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Ojima K.,Calpain Project | Ojima K.,Japan Science and Technology Agency | Ojima K.,National Institute of Livestock and Grassland Science NILGS | Kawabata Y.,Japan Science and Technology Agency | And 31 more authors.
Journal of Clinical Investigation | Year: 2010

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle-specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretch-induced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.

Ni K.,Zhengzhou University | Wang Y.,Zhengzhou University | Li D.,Zhengzhou University | Cai Y.,National Institute of Livestock and Grassland Science NILGS | Pang H.,Zhengzhou University
PLoS ONE | Year: 2015

There has been growing interest to develop forage rice as a new feed resource for livestock. This study was to characterize the natural population of lactic acid bacteria (LAB) and select potentially excellent strains for paddy rice silage preparation in China. One hundred and twenty-six strains were isolated and screened from paddy rice silage prepared using a small-scale fermentation system, and ninety-nine of these isolates were considered to be LAB based on their Gram-positive and catalase-negative morphology and the production of most of their metabolic products as lactic acid. These isolates were divided into eight groups (A-H) on the basis of their morphological and biochemical characteristics. The Group A to H strains were identified as Lactobacillus (L.) plantarum subsp. plantarum (species ratio: 8.1%), L. casei (5.1%), Leuconostoc (Ln.) pseudomesenteroides (11.1%), Pediococcus (P.) pentosaceus (24.2%), Enterococcus (E.) mundtii (12.1%), Lactococcus (Lc.) garvieae (15.2%), E. faecium (9.1%) and Lc. lactis subsp. lactis (15.2%) based on sequence analyses of their 16S rRNA and recA genes. P. pentosaceus was the most abundant member of the LAB population in the paddy rice silage. A selected strain, namely L. casei R 465, was found to be able to grow under low pH conditions and to improve the silage quality with low pH and a relatively high content of lactic acid. This study demonstrated that forage paddy rice silage contains abundant LAB species and its silage can be well preserved by inoculation with LAB, and that strain R 465 can be a potentially excellent inoculant for paddy rice silage. © 2015 Ni et al.

Ichikawa A.,Japan National Institute of Advanced Industrial Science and Technology | Tanikawa T.,Japan National Institute of Advanced Industrial Science and Technology | Akagi S.,National Institute of Livestock and Grassland Science NILGS | Ohba K.,Japan National Institute of Advanced Industrial Science and Technology
Journal of Robotics and Mechatronics | Year: 2011

We have developed automated cell cutting by high-precision microfluidic control using a high-response and high-precision syringe pump. A microfluidic chip containing 2 orthogonal microchannels was used for cutting animal cells softened by cytochalasin and aspirated and fixed in 1 microchannel, then a high-velocity microchannel flow was generated from another channel to cut the cell. To control microchannel flow precisely, we made a syringe pump with a minimum flow of 0.35 × 10 -3 μl/min and response time of 10 ms. The syringe pump was connected to the microfluidic chip by a thin, hard Teflon tube to reduce the pressure transmission delay between the syringe pump and microfluidic channel. A microbead control experiment depending on PI control using the syringe pump was conducted to check the microchannel flow delay. Bovine oocytes softened by cytochalasin were injected into the microfluidic chip and bisected by microscopic image volume measurement. This paper reports the automatic cell cutting strategy and system, a result of microbead positioning control, and a result of automatic cell cutting.

Usui F.,Shinshu University | Nakamura Y.,Shinshu University | Nakamura Y.,National Institute of Livestock and Grassland Science NILGS | Yamamoto Y.,National Institute of Genetics | And 4 more authors.
Journal of Poultry Science | Year: 2010

Numerous studies in mammalian species have recently been reported that many stem cells have an ability to efficiently efflux the vital DNA-binding dye Hoechst 33342, and it is called side population (SP) cells. However, few study have been reported on the avian SP cells. It could be possible that concentration of hematopoietic stem cells (HSCs) in birds since the characteristic of SP cells should be shared in various tissues and species. In this study, we first attempted the isolation of SP cells from chicken bone marrow and the assessment by gene expression and morphologic analyses. Bone marrow cells (BMCs) were flushed from the femurs and tibias of chicks aged at 10 days with PBS. The BMCs were layered on lymphocyte separation medium and centrifuged for excluding the erythrocytes. The separated cells were adjusted to 106/ml in HBSS. Hoechst 33342 were added (1.25μg/ml) and incubated 60 to 90 minutes at 37°C. Propidium iodide was added (2μg/ml) to exclude dead cells. The SP cells were isolated with flow cytometer. The sorted cells were stained with May-Gruenwald Giemsa (MG) for morphological analysis and RNA was extracted for gene expression analysis. The avian SP cells which was vanished by addition verapamil counld be separated. The percentage of SP cells in chicken bone marrow was about 2.6%. The morphological analysis by MG staining indicated that the SP cells had a larger nuclear and little cytoplasm which were typical characterisation of mouse HSCs. The pattern of gene expressions (CD34, c-Kit, CD4 and CD8) in SP cells also resembled that of the mouse HSCs. These results suggested that the HSCs could be enriched from avian bone marrow cells. Together with these results, it was concluded that SP is one of powerful tools for concentration of avian stem cells. © 2010 Japan Poultry Science Association.

Yamashita T.,National Institute of Livestock and Grassland Science NILGS | Yokoyama H.,Waste Recycling Research Team | Kanafusa S.,Waste Recycling Research Team | Ogino A.,National Institute of Livestock and Grassland Science NILGS | And 3 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2011

The nitrate-removal activity of a biofilm attached to a perlite carrier from an aerobic bioreactor used for treating dairy farm wastewater was examined by batch experiments under continuous aeration conditions. Despite aeration, the biofilm removed nitrate at a rate of 114.4 mg-N/kg-perlite/h from wastewater containing cow milk and manure. In a clone library analysis of the biofilm, bacteria showing high similarity to the denitrifying bacteria Thauera spp. were detected.

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