Takeki Hamasaki

Kinetic analysis of superoxide anion radical-scavenging and hydroxyl radical-scavenging activities of platinum nanoparticles

There are few reports on the physiological effects of metal nanoparticles (nps), especially with respect to their functions as scavengers for superoxide anion radical (O2(.-)) and hydroxyl radical (.OH). We tried to detect the scavenging activity of Pt nps using a hypoxanthine-xanthine oxidase system for O2(.-) and using a Fenton and a UV/H2O2 system for .OH. Electron spin resonance analysis revealed that 2 nm particle size Pt nps have the ability to scavenge O2(.-) and .OH. The calculated rate constant for the O2(.-)-scavenging reaction was 5.03 +/- 0.03 x 10(7) M (-1) s (-1). However, the analysis of the Fenton and UV/H 2O 2 system in the presence of Pt nps suggested that the .OH-scavenging reaction cannot be determined in both systems. Among particle sizes tested from 1 to 5 nm, 1 nm Pt nps showed the highest O2(.-)-scavenging ability. Almost no cytotoxicity was observed even after adherent cells (TIG-1, HeLa, HepG2, WI-38, and MRC-5) were exposed to Pt nps at concentrations as high as 50 mg/L. Pt nps scavenged intrinsically generated reactive oxygen species (ROS) in HeLa cells. Additionally, Pt nps significantly reduced the levels of intracellular O2(.-) generated by UVA irradiation and subsequently protected HeLa cells from ROS damage-induced cell death. These findings suggest that Pt nps may be a new type of antioxidant capable of circumventing the paradoxical effects of conventional antioxidants.

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca2+ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca2+ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus.

Suppressive effects of electrolyzed reduced water on alloxan-induced apoptosis and type 1 diabetes mellitus

Electrolyzed reduced water, which is capable of scavenging reactive oxygen species, is attracting recent attention because it has shown improved efficacy against several types of diseases including diabetes mellitus. Alloxan produces reactive oxygen species and causes type 1 diabetes mellitus in experimental animals by irreversible oxidative damage to insulin-producing β-cells. Here, we showed that electrolyzed reduced water prevented alloxan-induced DNA fragmentation and the production of cells in sub-G1 phase in HIT-T15 pancreatic β-cells. Blood glucose levels in alloxan-induced type 1 diabetes model mice were also significantly suppressed by feeding the mice with electrolyzed reduced water. These results suggest that electrolyzed reduced water can prevent apoptosis of pancreatic β-cells and the development of symptoms in type 1 diabetes model mice by alleviating the alloxan-derived generation of reactive oxygen species.

Extension of the Lifespan of Caenorhabditis elegans by the Use of Electrolyzed Reduced Water

Electrolyzed reduced water (ERW) has attracted much attention because of its therapeutic effects. In the present study, a new culture medium, which we designated Water medium, was developed to elucidate the effects of ERW on the lifespan of Caenorhabditis elegans. Wild-type C. elegans had a significantly shorter lifespan in Water medium than in conventional S medium. However, worms cultured in ERW-Water medium exhibited a significantly extended lifespan (from 11% to 41%) compared with worms cultured in ultrapure water-Water medium. There was no difference between the lifespans of worms cultured in ERW-S medium and ultrapure water-S medium. Nematodes cultured in ultrapure water-Water medium showed significantly higher levels of reactive oxygen species than those cultured in ultrapure water-S medium. Moreover, ERW-Water medium significantly reduced the ROS accumulation induced in the worms by paraquat, suggesting that ERW-Water medium extends the longevity of nematodes at least partly by scavenging ROS.

Mechanism of the Lifespan Extension of Caenorhabditis elegans by Electrolyzed Reduced Water—Participation of Pt Nanoparticles

Electrolyzed reduced water (ERW) contains a large amount of molecular hydrogen and a small amount of Pt nanoparticles (Pt NPs). We have found that ERW significantly extended the lifespan of Caenorhabditis elegans in a novel culture medium designated Water Medium. In this study, we found that synthetic Pt NPs at ppb levels significantly extended the nematode lifespan and scavenged reactive oxygen species (ROS) in the nematode induced by paraquat treatment. In contrast, a high concentration of dissolved molecular hydrogen had no significant effect on the lifespan of the nematode. These findings suggest that the Pt NPs in ERW, rather than the molecular hydrogen, extend the longevity of the nematode, at least partly by scavenging ROS.

