[en] It has been reported that the hexavalent chromium compound (Cr(VI)) can induce both p53-dependent and p53-independent apoptosis. While a considerable amount of information is available on the p53-dependent pathway, only little is known about the p53-independent pathway. To elucidate the p53-independent mechanism, the roles of the Ca²⁺-calpain- and mitochondria-caspase-dependent pathways in apoptosis induced by Cr(VI) were investigated. When human lymphoma U937 cells, p53 mutated cells, were treated with 20 μM Cr(VI) for 24 h, nuclear morphological changes and DNA fragmentation were observed. Production of hydroxyl radicals revealed by electron paramagnetic resonance (EPR)-spin trapping, and increase of intracellular calcium ion concentration monitored by digital imaging were also observed in Cr(VI)-treated cells. An intracellular Ca²⁺ chelator, BAPTA-AM, and calpain inhibitors suppressed the Cr(VI)-induced DNA fragmentation. The number of cells showing low mitochondrial membrane potential (MMP), high level of superoxide anion radicals (O₂^-), and high activity of caspase-3, which are indicators of mitochondria-caspase-dependent pathway, increased significantly in Cr(VI)-treated cells. An antioxidant, N-acetyl-L-cysteine (NAC), decreased DNA fragmentation and inhibited the changes in MMP, O₂^- formation, and activation of caspase-3 induced by Cr(VI). No increase of the expressions of Fas and phosphorylated JNK was observed after Cr(VI) treatment. Cell cycle analysis revealed that the fraction of G2/M phase tended to increase after 24 h of treatment, suggesting that Cr(VI)-induced apoptosis is related to the G2 block. These results indicate that Ca²⁺-calpain- and mitochondria-caspase-dependent pathways play significant roles in the Cr(VI)-induced apoptosis via the G2 block, which are independent of JNK and Fas activation. The inhibition of apoptosis and all its signal transductions by NAC suggests that intracellular reactive oxygen species (ROS) are important for both pathways in Cr(VI)-induced apoptosis of U937 cell

[en] Iodine-123-labeled 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid (¹²³I-BMIPP) is widely used to detect myocardial metabolic changes, but the preferred energy substrates in the myocardium would be expected to be altered in the presence of metabolic disorders such as diabetes mellitus (DM). We investigated the metabolism of branched-chain fatty acids in the myocardium of rats with DM. Streptozotocin-induced DM rats were examined 48 h (acute; AD) and 6 weeks (chronic; CD) after injection of streptozotocin. Hearts were excised 15 min or 60 min after injection of 0.185 MBq of ¹²⁵I-BMIPP, followed by homogenization in an EDTA-Tris buffer. The homogenates were subjected to differential centrifugation to obtain the mitochondrial (MF) and cytoplasmic (CF) fractions. Myocardial ¹²⁵I uptake tended to increase in the AD group, but the change was not significant. Myocardial ¹²⁵I uptake at 15 min was significantly lower in the CD group than in the control group, even in the insulin-treated rats (control (CC), 4.4±0.4; not treated (CDN), 3.3±0.5; insulin-treated (CDI), 3.4±0.4 x 10⁴ cpm/g, p<0.05 in each case). The ¹²⁵I count value corrected for the blood count (counts/min (cpm) per g of protein divided by blood cpm) in the MF decreased by 40% at 60 min in the CC group, but increased by 60% in the CDN group. The results of the present study suggest that the myocardial uptake of branched-chain fatty acids is decreased in rats with chronic diabetes, probably as a result of mitochondrial dysfunction. (author)

[en] Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.

[en] Highlights: • Isolated SHED and hDPSCs share features with MSCs. • Transplantation of SHED and hDPSCs induced bone formation. • SHED induced same amount of newly formed bone as hDPSCs and hBMSCs. • SHED is one of the best cell source candidates for reconstructing an alveolar cleft. Cleft lip and palate is the most common congenital anomaly in the orofacial region. Autogenous iliac bone graft, in general, has been employed for closing the bone defect at the alveolar cleft. However, such iliac bone graft provides patients with substantial surgical and psychological invasions. Consequently, development of a less invasive method has been highly anticipated. Stem cells from human exfoliated deciduous teeth (SHED) are a major candidate for playing a significant role in tissue engineering and regenerative medicine. The aim of this study was to elucidate the nature of bone regeneration by SHED as compared to that of human dental pulp stem cells (hDPSCs) and bone marrow mesenchymal stem cells (hBMSCs). The stems cells derived from pulp tissues and bone marrow were transplanted with a polylactic-coglycolic acid barrier membrane as a scaffold, for use in bone regeneration in an artificial bone defect of 4 mm in diameter in the calvaria of immunodeficient mice. Three-dimensional analysis using micro CT and histological evaluation were performed. Degree of bone regeneration with SHED relative to the bone defect was almost equivalent to that with hDPSCs and hBMSCs 12 weeks after transplantation. The ratio of new bone formation relative to the pre-created bone defect was not significantly different among groups with SHED, hDPSCs and hBMSCs. In addition, as a result of histological evaluation, SHED produced the largest osteoid and widely distributed collagen fibers compared to hDPSCs and hBMSCs groups. Thus, SHED transplantation exerted bone regeneration ability sufficient for the repair of bone defect. The present study has demonstrated that SHED is one of the best candidate as a cell source for the reconstruction of alveolar cleft due to the bone regeneration ability with less surgical invasion.