Molecular study on chicken tumor necrosis factor receptor-II and tumor necrosis factor receptor-associated factor-5

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the tumor necrosis factor receptor (TNFR) superfamily. In this study, we cloned and characterized two genes that encode chicken TNFR-II and TRAF5. The initial cDNA fragments were obtained by suppressive subtractive hybridization (SSH) of chicken spleen cells with or without lipopolysaccharide stimulation (Salmonella typhimurium SL1181 (RE-mutant)). The results showed that chicken TNFR-II is 1518 bp in length with an open reading frame (ORF) of 1386 bp having 31% homology with human TNFR-II. Expression analysis of chicken TNFR-II revealed that it is highly expressed in the spleen and bursa of Fabricius. The chicken cell lines IN24, MSB1 and 1104B express TNFR-II abundantly. The time course analysis of expression in spleen, bursa of Fabricius and IN24 cell line showed that TNFR-II is maximally expressed at 6 h after stimulation in bursa of Fabricius and after 8 h stimulation in the IN24 cell line. With regard to TRAF5, the complete sequence was 1936 bp in length with an ORF of 1671 bp that showed 71.3% homology with human TRAF5. Expression analysis showed that, among the tissues examined, TRAF5 was strongly expressed in spleen and bursa of Fabricius, while among the cell lines examined, it was maximally expressed in IN24. Thus, both genes were expressed in the same tissues and cell line among examined materials. These results suggest that chicken TNFR-II may interact with TRAF5 adaptor protein to complete its signal transduction pathway.     

Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.     

Usefulness of MRI compared with CT for diagnosis of mesenteric lymphoma in a dog

We evaluated the usefulness of MRI and compared it with CT for diagnosis of mesenteric lymphoma in a dog. The results in the plain CT, dynamic CT and plain MR (T1WI and T2WI) images suggested that the mass was a large single nodular lesion with abundant blood perfusion. On enhanced MRI(T1WI) , the mass was depicted as a tumor with adhesion to the gut wall. Exploratory laparotomy confirmed the mass was consistent with the findings on enhanced MRI. We think that MRI might be a useful imaging tool for diagnosis of canine mesenteric lymphoma.     

Simultaneous observation of the GnRH pulse generator activity and plasma concentrations of metabolites and insulin during fasting and subsequent refeeding periods in Shiba goats

The time course of GnRH pulse generator activity and plasma concentrations of energy substrates and insulin were simultaneously observed in female goats during 4-day fasting and subsequent refeeding in the presence or absence of estrogen for a better understanding of the mechanism of energetic control of gonadotropin secretion in ruminants. The GnRH pulse generator activity was electrophysiologically assessed with the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In estradiol-treated ovariectomized (OVX+E2) goats, the MUA volley intervals increased as fasting progressed. Plasma concentrations of non-esterified fatty acid and ketone body increased, while those of acetic acid and insulin decreased during fasting. The MUA volley intervals and plasma concentrations of those metabolites and insulin were restored to pre-fasting levels after subsequent refeeding. In ovariectomized (OVX) goats, changes in plasma metabolites and insulin concentrations were similar to those in OVX+E2 goats, but the MUA volley intervals were not altered. The present results demonstrated that fasting suppressed GnRH pulse generator activity in an estrogen-dependent manner. Changes in plasma concentrations of energy substrates and insulin during fasting were associated with the GnRH pulse generator activity in the presence of estrogen, but not in the absence of the steroid in female goats.     

Parathyroid hormone (1-34) improves bone mineral density and glucose metabolism in Spontaneously Diabetic Torii-Lepr(fa) rats

The Spontaneously Diabetic Torii-Lepr(fa) (SDT-fa/fa) rat, a model of obese type 2 diabetes, shows obesity, hyperglycaemia and low bone mineral density (BMD). The objective of this study is to evaluate the effects of parathyroid hormone (1-34) [PTH(1-34)] on BMD and glucose metabolism in the SDT-fa/fa rat. SDT-fa/fa rats showed obesity with hyperglycaemia and decreased serum osteocalcin levels and the tibial BMD. A 4-week treatment of PTH(1-34) (20 μg/kg/day) increased the serum osteocalcin levels and the tibial BMD, and decreased the serum glucose levels without changing the serum insulin levels. These findings indicate that PTH(1-34) improved not only BMD but also glucose metabolism in SDT-fa/fa rats. This study suggests that PTH(1-34) is a novel agent for the treatment of diabetic osteoporosis.     

Genetic studies for breeding of rice cultivars with superior grain appearance and lodging resistance from the rice cultivar ‘Emi-no-kizuna’

ABSTRACT

We conducted a quantitative trait locus (QTL) analysis of grain appearance (GA) and agronomic traits of rice, using 128 recombinant inbred lines derived from ‘Emi-no-kizuna’ and ‘Tomohonami’. We detected two promising QTLs associated with GA: qGA4 on chromosome 4 and qGA8 on chromosome 8. qGA4 contributed highly to the greater percentage of perfect grains of the Emi-no-kizuna genotype. In the same region, we detected other QTLs associated with panicle number and spikelet number per panicle. In near-isogenic lines (NILs) in which Emi-no-kizuna alleles were introgressed in the genomic region of only the semi-dwarf 1 (sd1) locus (NIL_1) and both the sd1 locus and qGA8 (NIL_2), respectively, the percentage of perfect grains was significantly higher and the percentages of milky white, basal white, and white back grains were significantly lower than in Tomohonami; and the percentages of milky white and white back grains of NIL_2 were significantly lower than those of NIL_1. These results suggest that introgression in the sd1 region could improve GA, and that the addition of qGA8 could further improve GA. The culm lengths of the NILs were significantly shorter than that of Tomohonami, indicating improved lodging resistance. Grain weight of NIL_2 was significantly smaller than that of NIL_1, suggesting that the effect of qGA8 could be pleiotropic, or the gene that underlies qGA8 could be linked with genes associated with grain weight.

