Automatic counting of FISH-labeled microbes by an LED illuminated detecting apparatus

ABSTRACT:   The Bioplorer (BP) (Matsushita Ecology Systems Co. Ltd, Kasugai, Aichi-ken, Japan) is an apparatus that consists of a light-emitting diode and optical and image analysis systems. This instrument has been developed and used for the semi-automatic counting of total microbes, mainly in food, cosmetics and industrial products. In this study, the applicability of BP to the detection and enumeration of eukaryotic and prokaryotic cells, after the fluorescent in situ hybridization (FISH) procedure, was examined. The cells of a yeast ( Candida albicans ) and a bacterium ( Escherichia coli ) were specifically labeled using an oligonucleotide probe with fluorochrome and then counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The numbers of cells after FISH treatment and DAPI staining agreed well, indicating that the BP optical device and image analysis system had enough sensitivity to detect and quantify eukaryotic and prokaryotic cells. Because of its simplicity and reliability, BP can be a new tool for quantification of specifically labeled target microorganisms, both in industrial products and natural samples.     

Distribution,growth and hatch date of juvenile Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> in the coastal area of the sea of Japan

ABSTRACT:   Mid-water trawl surveys were conducted from late August to late September in 1999 and 2004 in order to investigate the distribution pattern, hatch date, and growth of juvenile Pacific bluefin tuna Thunnus orientalis in the Sea of Japan. Juveniles were collected at the stations where ambient water temperature (mean water temperature from surface to 30 m deep, WT₀₋₃₀) was 23.4–25.9°C, and most of them were found in waters where WT₀₋₃₀ was higher than 24°C. Sampled juveniles ranged 108–280 mm fork length. Based on otolith analysis, they were estimated to grow to approximately 180 and 250 mm at 60 and 90 days old, respectively, and showed similar growth to that of Atlantic bluefin tuna in the Mediterranean Sea. The back-calculated hatch date of the samples was mostly in July and most juveniles spawned in the Sea of Japan.     

Factors Associated with Well-to-Well Variation in Nitrate Concentration of Groundwater in a Nitrate-Polluted District in Miyakonojo Basin, Southern Kyushu, Japan

This study monitored nitrate levels in well water and analyzed their association with well attributes (physicochemical parameters of water, location, livestock farming conditions) in a nitrate-polluted, animal industry district in central Miyakonojo Basin, southern Kyushu, Japan, to characterize the nitrate status of groundwater in the district. Water quality varied considerably among the wells, with nitrate-N concentration and natural nitrogen-15 abundance in nitrate-N (δ¹⁵N-NO₃) ranging from 1.1–44.6 mg L^?1 and 4.2–17.8‰, respectively. There was a significant positive correlation between nitrate-N concentration and δ¹⁵N-NO₃. Nitrate-N concentration was higher in wells located at higher elevations, having larger areas of livestock barns within a 100-m radius and with higher animal populations. Wells in pig farms showed a higher nitrate tendency than those in the other situations (cattle farm, poultry farm, non-livestock farm and non-farmer). The results show that the nitrate status of groundwater in the district has not been drastically changed since 1996, and the nitrate in the wells is of multiple origins (e.g. chemical fertilizer, animal wastes) with a tendency for higher contribution of animal wastes in more heavily polluted wells. The results also highlight a need for further regular monitoring of groundwater quality in the district particularly for wells; (1) located at higher elevations, (2) with a large area of livestock barns nearby, (3) in farms with a high animal population and/or (4) in pig farms.     

GeneScan analysis to detect clonality of T-cell receptor γ gene rearrangement in feline lymphoid neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. Polymerase chain reaction (PCR) amplification of the complementarity-determining region (CDR) 3 of the T-cell receptor (TCR) γ gene can be used to assess clonality of T-cell populations as a supportive diagnostic tool for T-cell neoplasms. Because the length variation in the TCRγ CDR3 is relatively small, false positive results may occur in non-neoplastic T-cell populations in the absence of high-resolution analytical methods for PCR products. In the present study, a PCR assay system was developed to detect clonal TCRγ gene rearrangement in feline lymphoid cells using GeneScan analysis. Thirty T-cell neoplasms, 27 B-cell neoplasms, and 34 non-neoplastic tissues were subjected to the newly developed TCRγ gene rearrangement analysis. Clonal TCRγ gene rearrangement was detected in 26 of 30 (87%) T-cell neoplasms, 2 of 27 (7%) B-cell neoplasms, and 1 of 34 (3%) non-neoplastic tissues. To compare GeneScan analysis with conventional PAGE and heteroduplex analysis, 20 clonal and 20 polyclonal samples were subjected to both analyses. Most of the results were concordant between the 2 analyses; however, several clonal peaks (bands) appeared as a single band when analyzed via conventional PAGE with heteroduplex analysis in 4 of the 20 (20%) clonal samples as a result of the difference in resolution. The PCR assay system to detect clonal TCRγ gene rearrangement in feline lymphoid cells, using GeneScan analysis, would be a useful molecular diagnostic tool for feline T-cell neoplasms, with high fidelity.     

Dynamic responsive characteristics of nailed plywood–timber joints under harmonic vibrations

Dynamic tests of nailed plywood–timber joints are conducted under harmonic vibrations from 2 to 7 Hz. The principal results are as follows: under dynamic loading, nailed plywood–timber joints may break in low-cyclic bending fatigue failure of nails besides the other failure modes typical under static loading. The dynamic response of nailed plywood–timber joints is clearly dependent upon both the input frequency and the acceleration. These responsive characteristics arise from the nonlinear load–slip relationships and the characteristic cyclic stiffness degradation of nailed joints; that is, the cyclic degradation of the equivalent linear stiffness decreases the resonant frequencies of the same joints, which results in a transition of dynamic responses. It indicates that frequency components of seismic waves resonant to the frequencies corresponding to safety-limit stiffness of nailed joints may lead them to critical failures, even if the accelerations do not exceed the accelerations equivalent to the static damage-limit resistance.     

