Real-time PCR for differential determination of the tomato wilt fungus, <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>, and its races

Five primer/probe sets to identify the tomato wilt pathogen, Fusarium oxysporum f. sp. lycopersici (FOL), and its three races selectively were designed based on the rDNA-intergenic spacer and avirulence genes. Real-time PCR using genomic DNA from mycelia and soil DNA with the primer/probe sets allowed the successful identification of FOL and its races.     

Flow cytometric analysis of thymocyte subpopulations in mice after whole-body X-irradiation.

To determined the cellular kinetics of thymocyte subpopulations in DBA1 mice after whole-body 6.8 Gy X-irradiation, they were analyzed for the expression of several cell surface antigens using flow cytometry. The results show that i) The majority of thymocytes rapidly depleted by irradiation was CD4+8+ cells. ii) radioresistant CD4+8- and CD4-8+ survived 18-48 hr after X-irradiation were considered to be relatively mature type, since they expressed high levels of CD3 and LECAM-1. iii) CD3-positive cells were detected in CD4-8- cells at 72 hr after irradiation.     

B220 is expressed on apoptotic thymocytes induced by X-irradiation.

CD45 is cell surface glycoprotein and expressed on all haematopoietic cells except mature erythrocytes and platelets. Eight isoforms of CD45 are generated by alternative splicing of exons 4-6. B220 including all three exons is expressed specifically on pan-B cell lineage. Recently, it was reported that B220 was expressed on apoptotic T cells induced by staphylococcal enterotoxin B (SEB). In the present study, we investigated the expression of B220 on murine thymocytes after whole-body X-irradiation. We used the forward light scattering of flow cytometry as a parameter of cell size, and defined two populations; FSChigh (normal cell size) and FSClow (correspond to apoptotic cell in size) fraction. B220+ cells in FSChigh fraction reached a maximum value (35%) at 18 hr after irradiation. In FSClow fraction, 40-60% cells were positive for B220 at any time points. These results suggest that B220 is expressed on thymocytes in the pre-apoptotic stage, because B220 was expressed on not only FSClow cells but also FSChigh cells.     

Immunoreactive changes in pituitary FSH and LH cells during seasonal reproductive and spawning cycles of female chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis>

The physiological functions of pituitary gonadotropins (GtHs) are well established in higher vertebrates, whereas those in teleosts are still poorly understood. To describe the role of GtHs during gonadal development of female chub mackerel Scomber japonicus, changes in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were investigated immunohistochemically during the seasonal reproductive and spawning cycles. FSH and LH cells were identified in the different cell types of the proximal pars distalis (PPD); FSH cells were located in the central PPD, whereas LH cells were localized along the border of the pars intermedia. To examine changes in FSH and LH cells, the percentage of FSH or LH cell-occupying area in the PPD was evaluated and represented as FSHβ-immunoreactive (ir) or LHβ-ir levels, respectively. FSHβ-ir levels increased significantly from immature to the completion of vitellogenesis, whereas LHβ-ir levels were maintained at high levels from early vitellogenesis to post-spawning. During the spawning cycle, which consisted of four stages from just after spawning to the next oocyte maturation, both FSHβ-ir and LHβ-ir levels showed no significant changes among different stages; however, LHβ-ir levels remained relatively high, and FSHβ-ir levels were constantly low. These results suggest that both FSH and LH may be involved in vitellogenesis and LH may act at final oocyte maturation in female chub mackerel, although the role of FSH during the spawning cycle is still unclear.     

Clonal structure in Ichthyobacterium seriolicida,the causative agent of bacterial haemolytic jaundice in yellowtail,Seriola quinqueradiata,inferred from molecular epidemiological analysis

Bacterial haemolytic jaundice caused by Ichthyobacterium seriolicida has been responsible for mortality in farmed yellowtail, Seriola quinqueradiata, in western Japan since the 1980s. In this study, polymorphic analysis of I. seriolicida was performed using three molecular methods: amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST) and multiple‐locus variable‐number tandem repeat analysis (MLVA). Twenty‐eight isolates were analysed using AFLP, while 31 isolates were examined by MLST and MLVA. No polymorphisms were identified by AFLP analysis using EcoRI and MseI, or by MLST of internal fragments of eight housekeeping genes. However, MLVA revealed variation in repeat numbers of three elements, allowing separation of the isolates into 16 sequence types. The unweighted pair group method using arithmetic averages cluster analysis of the MLVA data identified four major clusters, and all isolates belonged to clonal complexes. It is likely that I. seriolicida populations share a common ancestor, which may be a recently introduced strain.     

