Expression of genes related to corticotropin production and glucocorticoid feedback in corticotroph adenomas of dogs with Cushing's disease

Cushing's disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to negative feedback by glucocorticoids. In this study, we examined gene expression related to adrenocorticotropic hormone (ACTH) production and secretion, and the negative feedback by glucocorticoids in canine corticotroph adenoma. We used resected corticotroph adenomas from 10 dogs with Cushing's disease. In order to investigate the alteration of gene expression between corticotroph adenoma and normal corticotrophic cells, ACTH-positive cells in the anterior lobe were microdissected using a laser-capture microdissection system, and mRNA levels of proopiomelanocortin (POMC), corticotropin releasing hormone receptor 1 (CRHR1), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11 beta hydroxysteroid dehydrogenase (11HSD) type 1 and type 2 were determined using real-time RT-PCR. POMC, CRHR1, and 11HSD2 mRNA levels in corticotroph adenoma were greater than those in normal corticotrophic cells (POMC, 5.5-fold; CRHR1, 4.9-fold; 11HSD2, 4.2-fold, P<0.01, respectively). MR and 11HSD1 mRNA levels in corticotroph adenoma were lower than those in normal corticotrophic cells (MR, 2.2-fold; 11HSD1, 2.9-fold, P<0.01, respectively). GR mRNA levels did not differ between corticotroph adenoma and normal corticotrophic cells. Our results may help to understand the increased ACTH production and the resistance to negative feedback suppression by glucocorticoids in canine corticotroph adenomas. These changes in gene expression may have a role in the growth of canine corticotroph adenoma, and help elucidate the pathophysiology of dogs with Cushing's disease.     

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.     

Characteristic transfer of colostrum-derived biologically active substances into cerebrospinal fluid via blood in natural suckling neonatal pigs

The characteristic transfer of colostral components into cerebrospinal fluid (CSF) via serum after natural suckling has been studied by sodium dodecyl sulphate (SDS) electrophoresis, two-dimensional electrophoresis, immunoblot and enzyme linked immunosorbent assay methods in non-suckling pigs. Total protein concentrations in the serum increased immediately after first suckling, reached a peak value at 12 h, corresponding to a 2.3-fold compared with pre-suckling level. The protein concentration in CSF also increased and reached peak value at 6 h corresponding to 1.6-fold compared with presuckling level. IgG in serum not detected before suckling, increased steeply after suckling, IgG, IgM and IgA transported into the serum were observed in completely intact form by immunoblot method. The IgG transported into serum was quickly transferred into CSF after natural suckling in contrast to the case of bovine IgG. Serum concentration of transferrin was maintained at high level before suckling and was not changed by suckling. Transferrin also detected in CSF was not changed by suckling. Bovine lactoferrin (Lf) administered into the intestinal lumen was transported into serum (0.01%) and also detected in CSF after 6 h as undegraded form (3.1%). Thus, homologous IgG and bovine Lf are transported into CSF, suggesting that the transport of macromolecules into CSF is selective in neonatal pigs.     

Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.     

Potential application of simple easy-to-use insertion-deletion (InDel) markers in citrus cultivar identification

We previously developed insertion-deletion (InDel) markers that distinguish three genotypes (two homozygous and one heterozygous) of diverse citrus cultivars. These InDel markers were codominant and could be clearly detected by using simple agarose gel electrophoresis. We sought to establish a method for cultivar identification using these 28 InDel markers to genotype 31 citrus cultivars. The results revealed that a minimum of 6 markers were required to identify individuals using the three-genotype classification method. Furthermore, we found that a simple method for distinguishing between two genotypes (homozygous and heterozygous) could be used to identify individuals using a minimum of 7 markers. Our findings provide a basis for the development of simple and rapid citrus cultivar identification methods.     

Dehydroeffusol Rescues Amyloid β25–35-Induced Spatial Working Memory Deficit

Plant Foods for Human Nutrition - Amyloid β (Aβ) peptides produced from the amyloid precursor protein, a transmembrane protein, are neurotoxic and blocking the neurotoxicity may lead to...     

Corrosive-wear characteristics of diamond-coated cemented carbide tools

Continuous milling tests with diamond-coated and uncoated cemented carbide tools and polycrystalline diamond tools were carried out on air-dried and wet melapi (Shorea sp.) and western redcedar (Thuja plicata D. Don). These tools were examined for corrosive-wear characteristics of the tool-edge appearance, cutting-edge profile, edge recession, and cutting-power consumption. The tool surfaces were observed with a scanning electron microscope and were analyzed by energy-dispersive X-ray spectroscopy. Based on these examinations, the occurrence of corrosive wear while cutting wet woods was confirmed for the uncoated and polycrystalline diamond tools. In contrast, the coated tools did not exhibit corrosive wear, nor did delamitation or wear of the diamond film occur with any of the work materials. The diamond-coated tools showed high resistance not only to mechanical wear but also to corrosive wear.The results of this work were presented at the 48th annual meeting of the Japan Wood Research Society, Shizuoka, April 1998     

Preparation and characterization of fermented seaweed sauce manufactured from low-quality nori (dried and fresh fronds of <Emphasis Type="Italic">Pyropia yezoensis</Emphasis>)

The present study tested processes to manufacture fermented sauce from low-quality nori (dried and fresh fronds of Pyropia yezoensis). The nori sauce was prepared using three tanks with fresh or dried nori cultured in different conditions. In the present study enzymes were not added for the promotion of the degradation of nori, while in a previous study they were. The supernatants of culture mashes obtained from the three tanks were combined, and this low-quality nori sauce (LNS) was characterized and compared with sauces manufactured from high-quality nori, soy, and fish. The LNS showed low concentrations of total nitrogen compounds (0.20 g/100 ml) and free amino acids, and its taste showed a high sourness score as evaluated by a taste-sensing system. On the other hand, the LNS was rich in polysaccharides, which were observed to be readily degraded to lower molecular weight size sugars by heat treatment. The LNS showed little risk for heavy metal or allergen contamination. The obtained sauce product is expected to be commercially utilized as a component of low allergen-risk sauce products after blending with other seasonings without wheat or soy elements.