An efficient multiplex PCR suitable for large scale typing in linkage mapping.

The dissection of polygenic traits is made possible with the development of microsatellite markers. Linkage study of this kind involves many markers with tens of hundreds of samples. Although typing essentially contains only two steps: PCR amplification and gel electrophoresis. Such work is still heavy when a large number of samples had to be genotyped. Multiplex PCR may reduce the work, but one has to optimize the conditions from marker to marker. Here we describe a dye-compatible multiplex PCR that works under standardized condition without the need to pre-determine the combinational primer concentration and the time-consuming step to mix many samples with gel loading dye before electrophoresis. This successful protocol should greatly reduce the cost and labor for genetic study of polygenic traits.     

Central somatostatin-somatostatin receptor 2 signaling mediates lactational suppression of luteinizing hormone release via the inhibition of glutamatergic interneurons during late lactation in rats

Reproductive function is suppressed during lactation owing to the suckling-induced suppression of the kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and subsequent suppression of luteinizing hormone (LH) release. Our previous study revealed that somatostatin (SST) neurons mediate suckling-induced suppression of LH release via SST receptor 2 (SSTR2) in ovariectomized lactating rats during early lactation. This study examined whether central SST-SSTR2 signaling mediates the inhibition of ARC Kiss1 expression and LH release in lactating rats during late lactation and whether the inhibition of glutamatergic neurons, stimulators of LH release, is involved in the suppression of LH release mediated by central SST-SSTR2 signaling in lactating rats. A central injection of the SSTR2 antagonist CYN154806 (CYN) significantly increased ARC Kiss1 expression in lactating rats on day 16 of lactation. Dual in situ hybridization revealed that few ARC Kiss1-positive cells co-expressed Sstr2, and some of the ARC Slc17a6 (a glutamatergic neuronal marker)-positive cells co-expressed Sstr2. Furthermore, almost all ARC Kiss1-positive cells co-expressed Grin1, a subunit of N-methyl-D-aspartate (NMDA) receptors. The numbers of Slc17a6/Sstr2 double-labeled and Slc17a6 single-labeled cells were significantly lower in lactating dams than in non-lactating rats whose pups had been removed after parturition. A central injection of an NMDA antagonist reversed the CYN-induced increase in LH release in lactating rats. Overall, these results suggest that central SST-SSTR2 signaling, at least partly, mediates the suppression of ARC Kiss1 expression and LH release by inhibiting ARC glutamatergic interneurons in lactating rats.     

Green and black tea suppress hyperglycemia and insulin resistance by retaining the expression of glucose transporter 4 in muscle of high-fat diet-fed C57BL/6J mice

To investigate the preventive effects of tea on hyperglycemia and insulin resistance, male C57BL/6J mice were given a high-fat diet containing 29% lard and also green or black tea ad libitum for 14 weeks. Both teas suppressed body weight gain and deposition of white adipose tissue caused by the diet. In addition, they improved hyperglycemia and glucose intolerance by stimulating glucose uptake activity accompanied by the translocation of glucose transporter (GLUT) 4 to the plasma membrane in muscle. Long-term consumption of the high-fat diet reduced levels of insulin receptor β-subunit, GLUT4 and AMP-activated protein kinase α in muscle, and green and black tea suppressed these decreases. The results strongly suggest that green and black tea suppress high-fat diet-evoked hyperglycemia and insulin resistance by retaining the level of GLUT4 and increasing the level of GLUT4 on the plasma membrane in muscle.     