A simulation study for a linear measurement error model when error variances vary between measurements

When predicting scores in the Draize eye irritation test based on measurements of in vitro alternative tests, we are often faced with estimating parameters in a linear measurement error model with heterogeneous error variances. This article proposes a new statistical method for parameter estimation to address this issue. The proposed method is an extension of an earlier proposal that applied a linear measurement error model with homogeneous error variances, to cases with heterogeneous error variances. A simulation study to examine the performance of the proposed method was conducted in a framework that was adaptable to the data, which was obtained in a validation study of alternative methods to animal experiments conducted in Japan. The proposed method reduced the biases of estimates in comparison with an ordinary regression analysis method and three other methods under the assumption of homogeneous error variances. Although the proposed method did not fit the real data well, the resulting prediction formula was far better than those obtained by other methods.     

Nephrocalcinosis formation by soy isoflavones in male rats

Isoflavones (IFs), found in the form of both aglycones and glucosides in soybean foods, induce weak estrogenic activities. Although IFs have a number of health benefits, it was previously reported that IFs cause nephrocalcinosis (NC) in the kidney of male Fischer 344 (F344) rats. The present study aims to elucidate the safety of IFs by focusing on IF-induced NC formation in rats. Fermented soybean extract (FSE) containing 420 mg/g isoflavone aglycones was orally administered to male F344 and Sprague-Dawley (SD) rats for 28 days. FSE induced NC formation in the kidney of F344 rats, but not in SD rats. However, absorption of IFs did not differ between F344 and SD rats. NC formation and its severity of FSE were histologically compared with those of soybean extract (SE) containing 518 mg/g isoflavone glucosides in F344 rats. There were no differences in the number of NC formations and the extent of calcium deposit between FSE and SE groups. To examine the dose effect of FSE on NC formation, doses of 20, 140, or 1000 mg/kg FSE were administered to F344 rats for 90 days. NC formation was observed in the 140 and 1000 mg/kg groups. These results indicated that a high dose of oral administration of IFs induced NC formation depending on the strain of rat.     

Age validation,growth and annual reproductive cycle of chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis> off the waters of northern Kyushu and in the East China Sea

The age and growth of chub mackerel Scomber japonicus collected from the East China Sea and the northern waters off Kyushu between June 2000 and June 2001 were determined by observing the otolith surface after dipping it in xylene. The translucent and opaque zones on the otolith surface were identified, and the number of translucent zones was counted. Monthly changes in the frequency of fish with translucent zones on the otolith margin, and in the marginal increments, indicated that the translucent zones were formed between April and June. The seasonal pattern of annulus formation on the otolith became clear by observing the otoliths of fish with known ages, and the otolith formation in wild fish was consistent with that of fish with known ages. The mean gonadosomatic index of male and female fish was high from March to May, and spawning females were observed from mid-March to mid-May. The estimated ages were 1–5 years for males and 1–6 years for females. The von Bertalanffy growth parameters did not significantly differ between male and female. The model was obtained as FL _t=406×{1−exp[−0.372×(t+1.68)]     

Expression of LacZ gene in canine muscle by intramuscular inoculation of a plasmid DNA

DNA immunization induces systemic humoral and cellular immune responses to the antigen encoded by cDNA in a plasmid DNA. In the present study, a plasmid DNA encoding cDNA of beta-galactosidase (beta-gal), pCAGGS-lacZ, was inoculated intramuscularly to a healthy dog in order to evaluate location and duration of the gene expression. On day 7, the plasmid DNA was found by PCR in the muscle where the plasmid was injected. Furthermore, beta-gal expression was detected in the same muscle sample by beta-gal staining. However, the plasmid DNA was not detected in any samples collected on days 14, 21 and 28. The present results suggest that duration of the gene expression of beta-gal by the plasmid DNA is limited in the muscle in dogs and an efficacy for a gene expression should be evaluated depending on the gene inserted in the plasmid DNA for immunotherapy.