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.

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.

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 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.

Variation in the ontogeny of sex steroid levels between latitudinal populations of the medaka.

INTRODUCTION: Sex steroids mediate the expression of sexual dimorphism during ontogeny, and populations that differ in the magnitudes of sexual dimorphism may accordingly differ in the ontogenetic patterns of their sex steroid levels. The medaka, Oryzias latipes species complex, shows geographic variation in the magnitude of sexual dimorphism with respect to the lengths of their anal and dorsal fins; dimorphism is greater in low-latitude populations than in high-latitude populations. However, sexual differences in the ontogenetic dynamics of sex steroids, and its interpopulation variation, have not been examined. RESULTS: We measured testosterone (T), estradiol-17ß (E2), and 11-ketotestosterone (11-KT) concentrations throughout ontogeny of laboratory-reared fish from two latitudinal populations: Aomori (northern) and Okinawa (southern). In both populations, the levels of all three steroids were high during early ontogenetic stages and decreased with growth. After reaching about 15 mm in standard length, when sexual dimorphisms in fin lengths became apparent, steroid levels increased and tended to plateau. Sexual differences in the steroid levels were observed only in the later ontogenetic stages; T and 11-KT levels were higher in males, while E2 levels were higher in females. Accordingly, interpopulation differences also became clearer; the southern fish tended to show higher T levels and lower E2 levels than the northern fish. CONCLUSIONS: The ontogenetic patterns of sex steroid levels paralleled the ontogeny of anal and dorsal fins in the two latitudinal populations, suggesting that interpopulation variation in the degree of sexual dimorphisms in fin lengths is mediated by sex steroid-dependent regulation of fin elongation.

Induction of Phosphoenolpyruvate Carboxykinase (PEPCK) during Acute Acidosis and Its Role in Acid Secretion by V-ATPase-Expressing Ionocytes.

Vacuolar-Type H(+)-ATPase (V-ATPase) takes the central role in pumping H(+) through cell membranes of diverse organisms, which is essential for surviving acid-base fluctuating lifestyles or environments. In mammals, although glucose is believed to be an important energy source to drive V-ATPase, and phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis, is known to be activated in response to acidosis, the link between acid secretion and PEPCK activation remains unclear. In the present study, we used zebrafish larva as an in vivo model to show the role of acid-inducible PEPCK activity in glucose production to support higher rate of H(+) secretion via V-ATPase, by utilizing gene knockdown, glucose supplementation, and non-invasive scanning ion-selective electrode technique (SIET). Zebrafish larvae increased V-ATPase-mediated acid secretion and transiently expression of Pck1, a zebrafish homolog of PEPCK, in response to acid stress. When pck1 gene was knocked down by specific morpholino, the H(+) secretion via V-ATPase decreased, but this effect was rescued by supplementation of glucose into the yolk. By assessing changes in amino acid content and gene expression of respective enzymes, glutamine and glutamate appeared to be the major source for replenishment of Krebs cycle intermediates, which are subtracted by Pck1 activity. Unexpectedly, pck1 knockdown did not affect glutamine/glutamate catalysis, which implies that Pck1 does not necessarily drive this process. The present study provides the first in vivo evidence that acid-induced PEPCK provides glucose for acid-base homeostasis at an individual level, which is supported by rapid pumping of H(+) via V-ATPase at the cellular level.

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.

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.

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.

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.

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.

Body plan of turtles: an anatomical, developmental and evolutionary perspective.

The evolution of the turtle shell has long been one of the central debates in comparative anatomy. The turtle shell consists of dorsal and ventral parts: the carapace and plastron, respectively. The basic structure of the carapace comprises vertebrae and ribs. The pectoral girdle of turtles sits inside the carapace or the rib cage, in striking contrast to the body plan of other tetrapods. Due to this topological change in the arrangement of skeletal elements, the carapace has been regarded as an example of evolutionary novelty that violates the ancestral body plan of tetrapods. Comparing the spatial relationships of anatomical structures in the embryos of turtles and other amniotes, we have shown that the topology of the musculoskeletal system is largely conserved even in turtles. The positional changes seen in the ribs and pectoral girdle can be ascribed to turtle-specific folding of the lateral body wall in the late developmental stages. Whereas the ribs of other amniotes grow from the axial domain to the lateral body wall, turtle ribs remain arrested axially. Marginal growth of the axial domain in turtle embryos brings the morphologically short ribs in to cover the scapula dorsocaudally. This concentric growth appears to be induced by the margin of the carapace, which involves an ancestral gene expression cascade in a new location. These comparative developmental data allow us to hypothesize the gradual evolution of turtles, which is consistent with the recent finding of a transitional fossil animal, Odontochelys, which did not have the carapace but already possessed the plastron.

Characterization of novel RFamide peptides in the central nervous system of the brown hagfish: isolation, localization, and functional analysis.

RFamide (RFa) peptides play various important roles in the central nervous system in both invertebrates and vertebrates. However, there is no evidence of the existence of any RFamide peptide in the brain of hagfish, one of the oldest lineages of vertebrates. In this study, we sought to identify novel RFamide peptides from the brains of hagfish (Paramyxine atami). We identified four novel RFamide peptides, which had the C-terminal Pro-Gln-Arg-Phe-NH2 structure. cDNA cloning revealed that the identified RFamide peptides are encoded in two types of cDNA. Molecular phylogenetic analysis of the two precursors indicated that the hagfish RFamide peptides belong to the PQRFamide peptide group that includes mammalian neuropeptide FF and AF. Based on immunohistochemistry and in situ hybridization, hagfish PQRFamide peptide precursor mRNA and its translated peptides were localized in the infundibular nucleus of the hypothalamus. Immunoreactive fibers were terminated on blood vessels in the infundibular nucleus. Dense immunoreactive fibers were also observed in other brain regions. We further showed that one of the hagfish PQRFamide peptides significantly stimulated the expression of gonadotropin-ß mRNA in the cultured hagfish pituitary. These results indicate that the control mechanism of gonadotropin expression by a hypothalamic neuropeptide evolved in the agnathan brain. This is the first evidence describing the identification of RFamide peptides in the hagfish brain. This is also the first report showing the regulation of gonadotropin expression by a homolog of neuropeptide FF that belongs to the PQRFamide peptide group in any vertebrate.

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.

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.

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.

Comparative study of direct polymerase chain reaction, microscopic examination and culture-based morphological methods for detection and identification of dermatophytes in nail and skin samples.

The positive rates of dermatophytes isolated and identified by conventional methods are rather low. Moreover, clinical isolates sometimes show atypical morphology, and in such cases microscopic methods are not applicable for identification. The present study was performed to assess the utility of specific polymerase chain reaction (PCR)-based methods for Trichophyton rubrum and Trichophyton mentagrophytes as diagnostic tools for dermatophytoses. Both conventional morphological identification and specific PCR methods based on the nuclear ribosomal internal transcribed spacer (ITS)1 DNA sequence were performed to identify dermatophyte species from clinical specimens of patients who visited Kawasaki Social Insurance Hospital between 16 May and 17 August 2005. Specific PCR methods were also directly applied to clinical specimens, and the results of the two methods were compared. The clinical samples examined consisted of 126 skin scale specimens and 80 nail specimens. The positive rates of culture isolation from clinical specimens were 67% and 33% for skin scale and nail specimens, respectively. In contrast, PCR analysis yielded a positive rate of 100% for clinical isolates from both skin scales and nails, and rates of 95% and 99% were obtained by direct application to clinical specimens. The results of the present study indicated that specific PCR is highly advantageous as a diagnostic tool for detection and identification of dermatophytes on direct application to skin scale or nail specimens.