evolutionary origin of a functional gonadotropin in the pituitary of the most primitive vertebrate, hagfish.

Hagfish, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Hagfish have long been the enigma of vertebrate evolution not only because of their evolutionary position, but also because of our lack of knowledge on fundamental processes. Key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Here, the presence and identity of a functional glycoprotein hormone (GPH) have been elucidated from the brown hagfish Paramyxine atami. The hagfish GPH consists of two subunits, alpha and beta, which are synthesized and colocalized in the same cells of the adenohypophysis. The cellular and transcriptional activities of hagfish GPHalpha and -beta were significantly correlated with the developmental stages of the gonad. The purified native GPH induced the release of gonadal sex steroids in vitro. From our phylogenetic analysis, we propose that ancestral glycoprotein alpha-subunit 2 (GPA2) and beta-subunit 5 (GPB5) gave rise to GPHalpha and GPHbeta of the vertebrate glycoprotein hormone family, respectively. The identified hagfish GPHalpha and -beta subunits appear to be the typical gnathostome GPHalpha and -beta subunits based on the sequence and phylogenetic analyses. We hypothesize that the identity of a single functional GPH of the hagfish, hagfish GTH, provides critical evidence for the existence of a pituitary-gonadal system in the earliest divergent vertebrate that likely evolved from an ancestral, prevertebrate exclusively neuroendocrine mechanism by gradual emergence of a previously undescribed control level, the pituitary, which is not found in the Protochordates.

Glycoprotein hormone in the pituitary of hagfish and its evolutionary implications.

The pituitary gland is present in all vertebrates, from agnathans (jawless vertebrates) to mammals, but not in invertebrates. Reproduction in gnathostomes (jawed vertebrates) is controlled by two pituitary gonadotropins (GTHs), luteinizing hormone and follicle-stimulating hormone, which are part of the pituitary glycoprotein hormone (GPH) family. Hagfishes, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Accordingly, they are of particular importance in understanding the evolution of the pituitary GPHs and their functions related to vertebrate reproduction. Nevertheless, key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Our current report has revealed the first identification of a functional GPH composed of two subunits that possess gonadotropic action at the pituitary of brown hagfish. It seems most likely that an ancestral GPH gave rise to only one GTH in hagfish pituitary and that multiplicity of GPHs arose later during the early evolution of gnathostomes. This paper briefly summarizes the latest findings on the hagfish GPH from an evolutionary point of view.

Fatigue on Waking, Insomnia, and Workplace Relationship Problems May Help to Detect Suicidal Ideation among New Middle-Aged Primary Care Patients: A 6-Month Prospective Study in Japan.

Signs of suicidal depression often go undetected in primary care settings. This study explored predictive factors for depression with suicidal ideation (DSI) among middle-aged primary care patients at 6 months after an initial clinic visit. New patients aged 35-64 years were recruited from internal medicine clinics in Japan. Baseline characteristics were elicited using self-administered and physician questionnaires. DSI was evaluated using the Zung Self-Rating Depression Scale and the Profile of Mood States at enrollment and 6 months later. Multiple logistic regression analysis was conducted to calculate adjusted odds ratios for DSI. Sensitivity, specificity, and likelihood ratios for associated factors were calculated. Among 387 patients, 13 (3.4%) were assessed as having DSI at 6 months. Adjusted for sex, age, and related factors, significant odds ratios for DSI were observed for "fatigue on waking ≥1/month" (7.90, 95% confidence intervals: 1.06-58.7), "fatigue on waking ≥1/week" (6.79, 1.02-45.1), "poor sleep status" (8.19, 1.05-63.8), and "relationship problems in the workplace" (4.24, 1.00-17.9). Fatigue on waking, sleep status, and workplace relationship problems may help predict DSI in primary care. Because the sample size in this investigation was small, further studies with larger samples are needed to confirm our findings.

Correlation of in vitro activity and in vivo efficacy of itraconazole intravenous and oral solubilized formulations by testing Candida strains with various itraconazole susceptibilities in a murine invasive infection.

