Onset of nutrient consumption during early life stage digestive system development of two tuna species (Thunnus orientalis and Thunnus albacares).

To specify the timing of exogenous nutrient consumption in the larvae of two commercially important tuna species, the Pacific bluefin tuna (PBF) Thunnus orientalis and the yellowfin tuna (YFT) Thunnus albacares, the gene expressions of peptide transporter 1 (PEPT1) were examined. The mRNA expressions of PEPT1 first occurred at 2 days post hatching (dph) in PBF larvae and 3 dph for the YFT, and PEPT1 was found to only be expressed in the intestinal tract. The histological changes of the digestive tract of the YFT larvae were observed and compared to PBF larvae from a previous study. The intestines were developed at the hatching day for both species. It was found that the developmental timing of internal organs differed between the species, with the YFT showing an approximately one-day delay. The major organs such as liver, pancreas and gall bladder that excrete digestive enzymes appeared at 1 dph for PBF and 2 dph for YFT. The development of external morphological features was similar to organ development timings, with mouth-opening and first feeding starting at 2 dph for PBF, and 3 dph for YFT. Growth during the first month is rapid and variable for both species, ranging from 1.06 to 1.56 mm/d. Our findings provide new information about the early onset of feeding and larval development for the two species which would contribute to future aquaculture.

Phylogenetic study of the genus Kudoa (Myxozoa: Multivalvulida) with a description of Kudoa rayformis sp. nov. from the trunk muscle of Pacific sierra Scomberomorus sierra.

Kudoa rayformis n. sp. (Myxozoa; Multivalvulida) was observed in the trunk muscle of Pacific sierra Scomberomorus sierra caught off the coast of Tonosi, Panama. The species formed pseudocysts in myofibers and infection was subclinical. The myxospores possessed four polar capsules and spore valves, one of which had a distinct filamentous extension. This unique morphological characteristic of the myxospore validated this as a new species of Kudoa. Genetically, K. rayformis n. sp. is closest to K. inornata, with 98% and 91% similarity in 18S and 28S rDNA, respectively, but its spore shape was clearly distinct. The 18S rDNA and concatenated sequences from K. rayformis were used in molecular phylogenetic analyses of kudoids to examine the congruence of phylogeny with infection site tropism, spore morphology and cyst/pseudocyst formation. The results demonstrated that the phenotypic traits were correlated with the phylogeny of Kudoidae, and that the biological features of K. rayformis originated from the ancient Kudoidae as exhibited by the non-specific infection site tropism and the ability to infect muscle and form pseudocysts.

mRNA levels of kisspeptins, kisspeptin receptors, and GnRH1 in the brain of chub mackerel during puberty.

Kisspeptin (Kiss) and its cognate receptor (Kiss1R), implicated in the neuroendocrine control of GnRH secretion in mammals, have been proposed to be the key factors in regulating puberty. However, the mechanisms underlying the initiation of puberty in fish are poorly understood. The chub mackerel Scomber japonicus expresses two forms of Kiss (kiss1 and kiss2) and two Kiss receptor (kissr1 and kissr2) genes in the brain, which exhibit sexually dimorphic changes during the seasonal reproductive cycle. This indicates that the kisspeptin system plays an important role in gonadal recrudescence of chub mackerel; however, the involvement of the kisspeptin system in the pubertal process has not been identified. In the present study, we examined the mRNA expression of kiss1, kiss2, kissr1, kissr2, and gnrh1 (hypophysiotropic form) in the brain of a chub mackerel during puberty. In male fish, kiss2, kissr1 and kissr2 levels increased significantly at 14weeks post-hatch (wph), synchronously with an increase in type A spermatogonial populations in the testis; kiss2 and gnrh1 levels significantly increased at 22wph, just before the onset of meiosis in the testes. In female fish, kiss2 increased significantly at 14wph, synchronously with an increase in the number of perinucleolar oocytes in the ovary; kiss1 and kiss2 levels significantly increased concomitantly with an increase in the kissr1, kissr2, and gnrh1 levels at 24wph, just before the onset of vitellogenesis in oocytes. The present results suggest positive involvement of the kisspeptin-GnRH system in the pubertal process in the captive reared chub mackerel.

