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Articles by Y Kubota
Total Records ( 3 ) for Y Kubota
  S Tada , A Kitanaka , Y Kubota , M Ito and T. Taminato
  Background

We have previously reported an ultrasensitive fluorometric assay for measuring cellular cholesterol. Although this technique is reliable, the use of the assay has limitations due to the requirement for special equipment. It is therefore difficult to apply this assay for the routine determination of cellular cholesterol.

Methods

A colorimetric assay to measure cellular cholesterol was established that utilizes reagents widely used for the measurement of cholesterol in blood samples in conjunction with a random access chemistry analyser ARCHITECT c8000 that is also common in clinical laboratories.

Results

This colorimetric assay showed excellent linearity and recovery. The within-run coefficients of variation were less than 2.5%. The sensitivity of this method, with its detection limit of 1.29 µmol/L, was found to be superior to that of the fluorometric assay we have developed previously. In platelets obtained from patients with diabetes, both the free cholesterol and cholesterol ester content were significantly increased.

Conclusions

Using this technique, measurement of cellular cholesterol could be performed routinely without the requirement for special reagents and equipment.

  J Igarashi , K Shoji , T Hashimoto , T Moriue , K Yoneda , T Takamura , T Yamashita , Y Kubota and H. Kosaka
 

In vascular endothelial cells, specialized microdomains of plasma membrane termed caveolae modulate various receptor signal transduction pathways regulated by caveolin-1, a resident protein of caveolae. We examined whether transforming growth factor-β1 (TGF-β1), a multifunctional cytokine, alters expression levels of caveolin-1 and influences heterologous receptor signaling. Treatment of cultured bovine aortic endothelial cells (BAEC) with TGF-β1 induces marked decreases in caveolin-1 expression in a time- and dose-dependent fashion at both levels of protein and mRNA. A pharmacological inhibitor of activin receptor-like kinase 5 (ALK-5) counteracts caveolin-1 downregulation by TGF-β1, indicating the involvement of ALK-5 receptor subtype for TGF-β1. Sphingosine 1-phosphate (S1P) is a serum-borne angiogenic lipid growth factor that exerts a wide variety of biological actions. S1P modulates G protein-coupled S1P receptors, activating downstream molecules kinases AMP-activated protein kinase (AMPK), and Akt as well as a small G protein Rac1, ultimately to promote migration. Because S1P receptor signaling is associated with caveolae/caveolin-1, we examined whether pretreatment with TGF-β1 enhances effects of S1P on BAEC. Whereas S1P alone evokes robust BAEC responses to S1P, pretreatment with TGF-β1 leads to even higher magnitudes of S1P-elicited signaling responses and cell migration. Conversely, genetic knockdown of caveolin-1 using small interfering RNA mimics TGF-β1-induced promotion of BAEC responses to S1P. Collectively, these data demonstrate that TGF-β1 downregulates caveolin-1 of cultured endothelial cells, involving ALK-5 receptor subtype. Because downregulation of caveolin-1 by TGF-β1 promotes subsequent heterologous receptor signaling by S1P, these results may also identify novel point of cross-talk between cytokines and sphingolipids within endothelial signal transduction machineries.

  S Morikawa , Y Mabuchi , Y Kubota , Y Nagai , K Niibe , E Hiratsu , S Suzuki , C Miyauchi Hara , N Nagoshi , T Sunabori , S Shimmura , A Miyawaki , T Nakagawa , T Suda , H Okano and Y. Matsuzaki
 

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and can give rise to multiple mesenchymal lineages. Because MSCs have only been isolated from tissue in culture, the equivalent cells have not been identified in vivo and little is known about their physiological roles or even their exact tissue location. In this study, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of MSCs (PDGFR+Sca-1+CD45TER119) from adult mouse bone marrow. Individual MSCs generated colonies at a high frequency and could differentiate into hematopoietic niche cells, osteoblasts, and adipocytes after in vivo transplantation. Naive MSCs resided in the perivascular region in a quiescent state. This study provides the useful method needed to identify MSCs as defined in vivo entities.

 
 
 
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