OBJECTIVES: To examine whether in vitro antifungal susceptibility test results correlate with in vivo efficacy of two cyclodextrin-solubilized itraconazole formulations (intravenous and oral) against Candida in a murine model of invasive infection. METHODS: A selected set of 12 Candida spp. strains with various itraconazole susceptibilities were tested. We studied the efficacy of intravenous and oral itraconazole administered once daily at dosages of 0.63, 2.5, 10 and 40 mg/kg body weight in mice lethally infected with each tested strain. Survival of mice in each treated group was monitored daily until the death of all control mice and compared between groups. RESULTS: Survival of mice infected with 9 of 12 Candida strains with itraconazole MICs of ≤0.016-2.0 mg/L was significantly prolonged by treatment with intravenous itraconazole at dosages of 2.5 or 10 mg/kg and above. In contrast, the other three strains resistant to 8 mg/L itraconazole in vitro were refractory to the therapy, even at the highest itraconazole dosage (40 mg/kg). Closely similar in vivo data were obtained with the oral itraconazole therapy. The effective doses of the two itraconazole formulations increased with increasing itraconazole MICs for the infecting strains. CONCLUSIONS: The in vivo efficacy of intravenous and oral itraconazole correlated with the in vitro susceptibility data.

Photoperiodic regulation of pituitary thyroid-stimulating hormone and brain deiodinase in Atlantic salmon.

Seasonal timing is important for many critical life history events of vertebrates, and photoperiod is often used as a reliable seasonal cue. In mammals and birds, it has been established that a photoperiod-driven seasonal clock resides in the brain and pituitary, and is driven by increased levels of pituitary thyroid stimulating hormone (TSH) and brain type 2 iodothyronine deiodinase (DIO2), which leads to local increases in triiodothyronine (T3). In order to determine if a similar mechanism occurs in fish, we conducted photoperiod manipulations in anadromous (migratory) Atlantic salmon (Salmo salar) that use photoperiod to time the preparatory development of salinity tolerance which accompanies downstream migration in spring. Changing daylength from short days (light:dark (LD) 10:14) to long days (LD 16:8) for 20 days increased gill Na+/K+-ATPase (NKA) activity, gill NKAα1b abundance and plasma growth hormone (GH) levels that normally accompany increased salinity tolerance of salmon in spring. Long-day exposure resulted in five-fold increases in pituitary tshßb mRNA levels after 10 days and were sustained for at least 20 days. tshßb mRNA levels in the saccus vasculosus were low and not influenced by photoperiod. Increased daylength resulted in significant increases in dio2b mRNA levels in the hypothalamus and midbrain/optic tectum regions of the brain. The results are consistent with the presence of a photoperiod-driven seasonal clock in fish which involves pituitary TSH, brain DIO2 and the subsequent production of T3, supporting the hypothesis that this is a common feature of photoperiodic regulation of seasonality in vertebrates.

Changes in gene expression levels of somatolactin in the pituitary and morphology of gill mitochondria-rich cells in Mozambique tilapia after transfer to acidic freshwater (pH 3.5).

Mozambique tilapia, Oreochromis mossambicus, is easily acclimated to highly acidic water, and thus presents a useful model to unravel endocrine regulation of adaptation to acidic water in fish. We analyzed gene expression of somatolactin (sl), growth hormone (gh) and prolactin (prl), in the pituitary gland and size distribution of mitochondria-rich (MR) cells in the gills after transfer from normal freshwater (FW, pH 7.2) to acidified freshwater (AW, pH 3.5). Plasma osmolality drastically decreased until 2 days after transfer to AW, but had restored to normal after 1 week of acclimation, and this confirmed the excellent acid tolerance of tilapia. Expression levels of sl, gh and prl were all up-regulated during short-term exposure to AW. The expression of sl remained elevated up to 7 days after transfer; the expression of gh and prl was back to initial levels at that time. These findings point to an important and specific role of SL in adaptation to acid water in this tilapia, although temporal contribution of GH and PRL cannot be ruled out. The size distribution of branchial MR cells changed drastically during acclimation to AW. The mean MR cell size was 1.5-fold larger in the fish exposed to AW for 7 days compared to controls in FW. The gills and their MR cells are a likely site of important acid-base regulation, and SL may change ion-transport functions of MR cells to correct plasma osmotic balance disturbed by acid exposure.

cDNA cloning and isolation of somatolactin in Mozambique tilapia and effects of seawater acclimation, confinement stress, and fasting on its pituitary expression.