Abbreviations: ANOVA: analysis of variance; AT20: mean air temperature in the 20 days after heading; BW: basal white grain; CL: culm length; DAH: days after heading; GA: grain appearance; GW: 1000-grain weight; LOD: logarithm of odds; MW: milky white grain; NIL: near-isogenic line; PG: perfect grain; PL: panicle length; PN: panicle number; PTSN: putative total spikelet number; PVE: percentage of phenotypic variation explained; QTL: quantitative trait locus; RIL: recombinant inbred line; SN: spikelet number per panicle; SNP: single nucleotide polymorphism; WB: white back grain     

Identification and species-typing of wood rotting fungi using melting curve analysis

A method for identification and typing of wood rotting fungi using the melting temperature [T(m)] of DNA fragments coding rRNA (rDNA) was examined. The T(m)s of four DNA fragments, inter transcribed spacer (ITS) I, ITS II, and two partial fragments of 28S rDNA from each of 20 species of wood rotting fungi, were measured by melting curve analysis. The T(m) variation was large enough between species to enable identification based on the T(m) values. A pair of T(m)s of the ITS I region (between the primers ITS1 and ITS2) and the ITS II region (between the primers ITS3 and ITS4) had the highest resolution for identifying wood rotting fungi. To assess about the diversity of the T(m), intraspecific diversity of these DNA fragment sequences was evaluated using test strain sequences and data from the GenBank/EMBL/DDBJ biological database. The intraspecific diversity of T(m) was considered to be small because the nucleotide diversity of each fragment was small within the species.     

Improvement of antifungal activity of 10-undecyn-1-ol by inclusion complexation with cyclodextrin derivatives

The inclusion complexation behavior between 10-undecyn-1-ol and cyclodextrin (CD) derivatives, namely, randomly methylated beta-CD (RM-beta-CD) and hydroxypropyl-beta-CD (HP-beta-CD), was studied in terms of solubility improvement, apparent stability constant, and the inclusion ratios of the resultant inclusion complexes. The aqueous solubility of 10-undecyn-1-ol was greatly improved through complexation with the CD derivatives. RM-beta-CD is comparatively more efficient in solubilizing 10-undecyn-1-ol with an apparent stability constant outstripping that of HP-beta-CD by about an order of magnitude. Comparative in vitro evaluations of the growth inhibition effects of inclusion complex solutions toward Rosellinia necatrix, a phytopathogenic fungus, were performed. In comparison with the positive control, appreciable improvements of the antifungal activity of 10-undecyn-1-ol through the addition of CD derivatives were observed visually. The improvement was evaluated in terms of area covered by the mycelia of Rosellinia necatrix and their growth rate. RM-beta-CD was proven to be more effective compared to HP-beta-CD with regard to the reduction of both fungal mycelium-covered area and growth rate constant, presumably owing to greater solubility enhancement by RM-beta-CD and thus the bioavailability of 10-undecyn-1-ol. Inclusion complexation of 10-undecyn-1-ol with CD derivatives suggests a potential means for production of an environmentally friendly 10-undecyn-1-ol-based fungicide to counteract R. necatrix.     

Loop-mediated isothermal amplification for the rapid, sensitive, and specific detection of the O9 group of Salmonella in chickens

In the present study, the loop-mediated isothermal amplification (LAMP) assay was developed to amplify the fragments of the O9 Salmonella-specific insertion element and evaluated in the laboratory for its potential use in a field situation, such as poultry farms. Among the bacteria tested, a positive reaction was observed only for 128 strains of 6 serovars of the O9 group Salmonella, such as Enteritidis (SE) and Pullorum. The detection limit of the LAMP assay was 10(3)CFU/ml, which was more sensitive than that of the polymerase chain reaction (PCR) assay with the same target gene (10(6)CFU/ml). The final results were obtained within 30 min for the LAMP assay, while the PCR assay needed a total of 120 min. When the LAMP assay was applied to the enrichment broth mixed with cecal dropping samples either spiked with SE in vitro or excreted by SE-inoculated hens, the results were comparable to those of the conventional plating method including 2 separate enrichments. In conclusion, the LAMP assay developed in the present study is an effective method for the specific detection of the O9 group Salmonella serovars, including SE.     

Molecular detection of Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' Infection in cats by direct PCR using whole blood without DNA extraction

Detection of hemotropic Mycoplasma spp. infection was attempted in cats by PCR using whole blood without DNA extraction. A total 46 of 54 (85%) cats with suspected Mycoplasma spp. infection showed a positive reaction, corresponding completely with the results of standard PCR testing. The direct PCR assay was sensitive enough to detect more than 0.0061% parasitemia for ;C. M. haemominutum' and 0.0075% parasitemia for M. haemofelis. These data indicate that the direct PCR assay might be sufficient for use as a tool in clinical examinations.