Removal efficiency of radioactive cesium and iodine ions by a flow-type apparatus designed for electrochemically reduced water production.

The Fukushima Daiichi Nuclear Power Plant accident on March 11, 2011 attracted people’s attention, with anxiety over possible radiation hazards. Immediate and long-term concerns are around protection from external and internal exposure by the liberated radionuclides. In particular, residents living in the affected regions are most concerned about ingesting contaminated foodstuffs, including drinking water. Efficient removal of radionuclides from rainwater and drinking water has been reported using several pot-type filtration devices. A currently used flow-type test apparatus is expected to simultaneously provide radionuclide elimination prior to ingestion and protection from internal exposure by accidental ingestion of radionuclides through the use of a micro-carbon carboxymethyl cartridge unit and an electrochemically reduced water production unit, respectively. However, the removability of radionuclides from contaminated tap water has not been tested to date. Thus, the current research was undertaken to assess the capability of the apparatus to remove radionuclides from artificially contaminated tap water. The results presented here demonstrate that the apparatus can reduce radioactivity levels to below the detection limit in applied tap water containing either 300 Bq/kg of 137Cs or 150 Bq/kg of 125I. The apparatus had a removal efficiency of over 90% for all concentration ranges of radio–cesium and –iodine tested. The results showing efficient radionuclide removability, together with previous studies on molecular hydrogen and platinum nanoparticles as reactive oxygen species scavengers, strongly suggest that the test apparatus has the potential to offer maximum safety against radionuclide-contaminated foodstuffs, including drinking water.

Suppressive effects of electrochemically reduced water on matrix metalloproteinase-2 activities and in vitro invasion of human fibrosarcoma HT1080 cells

It has been demonstrated that hydrogen peroxide (H2O2) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H2O2 in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H2O2-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH2-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H2O2-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect.

Anti-diabetes effect of water containing hydrogen molecule and Pt nanoparticles

) and scavenges reactive oxygenspecies (ROS) to protect DNA from oxidative damage[1]. ERW also contains small amounts of Pt nanoparti-cles (NPs) and elongates the lifespan ofC. elegans [2].Pt NPs are newly recognized multi-functional ROS sca-vengers [3]. ERW exhibits anti-diabetes effectsin vitroand in vivo [4-6][7]. We proposed mineral nanoparticleactive hydrogen reduced water hypothesis to explain theactivation mechanism of H

Electrochemically Reduced Water Protects Neural Cells from Oxidative Damage

Aging-related neurodegenerative disorders are closely associated with mitochondrial dysfunction and oxidative stresses and their incidence tends to increase with aging. Brain is the most vulnerable to reactive species generated by a higher rate of oxygen consumption and glucose utilization compared to other organs. Electrochemically reduced water (ERW) was demonstrated to scavenge reactive oxygen species (ROS) in several cell types. In the present study, the protective effect of ERW against hydrogen peroxide (H2O2) and nitric oxide (NO) was investigated in several rodent neuronal cell lines and primary cells. ERW was found to significantly suppress H2O2 (50–200 μM) induced PC12 and SFME cell deaths. ERW scavenged intracellular ROS and exhibited a protective effect against neuronal network damage caused by 200 μM H2O2 in N1E-115 cells. ERW significantly suppressed NO-induced cytotoxicity in PC12 cells despite the fact that it did not have the ability to scavenge intracellular NO. ERW significantly suppressed both glutamate induced Ca2+ influx and the resulting cytotoxicity in primary cells. These results collectively demonstrated for the first time that ERW protects several types of neuronal cells by scavenging ROS because of the presence of hydrogen and platinum nanoparticles dissolved in ERW.

A rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.