Somatolactin (SL) is a member of the growth hormone (GH)/prolactin (PRL) family of pituitary hormones, and is found in a variety of teleost species. Somatolactin is thought to be involved in a wide range of physiological actions, including reproduction, stress response, the regulation of Ca(2+) and acid-base balance, growth, metabolism, and immune response. We report here on the cDNA structure of SL from the pituitary of Mozambique tilapia, Oreochromis mossambicus, and its gene expression in response to seawater acclimation, stress, and fasting. Tilapia SL cDNA (1573bp long) encoded a prehormone of 230 amino acids. Sequence analysis of purified SL revealed that the prehormone is composed of a signal peptide of 23 amino acids and a mature protein of 207 amino acids, which has a possible N-glycosylation site at position 121 and seven Cys residues. Tilapia SL shows over 80% amino acid identity with SLalpha of advanced teleosts such as medaka and flounder, and around 50% identity with SLbeta of carp and goldfish. Acclimation to seawater had no effect on pituitary expression of SL or on hepatic expression of the putative tilapia SL receptor (GHR1). By contrast, seawater acclimation resulted in significant increases in pituitary GH expression and in hepatic expression of tilapia GH receptor (GHR2). Confinement stress had no effect on pituitary expression of either SL or GH, or on hepatic expression of GHR1, whereas a significant increase was seen in GHR2 expression in the liver. Fasting for 4 weeks resulted in significant reductions in SL transcripts both in fresh water and seawater. It is highly likely that SL is involved in metabolic processes in tilapia along with the GH/IGF-I axis.

Histopathological study on experimental endophthalmitis induced by bloodstream infection with Candida albicans.

To investigate the details of the pathophysiology of endogenous fungal endophthalmitis (EFE), we performed sequential histological and ophthalmoscopic examination on a rabbit model comparing immunocompromised EFE developed using a steroid with an immunocompetent one intravenously inoculated with Candida albicans. The ophthalmoscopic examination and histological analysis of the retina in both groups demonstrated that lesions appear on the equator of the eyeball and then spread toward the posterior pole. It has been speculated that, because of the unique innate vasculature system of the equator, there is a sudden, decrease of shear stress in rheologically, resulting in adhesion of yeast cells to the endothelial cells. Histological examination revealed that the degree of polymorphonuclear leukocyte (PMN) infiltration was equivalent in the two groups. However, the appearance of PMN was delayed and the number of fungi was higher in the state of hyphae and/or pseudohyphae in the steroid-treated group. Furthermore, the eyeball was found to be the second earliest organ involved in candidemia. Our results indicate that ophthalmic examination is useful to monitor the development and systemic involvement of endophthalmitis in patients with candidemia.

Combined effect of heat, essential oils and salt on fungicidal activity against Trichophyton mentagrophytes in a foot bath.

This work was originally undertaken to determine the effective conditions of essential oils against Trichophyton mentagrophytes in vitro for the treatment of tinea pedis in a foot bath. Agar blocks implanted with T. mentagrophytes were immersed in 0.1% aqueous agar containing two-fold dilutions of essential oils with or without sodium chloride at 27 degrees C, 37 degrees C and 42 degrees C for 10 and 20 min. The number of surviving mycelia on the agar blocks was determined from the standard curves of the colony diameter and original inocula of the conidia. At the same time, the thermal effect on the cellular morphology was examined using SEM. Most fungal mycelia (99.7%) were killed after treatment at 42 degrees C for 20 min without essential oil. The fungicidal activity of essential oils was markedly enhanced by treating at 42 degrees C for 20 min as compared with that at 27 degrees C, showing 1/4 - 1/32-fold reduction of minimum fungicidal concentration (MFC to kill 99.99%). The order of the fungicidal activity of 11 essential oils was oregano, thyme thymol, cinnamon bark > lemongrass > clove, palmarose, peppermint, lavender > geranium Bourbon, tea tree > thyme geraniol oils. MFCs were further reduced to 1/2 - 1/8 by the addition of 10% sodium chloride. The salt effect was explained, at least partly, by an increase in mycelial adsorption of antifungal constituents in the presence of sodium chloride. Considerable hyphal damage was done at 27 degrees C by the essential oils, but no further alteration in morphology of the hyphae treated at 42 degrees C with or without oil was observed by SEM. The inhibitory effect of heat and oils was also observed against mycelia of T. rubrum and conidia of T. mentagrophytes. Thermotherapy combined with essential oils and salt would be promising to treat tinea pedis in a foot bath.