Evaluation of the prognostic significance of BCL6 gene expression in canine high-grade B-cell lymphoma

The clinical usefulness of BCL6 gene expression was evaluated as a prognostic indicator in dogs with high-grade B-cell lymphoma. Forty-four dogs were diagnosed with centroblastic or B-cell immunoblastic type lymphoma according to the updated Kiel classification. BCL6 mRNA expression was measured by real-time PCR and its relationship with prognosis was analyzed. Progression-free and overall survival was not significantly different between the high BCL6 expression group (higher than the median) and the low BCL6 expression group (lower than the median) (P=0.99 and P=0.61, respectively). No correlation between BCL6 and prognosis was observed in this study, which is inconsistent with findings reported for human diffuse large B-cell lymphoma. BCL6 protein expression was not detected in the 11 dogs evaluated by immunohistochemistry. Furthermore, BCL6 protein expression was assessed in 13 archived paraffin-embedded high-grade canine lymphoma tissues and all were also negative. The results suggest that most canine high-grade B-cell lymphomas correspond to human diffuse large B-cell lymphoma with no immunohistochemical expression of BCL6.     

Detection of QTLs for traits associated with pre-harvest sprouting resistance in bread wheat (Triticum aestivum L.)

Pre-harvest sprouting (PHS) is one of the serious problems for wheat production, especially in rainy regions. Although seed dormancy is the most critical trait for PHS resistance, the control of heading time should also be considered to prevent seed maturation during unfavorable conditions. In addition, awning is known to enhance water absorption by the spike, causing PHS. In this study, we conducted QTL analysis for three PHS resistant related traits, seed dormancy, heading time and awn length, by using recombinant inbred lines from ‘Zenkouji-komugi’ (high PHS resistance) × ‘Chinese Spring’ (weak PHS resistance). QTLs for seed dormancy were detected on chromosomes 1B (QDor-1B) and 4A (QDor-4A), in addition to a QTL on chromosome 3A, which was recently cloned as TaMFT-3A. In addition, the accumulation of the QTLs and their epistatic interactions contributed significantly to a higher level of dormancy. QDor-4A is co-located with the Hooded locus for awn development. Furthermore, an effective QTL, which confers early heading by the Zenkouji-komugi allele, was detected on the short arm of chromosome 7B, where the Vrn-B3 locus is located. Understanding the genetic architecture of traits associated with PHS resistance will facilitate the marker assisted selection to breed new varieties with higher PHS resistance.     

The effect of M‐phase stage‐dependent kinase inhibitors on inositol 1,4,5‐trisphosphate receptor 1 (IP3R1) expression and localization in pig oocytes

At fertilization, inositol 1,4,5‐trisphosphate receptor type 1 (IP₃R1) has a crucial role in Ca²⁺ release in mammals. Expression levels, localization and phosphorylation of IP₃R1 are important for its function, but it still remains unclear which molecule(s) regulates IP₃R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP₃R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP₃R1 were formed in the cortex of the oocyte except in a ring‐shaped band of cortex adjacent to the spindle. However, no such clusters of IP₃R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP₃R1 localization, phosphorylation and expression using M‐phase stage‐dependent kinase inhibitors. Our results show that treatments with roscovitine (p34^cdc2 kinase inhibitor) or U0126 (mitogen‐activated protein kinase inhibitor) did not affect IP₃R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI‐2536, an inhibitor of polo‐like kinase 1 (Plk1), dramatically decreased the expression level of IP₃R1 in pig oocytes in a dose‐dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP₃R1 expression in pig oocytes.     

Effects of Controlled-Release Fertilizers and Their Application Methods on Germination and Seedling Growth of Dent and Sweet Corns

Effects of controlled-release fertilizers (CRFs) (C-AS, polyolefin coated ammonium sulfate, 50-day-type; Dd-LP, polyolefin coated urea with dicyandiamide, 40-day-type; C-ANP, polyolefin coated ammonium nitrate phosphate, 40-day-type; and C-DAP, polyolefin coated diammonium acid phosphate, 40-day-type), ammonium sulphate and no fertilizer control, and their application methods (spot, band, surface and mixed) on germination and seedling development of sweet corn (Zea mays L.var. saccharata Sturt.) and dent corn (Zea mays L.var. indentata Sturt.) were investigated in a greenhouse. Under co-situs application (band and spot) of CRFs, there were no obvious differences in the germination speed and rate for both dent corn and sweet corn relative to control. Mortality rates of sweet corn seedlings under co-situs application were high in experiment 1, but were very low in experiment 2, because the environmental conditions were different in the two experiments. That is, under lower temperature and weaker sunlight, young seedlings easily die due to high soil nutrient concentration and slow growth speed of corn. Shoot weight of both dent and sweet corn did not greatly decrease in experiment 1. In experiment 2, there were no significant differences in shoot and root weight of both corns between cositus and surface or mixed application methods. However, with spot and band application of ammonium sulfate, shoot and root weight were significantly reduced. Soil EC and pH were considerably affected by co-situs application, especially at the fertilizer application site. For both dent and sweet corn, EC in the 0-3 cm soil was significantly higher under co-situs application and surface application than that under mixed application, whereas in the 3-6 cm soil depth the situation was reversed. Compared with control, mixed application of CRFs decreased soil pH slightly (0-3 cm depth) or greatly (3-6 cm depth).