We have previously reported that Saccharomyces cerevisiae has three glutathione peroxidase homologues (GPX1, GPX2, and GPX3) (Inoue, Y., Matsuda, T., Sugiyama, K., Izawa, S., and Kimura, A. (1999) J. Biol. Chem. 274, 27002-27009). Of these, the GPX2 gene product (Gpx2) shows the greatest similarity to phospholipid hydroperoxide glutathione peroxidase. Here we show that GPX2 encodes an atypical 2-Cys peroxiredoxin which uses thioredoxin as an electron donor. Gpx2 was essentially in a reduced form even in mutants defective in glutathione reductase or glutaredoxin under oxidative stressed conditions. On the other hand, Gpx2 was partially oxidized in a mutant defective in cytosolic thioredoxin (trx1⌬trx2⌬) under non-stressed conditions and completely oxidized in tert-butyl hydroperoxide-treated cells of trx1⌬trx2⌬ and thioredoxin reductase-deficient mutant cells. Alanine scanning of cysteine residues of Gpx2 revealed that an intramolecular disulfide bond was formed between Cys 37 and Cys 83 in vivo. Gpx2 was purified to determine whether it functions as a peroxidase that uses thioredoxin as an electron donor in vitro. Gpx2 reduced H 2 O 2 and tert-butyl hydroperoxide in the presence of thioredoxin, thioredoxin reductase, and NADPH (for H 2 O 2 , K m ‫؍‬ 20 M, k cat ‫؍‬ 9.57 ؋ 10 2 s ؊1 ; for tert-butyl hydroperoxide, K m ‫؍‬ 62.5 M, k cat ‫؍‬ 3.68 ؋ 10 2 s ؊1 ); however, it showed remarkably less activity toward these peroxides in the presence of glutathione, glutathione reductase, and NADPH. The sensitivity of yeast cells to tert-butyl hydroperoxide was found to be exacerbated by the co-existence of Ca 2؉ , a tendency that was most obvious in gpx2⌬ cells. Although the redox state of Gpx2 was not affected by Ca 2؉ , the Gpx2 level was markedly increased in the presence of both tert-butyl hydroperoxide and Ca 2؉ . Gpx2 is likely to play an important role in the protection of cells from oxidative stress in the presence of Ca 2؉

Bone marrow derived human mesenchymal stem cells (hMSCs) have attracted great interest from both bench and clinical researchers because of their pluripotency and ease of expansion ex vivo. However, these cells do finally reach a senescent stage and lose their multipotent potential. Proliferation of these cells is limited up to the time of their senescence, which limits their supply, and they may accumulate chromosomal changes through ex vivo culturing. The safe, rapid expansion of hMSCs is critical for their clinical application. Chromosomal aberration is known as one of the hallmarks of human cancer, and therefore it is important to understand the chromosomal stability and variability of ex vivo expanded hMSCs before they are used widely in clinical applications. In this study, we examined the effects of culturing under ambient (20%) or physiologic (5%) O2 concentrations on the rate of cell proliferation and on the spontaneous transformation of hMSCs in primary culture and after expansion, because it has been reported that culturing under hypoxic conditions accelerates the propagation of hMSCs. Bone marrow samples were collected from 40 patients involved in clinical research. We found that hypoxic conditions promote cell proliferation more favourably than normoxic conditions. Chromosomal aberrations, including structural instability or aneuploidy, were detected in significantly earlier passages under hypoxic conditions than under normoxic culture conditions, suggesting that amplification of hMSCs in a low-oxygen environment facilitated chromosomal instability. Furthermore, smoothed hazard-function modelling of chromosomal aberrations showed increased hazard after the fourth passage under both sets of culture conditions, and showed a tendency to increase the detection rate of primary karyotypic abnormalities among donors aged 60 years and over. In conclusion, we propose that the continuous monitoring of hMSCs will be required before they are used in therapeutic applications in the clinic, especially when cells are cultured under hypoxic conditions.

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The MeOH extract of stems of Actinidia arguta promoted proliferation of cultured bone marrow cells and stimulated formation of myeloid colonies from bone marrow cells. (+)-Catechin ( 1) and (-)-epicatechin ( 2) were isolated as active compounds from the MeOH extract. Compounds 1 and 2 stimulated the cell proliferation in a concentration-dependent manner in the range of 1 to 100 mg/mL. Compounds 1 and 2 also stimulated formation of myeloid colonies and enhanced the effect of interleukin-3 (IL-3) to increase the number of colony forming-units in culture (CFU-c). In an ex vivo experiment using a model mouse of decreasing bone marrow functions, orally administrated 1 (100 mg/kg/day) stimulated IL-3-induced CFU-c formation of the bone marrow cells.