Gonadal development and gonadotropin gene expression during puberty in cultured chub mackerel (Scomber japonicus).

Understanding puberty is important for establishing aquaculture in fish. In this study, we analyzed the timing and completion of pubertal development along with changes in pituitary gonadotropin genes (fshb and lhb) in cultured chub mackerel (Scomber japonicus). At 45 days post-hatching (dph), gonadal sex differentiation was observed. The onset of puberty occurred at 192 dph in females with the start of vitellogenesis, whereas it occurred at 164 dph in males, with the beginning of spermatogenesis (proliferation and differentiation of germ cells). The completion of puberty was at 326 dph in females when vitellogenesis completed, and it was at 338 dph in males during spermiation. All fish sampled during the spawning season completed pubertal development. In the pituitary of female fish, fshb expression was activated during early secondary growth and was maintained high throughout vitellogenesis, whereas lhb expression was highest at the completion of vitellogenesis. In male fish, fshb and lhb expression were activated from the onset of spermatogenesis and further activated during late pubertal development; fshb remained high between late spermatogenesis and spermiation, whereas lhb was highest during spermiation.

Vaccine efficacy of Mycobacterium bovis BCG against Mycobacterium sp. infection in amberjack Seriola dumerili.

Mycobacteriosis, caused by the intracellular parasitism Mycobacterium sp., causes economic damages to aquaculture production in Japan, particularly in seriola fish production. Antibiotics are not effective against Mycobacterium sp. and so a potent vaccine is needed. We previously reported that BCG vaccine (Mycobacterium bovis BCG) induces adaptive immunity against Mycobacterium sp. in Japanese flounder, Paralichthys olivaceus. In a phylogenetic tree, the genes for a major antigen, the Ag85 complex, in Mycobacterium sp. TUMSAT-Msp001 are closely related to homologues in Mycobacterium ulcerans. M. bovis BCG was detected until 7 days post-injection at the injection site (muscle) and 28 days post-vaccination in spleen. Cumulative mortality of amberjack, Seriola dumerili vaccinated intramuscularly (i.m.) and intraperitoneally (i.p.) with M. bovis BCG was 32.3% and 59.5% respectively, at 24 days post-infection of Mycobacterium sp., compared to 97.8% in PBS-injected fish. The bacterial counts of Mycobacterium sp. in spleen of both i.m.-and i.p.-vaccinated fish (6.2 x 10³ and 1.3 x 104 CFU/mg tissue, respectively) at 20 days post-infection were significantly lower (P < 0.01) than those of PBS-injected fish (8.0 x 106 CFU/mg). Furthermore, Immersion challenge with Mycobacterium sp. TUMSAT Msp-001 showed 50% RPS value in BCG i.m.-vaccinated fish at the end of the experiment. These results support our previous study using Japanese flounder and suggest that BCG vaccine is also effective against Mycobacterium sp. infection in amberjack.

A close relationship between androgen levels and eumelanogenesis in the teleost red seabream (Pagrus major): Quantitative analysis of its seasonal variation and effects of oral treatment with methyl-testosterone.

Male and female teleost seabream (Pagrus major) were examined for seasonal variation of eumelanin, pheomelanin, 11-ketotestosterone (11KT, fish androgen), lightness (L* value) and Gonad Somatic Index (GSI: gonad mass/body massx100). In males, levels of pyrrole-2,3,5-tricarboxylic acid (a marker of eumelanin), 11KT and the GSI increased sharply from September and plateaued in March and April when the fish are sexually mature. These results are consistent with the lightness of their body color. Using the data from males, a high correlation was observed for all combinations of those four variables (PTCA, 11KT, lightness and GSI). In females, little change was observed in those variables except for the GSI. 4-Amino-3-hydroxyphenylalanine (a marker of pheomelanin) was also analyzed, but it was below the detection limit at all times. Oral treatment of juvenile red seabream with synthetic androgen methyl-testosterone for 2 months induced eumelanin accumulation about 3 times higher than the control. These data show that there is a close relationship between androgen levels and eumelanin accumulation in teleosts. This is the first report that androgen affects melanin accumulation in a dose-dependent manner.