Promotion of liver and kidney carcinogenesis by ethyl tertiary-butyl ether (ETBE) in male Wistar rats

Tumor-promoting effects of ethyl tertiary-butyl ether (ETBE) were investigated in a 2-stage carcinogenesis bioassay with regard to hepatic and renal carcinogenesis in rats. Male 6-week-old Wistar rats were given drinking water containing N-ethyl-N-(2-hydroxyethyl)nitrosamine (EHEN), as an initiator, at a dose of 500 ppm for 2 weeks. Starting one week thereafter, the animals were administered ETBE at dose levels of 0 (control), 100, 300, 500 or 1,000 mg/kg/day by gavage for 19 weeks from week 4 to 22. Necropsy of all rats was performed at week 23, and livers and kidneys were examined histopathologically. Incidences of hepatocellular adenomas, and those of combined hepatocellular adenomas and carcinomas were significantly elevated in rats given 1,000 mg/kg/day ETBE, but not 100‒500 mg/kg/day ETBE, and there was a significant increase in the average numbers of lesions. No significant differences in incidences and average numbers of renal tubule neoplasms were found in rats administered 100‒1,000 mg/kg/day ETBE. However, the average numbers of atypical tubule hyperplasias, considered to be preneoplastic lesions, were significantly increased in rats given ETBE at 1,000 mg/kg/day, but not in rats given 500 mg/kg/day or lower doses. Thus, these results imply that ETBE has hepatic and renal tumor-promoting activities that affect EHEN-induced carcinogenesis in male rats, and the no-observed-effect level is 500 mg/kg/day under the present experimental conditions.     

Anti-CD71 antibody immunohistochemistry in the diagnosis of acute myeloid leukemia,subtype acute erythroid leukemia with erythroid dominance (AML M6-Er), in a retrovirus-negative cat

CD71 is an immunohistochemical marker used in diagnosing acute myeloid leukemia (AML) M6-Er in humans; however, to our knowledge, it has not been reportedly used for immunohistochemistry in veterinary medicine. We evaluated the pathologic features of AML M6-Er in a retrovirus-negative cat and used CD71 to support the diagnosis. A 4-y-old spayed female Scottish Fold cat was presented with lethargy, anorexia, and fever. Whole-blood PCR assay results for pro feline leukemia virus/pro feline immunodeficiency virus and feline vector-borne diseases were negative. Early erythroid precursors were observed in the peripheral blood smear. Fine-needle aspiration of the enlarged spleen and splenic lymph node showed many early erythroid precursors. Bone marrow aspirate smears revealed erythroid hyperplasia with 68.4% erythroid lineage and 3.6% rubriblasts. Dysplastic cells infiltrated other organs. The patient was diagnosed with myelodysplastic syndrome, progressing to the early phase of AML M6-Er. The patient died on day 121 despite multidrug treatments. Postmortem examination revealed neoplastic erythroblasts infiltrating the bone marrow and other organs. Neoplastic cells were immunopositive for CD71 but immunonegative for CD3, CD20, granzyme B, von Willebrand factor, CD61, myeloperoxidase, and Iba-1. Although further studies are necessary for the application of CD71, our results supported the morphologic diagnosis of AML M6-Er.     

High sensitivit to streptozotocin in herbivorous voles,Microtus arvalis,compared to mice

Herbivorous voles, Microtus arvalis, have characteristics similar to herbivores in that their hepatic glycolytic enzyme activities are relatively low. The effects of a single low dose (100 mg/kg body weight) of streptozotocin (STZ) in voles were studied and the difference in sensitivity to or toxicity of STZ in voles and C57BL/6 mice was compared. In voles which received STZ, the cumulative incidence of glycosuria reached 53% by 4 weeks after administration. The diabetic voles showed marked increas in their blood glucose and plasma free fatty acid concentrations and a significant decrease in plasma immunoreactive insulin concentrations. Their hepatic hexokinase, glucokinase, glutathione peroxidase and glutamate dehydrogenase activities decreased significantly and lesions were widely observed in the liver, kidney and pancreas. The activities of glutathione peroxidase, a scavenger of H₂O₂, decreased significantly in their liver and pancreas. These changes were not observed in C57BL/6 mice which received STZ. The higher sensitivity to and toxicity of STZ in voles than in mice are considered to be caused by the characteristically low activities of glycolytic enzymes and glutathione peroxidase in the tissues of voles. Voles may be a good model for studying the mechanisms of cytotoxicity by STZ in herbivorous animals.Abbreviations GK glucokinase - GIDH glutamate dehydrogenase - GSH-px glutathione peroxidase - HE haematoxylin-eosin - HK nexokinase - IRI immunoreactive insulin - STZ streptozotocin     

