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Biomedical subjects

Naoya Sakamoto

Publications and source records attributed to Naoya Sakamoto.

26 records · Page 2Linked to original sources
PubMed2003

Hepatitis C virus nonstructural protein 5A inhibits tumor necrosis factor-alpha-mediated apoptosis in Huh7 cells.

To analyze the influence of hepatitis C virus nonstructural protein 5A (NS5A) on apoptosis, we established Huh7 cells that stably express NS5A, and induced apoptosis using tumor necrosis factor (TNF)-alpha. The viability of control Huh7 cells was reduced to 40%, compared with untreated cells, after TNF-alpha treatment, whereas that of Huh7-NS5A cells was reduced only to 80%. DNA fragmentation also decreased to <50% in Huh7-NS5A compared with control cells. Nuclear factor-kappaB activation was the same in both cell types, whereas caspase-8, -9, and -3 activity was decreased in Huh7-NS5A cells, compared with control cells, which indicates that the inhibition of caspase-8 activation is responsible for the antiapoptotic effect of the NS5A protein. The coexpression of NS5A did not inhibit apoptosis induced by caspase-8 or Fas-associating death domain protein expression. These findings suggest that the NS5A protein inhibits the apoptotic effect of TNF-alpha upstream of caspase-8 in the apoptosis cascade.

Apoptosis↗
PubMed2003

Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs.

Small interfering RNAs (siRNAs) efficiently inhibit gene expression by RNA interference. Here, we report efficient inhibition, by both synthetic and vector-derived siRNAs, of hepatitis C virus (HCV) replication, as well as viral protein synthesis, using an HCV replicon system. The siRNAs were designed to target the 5' untranslated region (5' UTR) of the HCV genome, which has an internal ribosomal entry site for the translation of the entire viral polyprotein. Moreover, the 5' UTR is the most conserved region in the HCV genome, making it an ideal target for siRNAs. Importantly, we have identified an effective site in the 5' UTR at which approximately 80% suppression of HCV replication was achieved with concentrations of siRNA as low as 2.5 nM. Furthermore, DNA-based vectors expressing siRNA against HCV were also effective, which might allow the efficient delivery of RNAi into hepatocytes in vivo using viral vectors. Our results support the feasibility of using siRNA-based gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

5' Untranslated Regions↗
PubMed2003

Oxygen tension modulates Ca2+ response to flow stimulus in endothelial cells exposed to hydrostatic pressure.

The effects of oxygen gas tensions and hydrostatic pressure on intracellular calcium, [Ca2+]i, response to a flow stimulus in endothelial cells were investigated. Cultured bovine aortic endothelial cells (BAECs) were exposed to a hydrostatic pressure of 100 mmHg under low oxygen gas tensions and were subsequently subjected to a 1 minute mechanical stimulation of fluid shear stress of 20 dynes/cm2. The [Ca2+]i response in BAECs was measured using a fluorescent indicator, Calcium Green-1. The maximum intensity for low oxygen tension was significantly lower than that for normal oxygen tension, which provides evidence that low oxygen tension regulates cellular functions downward. Moreover, preloading of hydrostatic pressure also reduced the increases in [Ca2+]i. These results suggest that BAECs in venous system, where oxygen tension and hydrostatic pressure are lower than those in arterial system, may be less sensitive to fluid flow. A separate observation showed that low oxygen tension did not significantly affect the cell morphology. In contrast, BAECs exposed to hydrostatic pressure showed marked elongation with no predominant orientation and the F-actin filament distributions were rearranged, indicating centrally located thick stress fibers. For better understanding of endothelial cell physiology, it is very important to elucidate the effect of oxygen gas tensions together with mechanical environment on endothelial cell responses.

Animals↗
PubMed2002

Role of sarcolemmal K(ATP) channels in cardioprotection against ischemia/reperfusion injury in mice.

Recently it has been postulated that mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels rather than sarcolemmal K(ATP) (sarcK(ATP)) channels are important as end effectors and/or triggers of ischemic preconditioning (IPC). To define the pathophysiological significance of sarcK(ATP) channels, we conducted functional experiments using Kir6.2-deficient (KO) mice. Metabolic inhibition with glucose-free, dinitrophenol-containing solution activated sarcK(ATP) current and shortened the action potential duration in ventricular cells isolated from wild-type (WT) but not KO mice. MitoK(ATP) channel function was preserved in KO ventricular cells. In anesthetized mice, IPC reduced the infarct size in WT but not KO mice. Following global ischemia/reperfusion, the increase of left ventricular end-diastolic pressure during ischemia was more marked, and the recovery of contractile function was worse, in KO hearts than in WT hearts. Treatment with HMR1098, a sarcK(ATP) channel blocker, but not 5-hydroxydecanoate, a mitoK(ATP) channel blocker, produced a deterioration of contractile function in WT hearts comparable to that of KO hearts. These findings suggest that sarcKATP channels figures prominently in modulating ischemia/reperfusion injury in the mouse. The rapid heart rate of the mouse (>600 beats per minute) may magnify the relative importance of sarcK(ATP) channels during ischemia, prompting caution in the extrapolation of the conclusions to larger mammals.

Animals↗