Developing 23 new polymorphic microsatellite markers and simulating parentage assignment in the Pacific bluefin tuna, Thunnus orientalis.

Twenty-three new polymorphic microsatellite markers were isolated in the Pacific bluefin tuna, Thunnus orientalis. Each locus comprised three to 34 alleles. The expected and observed heterozygosities ranged between 0.46 and 0.96 and between 0.44 and 0.97, respectively. The Kto9, Kto11, and Kto42 markers demonstrated significant deviation from Hardy-Weinberg equilibrium; high null allele frequencies (0.08-0.14) were observed in the deviating group. From the results of simulation of parentage assignment, a combination of four loci (i.e. Kto15, Kto23, Kto38, and Kto39) was considered the best for parentage assignment.

Generation of transgenic medaka expressing claudin7-EGFP for imaging of tight junctions in living medaka embryos.

The tight junction (TJ) is a specialized cell-cell adhesion structure in epithelial and endothelial sheets unique to the chordates and functions as a barrier of fluidal diffusion across the cell sheets. In order to study the dynamics of TJ formation in vivo during embryogenesis, we have generated a transgenic medaka line that expresses claudin-7 protein fused to enhanced green fluorescent protein under the regulation of the red seabream beta-actin promoter in transparent medaka embryos. Claudins contain four transmembrane domains and have been identified as the key molecules that dictate the function of TJs. This transgenic medaka line will thus be useful for imaging of TJs in living embryos and hence in screening for mutations affecting cell-cell adhesion.

Application of the arming system for the expression of the 380R antigen from red sea bream iridovirus (RSIV) on the surface of yeast cells: a first step for the development of an oral vaccine.

The cell surface is a functional interface between the inside and the outside of the cell. Moreover, cells have systems for anchoring surface specific proteins and for confining surface proteins to particular domains on the cell surface. For use in bioindustrial processes applied to oral vaccination, we consider that cell-surface display systems must be useful and that the yeast Saccharomyces cerevisiae, the most suitable microorganism for practical purposes, is available as a host for genetic engineering because it can be subjected to many genetic manipulations. In particular, the rigid structure of the cell makes the yeast suitable for several of the applications. In this study, we describe the expression of one of the target antigens, 380R, from the red sea bream iridovirus (RSIV), which is one of the most common viral diseases in the cultured marine fish Pagrus major in Japan, using the arming yeast system and aiming at its application for oral vaccination. We first performed the molecular cloning and expression of the 380R antigen from RSIV in Escherichia coli. The nucleotide sequence of the 380R antigen was composed of an open reading frame (ORF) of 1360 bp encoding a protein of 453 residues. To prepare a specific antibody against the 380R antigen, the recombinant protein was overexpressed and purified in E. coli. As a result of indirect immunofluorescence with the specific antibody, we could observe the expression of the 380R antigen on the surface of the yeast cells. Thus, we have successfully prepared the source of an oral vaccine using cell-surface display technology in yeast.

The histological analysis, colorimetric evaluation, and chemical quantification of melanin content in 'suntanned' fish.

Human melanocytes respond to UV irradiation by increasing the synthesis of melanin. While much is now understood of the pathways governing this process and the nature of the melanin synthesized, little is known of melanins produced by lower vertebrates and their capacity to respond to UV. Here we report that a fish, red seabream, can undergo 'suntanning'. Histological, colorimetric and chemical assays were performed for suntanned red seabream fish bred in net cages to analyse the melanins and compared with shaded or wild red seabream fish. For color evaluation, the L* values of suntanned fish were dramatically lower than those in the other two groups. Pyrrole-2,3,5-tricarboxylic acid (PTCA), an indicator of eumelanin, was detected in suntanned fish at five times higher levels than in shaded or wild fish while 4-amino-3-hydroxyphenyl-alanine (4-AHP), a marker for pheomelanin, could not be detected in any of the samples. Histological analysis showed that melanocytes in the suntanned skin enlarged and increased in number to form a monolayer at the surface of the skin. Analysis of L* values and PTCA levels showed quite a high correlation coefficient (r = -0.843). When comparing shaded and wild red seabream fish, the scores were closer but some significant differences were still found in some body areas. These results indicate that eumelanin accumulates in suntanned fish during the increase in skin color, which is induced by sunlight, presumably by ultraviolet radiation.