Rapid identification of Trichophyton tonsurans by specific PCR based on DNA sequences of nuclear ribosomal internal transcribed spacer (ITS) 1 region.

BACKGROUND: Trichophyton tonsurans, a dermatophyte implicated in an international epidemic of tinea capitis, was also found to be responsible for infecting wrestling and Judo athletes nationwide in Japan since 2001. OBJECTIVE: A rapid and highly accurate means of identifying this pathogen has been required to control the infection. We have developed a T. tonsurans-specific PCR method based on the DNA sequences of the nuclear ribosomal internal transcribed spacer 1 region. SUBJECTS: Eighteen species of six genera of standard strains and 75 strains of clinically isolated Trichophyton species were used in this study. METHODS: A T. tonsurans-specific PCR primer pair (tonsF1 and tonsR1) was designed on the nuclear ribosomal internal transcribed spacer 1 region, located between 18S and 5.8S rDNA. Fungal DNA was extracted from the colonies grown on culture plates, and the specificity of the PCR primers was tested. RESULTS: The specific PCR product was amplified from the standard strain of T. tonsurans and from five strains isolated from black dot ringworms, but there was no band from the 70 clinical isolates of other Trichophyton species. This T. tonsurans-specific PCR method was able to detect 10 pg of T. tonsurans genomic DNA with ethidium bromide staining. CONCLUSIONS: A PCR identification system specific for T. tonsurans is rapid, sensitive, and specific.

[Studies on the correlation of the method currently used by the Japanese Society for Medical Mycology and its modified method with the NCCLS M27-A2 microdilution method for azole susceptibility testing of Candida species].

To evaluate the currently used Japanese Society for Medical Mycology (JSMM) method for testing the azole susceptibility of yeasts, the activities of fluconazole and itraconazole were tested against recently collected clinical isolates of Candida spp. (n=946) and compared with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 microdilution reference method. Favorable correlation with the M27-A2 method was not seen for isolates of C. albicans, C. tropicalis or other Candida spp., particularly their trailing-growth isolates. However, the degree of correlation and agreement of MIC values were markedly improved when testing was performed by the modified JSMM method in which the end-point to be read was changed from IC80 (for the current JSMM method) to IC50. These results suggest that there is an urgent need to revise the current JSMM method.

A novel method to estimate the contribution of the vapor activity of essential oils in agar diffusion assay.

By the combined use of agar diffusion, agar vapor and agar vapor-inhibitory assays, contribution of the vapor activity of essential oils was quantitatively estimated. The test organisms were Trichophyton mentagrophytes and Aspergillus fumigatus. Agar vapor assay was used to confirm the vapor activity of the oils. The parameter delta defined as a contribution index of the vapor activity was calculated by (1 - b-c/a-c) x 100, where a is inhibitory diameter in the diffusion assay, b is inhibitory diameter in the vapor-inhibitory assay and c is diameter of the sealed ring in the vapor-inhibitory assay (21 mm). Many of the essential oils examined showed a delta value near 100, thus providing the major contribution of the vapor activity to the inhibitory diameter. Essential oils containing aldehyde as major constituent showed low delta value, indicating the major inhibition was due to agar diffusion. Major essential oil components behaved similarly; the delta value was increased in the following order: aldehyde < phenol < alcohol < ester, oxide, hydrocarbon, indicating the enhanced contribution of the vapor activity in that order.