Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.     

Synovial fluid total protein concentration as a possible marker for canine idiopathic polyarthritis

Idiopathic polyarthritis (IPA) is a very common inflammatory arthropathy in the dog. Canine IPA is diagnosed mainly by detecting increased number of leukocytes in the synovial fluid (SF), which is easily influenced by glucocorticoid therapy. We obtained 31 SF samples from 24 IPA dogs prior to (n=19) and/or after (n=12) 1 to 10 weeks of glucocorticoid therapy. The SF total protein concentrations of IPA dogs were significantly higher than those of dogs with non-arthritis diseases (n=34) and healthy controls (n=10). Our data revealed that the SF total protein concentrations are not influenced by several weeks of glucocorticoid therapy. Hence, the SF total protein concentration is applicable as a diagnostic marker of canine IPA even when the patients are receiving glucocorticoid therapy.     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

Establishment of a Conditional Transgenic System Using the 2A Peptide in the Female Mouse Germline

Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken β-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.     

Carbon source and trophic position of pelagic fish in coastal waters of south-eastern Izu Peninsula,Japan, identified by stable isotope analysis

ABSTRACT:   It is important to clarify trophic dynamics in marine ecosystems for management of the fishing ground. Organic carbon sources and trophic position of pelagic fishes in the coastal waters of the south-eastern Izu Peninsula, Japan, were examined on the basis of carbon and nitrogen stable isotope distributions. The δ¹³C of the fishes was mostly distributed from −19 to −16‰ for nektonic fishes (13 species of adults and immatures) and planktonic fishes (10 species of larvae and juveniles), close to the δ¹³C values of particulate organic matter and planktonic decapods. These δ¹³C signatures for the inhabitants of the water column were in contrast with the high δ¹³C values (mainly −16 to −13‰) for demersal fishes of Scorpaeniformes and benthic polychaetes collected in the surf zone. These results indicate that nektonic and planktonic fishes depend on phytoplankton for carbon supply. The δ¹⁵N signatures suggest that the trophic position ranged 3.1–4.5 for the nektonic fishes and 2.9–3.7 for the planktonic fishes, premised on trophic level 3 for larval Japanese anchovy Engraulis japonicus . Thus, planktivorous fishes should be mainly assigned to trophic levels 3 and 4 in this area.     

An economic value for ecosystem-based disaster risk reduction using paddy fields in the kasumitei open levee system

Paddy and Water Environment - In this article, we examined the value of the traditional Japanese flood control measure that uses a discontinuous levee (kasumitei). By avoiding the use of...     

Characterization of vitamin B12 compounds from commercially available fish roe products

Although fish roes (or the egg-laden ovary) are seafood products with high nutritional value and are considered abundant in vitamins including vitamin B₁₂, nevertheless, the detailed properties of vitamin B₁₂ have not been fully elucidated in fish roe products. Vitamin B₁₂ content was determined using C18-reversed-phase high-performance liquid chromatography after purification of samples from immunoaffinity columns. Salmon egg-laden ovary products (sujiko), salmon roe products (ikura), dried mullet roe products (bottarga), and white sturgeon roe products (caviar) were found to contain substantial levels of vitamin B₁₂ (more than approximately 15 µg/100 g wet weight). Interestingly, high levels of vitamin B₁₂ per 100 g wet weight have been measured in pollack roe products (tarako) and flying fish roe products (tobiko). Liquid chromatography–electrospray-ionization/tandem mass spectrometry analysis revealed that vitamin B₁₂ was the predominant corrinoid in the evaluated fish roe products examined, while no inactive corrinoid compounds were identified. These results suggest that commercially available fish roe products could be an important source of vitamin B₁₂ for use as a dietary product for humans.

     

Evaluation of resistance to Fusarium head blight in indigenous Japanese species of Agropyron (Elymus)

General (GSI) and specific (SSI) selection indices were constructed for three open-mating populations of sugarcane (Saccharum spp.) seedlings and their ratoon stages. Though specific indices were found to be the most effective in improving mean brix yield in their own source populations, but the GSI was better to SSI in other populations as indicated by higher mean values and more number of superior clones at selection and evaluation stages. This indicated the wider applicability of the GSI over SSI. Inspite of better expected genetic advance through GSI in ratoon crop, the actual gain in brix yield attained, through GSI, was better in seedlings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Infection and colonization of aseptically micropropagated sugarcane seedlings by nitrogen-fixing endophytic bacterium, Herbaspirillum sp. B501gfp1

In this study, we used Herbaspirillum sp. B501gfp1 (B501gfp1), an isolate from wild rice, to investigate the interaction between a non-host nitrogen-fixing endophytic bacterium and micropropagated sugarcane plants under aseptic condition. Two Japanese sugarcane plants (Saccharum sp.) cultivars (cvs) NiF8 and Ni15 were inoculated using B501gfp1 in two inoculum doses of 10⁸ and 10² bacterial-cells-per-milliliter suspension. The results showed that bacterial cells colonized both the root and stem tissues, and colonization was apparent in the intercellular spaces. Higher bacterial numbers were detected in plant tissues inoculated with the higher inoculum concentration treatment. Bacterial numbers also varied between the two cultivars, with the higher values determined in cv Ni15. This study provides evidence that Herbaspirillum sp. B501gfp1, a rice isolate, could colonize sugarcane tissues, suggesting non-specificity of host plant among endophytes.