Molecular cloning and characterization of alpha-class glutathione S-transferase genes from the hepatopancreas of red sea bream, Pagrus major.

Two distinct cDNAs corresponding to GSTA1 and GSTA2 genes encoding glutathione S-transferases (GSTs) from the hepatopancreas of red sea bream, Pagrus major were cloned and sequenced. A comparison of the nucleotide sequences of GSTA1 and GSTA2 revealed 98% identity and their derived amino acid sequences had 96% similarity. Both genes could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. Genomic DNA cloning showed that both GSTA1 and GSTA2 genes consisted of six exons and five introns. In a comparison of genomic DNAs, the structures of GSTA1 and GSTA2 differed. In addition, Southern-blot analysis indicated that at least two kinds of alpha-class GSTs existed in the P. major genome. In order to biochemically characterize the recombinant enzymes (pmGSTA1-1 and pmGSTA2-2), both clones were highly expressed in Escherichia coli. The purified pmGSTA1-1 and pmGSTA2-2 exhibited glutathione conjugating activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide, while neither pmGSTs show detectable activity toward 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal, or p-nitrobenzyl chloride. Despite their high level of amino acid sequence identity, the pmGSTs had quite different enzyme-kinetic parameters.

A new class of glutathione S-transferase from the hepatopancreas of the red sea bream Pagrus major.

To elucidate drug deposition and metabolism in cultured marine fishes, in a previous study we isolated and purified the GSTs (glutathione S-transferases) from the hepatopancreas of the red sea bream Pagrus major that contained 25 and 28 kDa GST subunits. The 25 kDa GST subunits encoded by two genes (GSTA1 and GSTA2) have been identified as Alpha-class GSTs. In the present study, we performed the molecular cloning and characterization of the GSTR1 gene encoding the 28 kDa GST subunit from the Pa. major hepatopancreas. The nucleotide sequence of GSTR1 was composed of an ORF (open reading frame) of 675 bp encoding a protein of 225 residues with a predicted molecular mass of 25.925 Da. A search of the BLAST protein database revealed that the deduced amino acid sequence of GSTR1 was structurally similar to that of GSTs derived from other fishes such as largemouth bass (Micropterus salmoides) and plaice (Pleuronectes platessa). The genomic DNA containing the GSTR1 gene was found to consist of six exons and five introns quite distinct from mammalian Theta-class GSTs. We have purified and characterized the recombinant GSTR1 enzyme (pmGSTR1-1) which showed activity only towards 1-chloro-2,4-dinitrobenzene, although it had no detectable activity towards cumene hydroperoxide, 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal and p-nitrobenzyl chloride. Moreover, pmGSTR1-1 revealed remarkable heat instability (melting temperature Tm=30.3+/-0.11 degrees C). Collectively, our results indicated that the characteristic GST genes including GSTR1 have been conserved and functional in fishes. Therefore we designate them 'Rho-class', a new class of GSTs.

Further investigation of the epitope recognized by the new monoclonal antibody 2C9.

OBJECTIVE: We established a new monoclonal antibody (2C9) that reacted with prostate tissue. The immunohistochemical reactivity of this antibody is similar to anti-prostate-specific membrane antigen (PSMA). Herein, we report the antigenic determinant of 2C9 antibody. METHODS: The reactivity of the antibody was characterized by immunohistochemical staining and the antigen target was characterized by amino acid sequencing after immuno-affinity purification from an LNCaP cell lysate and cloning of a cDNA using a mammalian expression cDNA cloning system. RESULTS: The amino acid and nucleotide sequences for the antigen molecule recognized with 2C9 monoclonal antibody demonstrated identity with PSMA. CONCLUSION: The target molecule of the 2C9 monoclonal antibody is PSMA, pointing to future diagnostic and therapeutic applications.

Vesicourethral anastomotic suture placement during radical prostatectomy using Maniceps.