Genotyping of Japanese field isolates of Mycoplasma synoviae and rapid molecular differentiation from the MS-H vaccine strain

Mycoplasma synoviae is an important causative agent of avian mycoplasmosis. In the present study the conserved domain of the variable lipoprotein and hemagglutinin (vlhA) gene of M. synoviae was sequenced and analyzed for 19 field strains of M. synoviae isolated from chickens across Japan. This analysis revealed that there were at least nine genotypes of M. synoviae present in Japan. Furthermore, we found a single nucleotide polymorphism (SNP) within this region in all the Japanese isolates, and based on this finding, we established a PCR method with cycling probe technology to differentiate between these field isolates and the live M. synoviae vaccine strain Mycoplasma synoviae-H (MS-H).     

Properties of soil humic substances in fractions obtained by sequential extraction with pyrophosphate solutions at different pHs

Abstract

Humic acids (HAs) obtained from four different soils by sequential extraction with pyrophosphate solutions at different pH values (i.e. at pH 3, 5, 7, 9, 11, and 13), were examined by ultraviolet and visible (UV-VIS) spectroscopy and elemental analysis. UV-VIS spectra from 230 to 700 nm were measured, and absorbance coefficients of 1% humic acid solutions at 600 nm (E ^1% ₆₀₀) and Δlog K were calculated. The shape of the spectra of humic acids depended on the soil types rather than on the pH values. The (E ^1% ₆₀₀) values were higher in the neutral region, and lower in the alkaline region, for each pH value they decreased in the following order: buried Andosol > Andosol > Histosol ≧ Cambisol. The Δlog K values for each pH value were higher in the following order: Histosol> Cambisol > buried Andosol≧Andosol. Although the linear correlation was found to be significant between the E ^1% ₆₀₀ and Δlog K values, the relation between these optical properties and pH values differed among the soil types. However, in the neutral region, the higher E ^1% ₆₀₀ values of humic acids from buried Andosol and Andosol and the lower Δlog K values of humic acids from Cambisol and Histosol suggest that the humification degree was higher in the neutral region. Data of elemental composition and atomic ratios were obtained for each humic acid. The H/C ratios in the four soils increased gradually with increasing pH values from 5 to 13, and those for each pH value were commonly higher in the following order: Cambisol> Histosol> Andosol> buried Andosol. The O/C and OIH ratios decreased with increasing pH values. The results of the H/C versus O/C diagram suggested that decarboxylated humic acids were extracted at the higher pH values. A highly significant correlation coefficient was obtained between the H/C and OIH ratios (r= ?0.891***). Results showing that the decrease of the pH values corresponded to the order of the humic acids in the correlation curve in the H/C versus O/H diagram suggest that the humic acids with a higher content of unsaturated bonds and a higher degree of oxidation were extracted at the lower pH values within the range from 5 to 13.     

Ovicidal activity of juvenile hormone mimics in the bean bug,Riptortus pedestris

Insect juvenile hormone (JH) mimics (JHMs) are known to have ovicidal effects if applied to adult females or eggs. Here, we examined the effects of exogenous JHMs on embryonic development of the bean bug, Riptortus pedestris. The expression profiles of JH early response genes and JH biosynthetic enzymes indicated that JH titer was low for the first 3 days of the egg stage and increased thereafter. Application of JH III skipped bisepoxide (JHSB₃) or JHM on Day 0 eggs when JH titer was low caused reduced hatchability, and the embryos mainly arrested in mid- or late embryonic stage. Application of JHMs on Day 5 eggs also resulted in an arrest, but this was less effective compared with Day 0 treatment. Interestingly, ovicidal activity of synthetic JHMs was much lower than that of JHSB₃. This study will contribute to developing novel insecticides that are selective among insect species.