Comparative proteomic analysis of liver mitochondrial proteins derived from cloned adult pigs reconstructed with Meishan pig fibroblast cells and European pig enucleated oocytes

Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P<0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.     

Epidemiology of hepatitis E virus in indoor-captive cynomolgus monkey colony

A serological survey of hepatitis E virus (HEV) antibody was conducted using 202 adult captive cynomolgus monkeys, who did not show any clinical signs of acute hepatitis. Out of these, 44 monkeys were sero-positive for anti-HEV IgG and all monkeys were negative for anti-HEV IgM. All positive monkeys came from either Vietnam or China, but none from the Philippines, Indonesia, or our facility. Selected 12 monkeys out of positive monkeys from Vietnam, including 9 positive and 3 negative, revealed mostly within the reference ranges for alanine aminotransferase (ALT) and asparatate aminotransferase (AST) by serum biochemistries. Their titers of anti-HEV IgG did not correlate with the concentrations of ALT and AST. Moreover, HEV-RNA could not be detected from any fecal specimens of the 12 monkeys. Thus, monkeys with anti-HEV IgG sero-positive did not seem to be source of the HEV-pollution, because 1) sero-positive monkeys did not excrete HEV-RNA from their feces, and 2) monkeys from the Philippines and Indonesia have remained to be sero-negative for anti-HEV IgG, even if the monkeys were kept in same animal room of our facility. From these results, it could be inferred that primary infection of HEV occurred in the exported countries, but not in our colony. The contamination of HEV in indoor-captive monkeys could be prevented by precise quarantine tests, including ELISA for detecting anti-HEV and RT-PCR for HEV RNA.     

Amputation for histiocytic sarcoma in a cat

A 9-year-old spayed female domestic shorthair cat presented with a skin lesion of the left tarsus. The lesion was biopsied and, based on the microscopic appearance and immunohistochemical characteristics, histiocytic sarcoma was diagnosed. Amputation was performed with improved demeanor seen postoperatively. However, between 44 and 60 days following the surgery, relapse of skin lesions appeared in multiple locations, including at the previous amputation site, and euthanasia was elected. This is the first report of a histiocytic sarcoma treated with amputation in a cat.     

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.     

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