INTRODUCTION: A new method of using a semiautomatic device (Maniceps) for precise vesicourethral anastomosis during radical retropubic prostatectomy is described. METHODS: A total of 15 patients underwent radical retropubic prostatectomy with this technique. The follow-up period ranged from 6 to 16 months. The retrograde urethrocystography was performed at 2 weeks. Patients were evaluated specifically for anastomotic time, anastomotic obstruction and urinary continence. RESULTS: The average anastomotic time was 8.1 (range 5-12) min. The catheter of all patients could be removed at 2 weeks. Anastomotic obstruction occurred in 1 patient (6.7%), and 14 patients (93.3%) were completely continent of urine. CONCLUSION: Maniceps is a useful procedure to carry out vesicourethral anastomosis safely, easily and surely, and it has provided good surgical results in our experience.

Clinical features of renal cell carcinoma less than 25 millimeters in diameter.

BACKGROUND: We retrospectively investigated the clinicopathological features and prognosis of patients who underwent surgical treatment at our department for renal cell carcinoma (RCC) less than 25 mm in diameter. METHODS: Of the 158 patients who underwent surgical treatment between April 1975 and April 1998, 16 (17 kidney, 10.1%) were included in this study. The study included 11 men and 5 women (ratio: 2.2). The age range was 35-76 years (average: age 53). The right kidney was involved in 9, left kidney in 6 and bilateral kidneys in 1 patient. The follow-up period was 26-157 months (mean: 86 months). RESULTS: Thirteen tumors (81.2%) were incidental carcinomas. No patients had a tumor of rapid growing type. Radical nephrectomy was performed for 12 kidneys (70.6%), simple nephrectomy for 2 (11.8%) and partial nephrectomy for 3 (17.8%). Seven patients (43.7%) received interferon-alpha as postoperative adjuvant therapy. All tumors were pathologically classified as expansive type; 11 (64.8%) as clear cell carcinoma; 3 (17.6%) cyst-associated, and 3 (17.6%) papillary. Nine (52.9%) tumors were grade 1, and 8 (47.1%) were grade 2. Fourteen patients were pNo and V(-). The 5- and 10-year survival rates were excellent (100%). CONCLUSION: The features of small RCCs less than 25 mm were as follows: many tumors were incidental to clear cell carcinomas; all tumors were low grade, low stage and expansive type; no tumors showed acute phase reactants; and few tumors were of the solid type. Thus, the prognosis seemed to be excellent.

Ammonium acid urate urinary stone caused by a low-caloric diet: a case report.

A 32-year-old woman complained of right back pain and pyuria. The plain radiograph (KUB) and drip infusion pyelography (DIP) demonstrated a right renal stone and hydronephrosis. The stone was successfully treated using extracorporeal shock wave lithotripsy. Infrared spectrophotometry revealed that the stone was composed of pure ammonium acid urate. The patient had a 3-year history of excessive anorexia. The low-caloric diet was considered to have caused the disease.

[A case of self-mutilation of testis].

A 22-year old man was admitted to the emergency room of our hospital with bleeding from scrotal wound. He had resected his own scrotum and both testes by himself with a small knife, since he thought that his testes had developed necrosis. He had thrown his scrotal skin and both testes away in a lavatory. We closed the wide wound surface and the surrounding skin after ligation of the spermatic cord. The post-operative course was uneventful. The patient was referred to the psychiatric department of our hospital and received supportive psychotherapy under diagnosis of schizophrenia. This is the 23rd case to be reported as self-injury of the external genitalia and the 7th case limited to testis in the Japanese literature.

Effective treatment of bladder tumor-bearing mice by direct delivery of bleomycin using electrochemotherapy.

We investigated the cytotoxicity on the combination of bleomycin (BLM) with electric pulses against transplanted bladder tumors in nude mice. Loading with high-voltage electric pulse after injection of BLM was associated with a decrease in tumor size; tumor size became smaller and disappeared 8 days after the treatment. Such complete regression was achieved with one treatment using BLM of one-tenth the lethal dose (LD(50)) combined with electric pulses. On day 20, regrowth was observed at a much slower rate than at lower concentrations of BLM. Pathological examinations of the tumor tissues after treatment revealed disruption of the nucleus in the exposed tissues on day 2. Furthermore, a decrease in number of the stained nuclei and cytoplasmic lysis were observed on day 4. These results showed that electric pulses was an effective tool to deliver drugs into bladder tumor cells and an effective treatment method for bladder tumors.