Atsuhiro Kanayama

Platinum nanoparticle is a useful scavenger of superoxide anion and hydrogen peroxide

Bimetallic nanoparticles consisting of gold and platinum were prepared by a citrate reduction method and complementarily stabilized with pectin (CP-Au/Pt). The percent mole ratio of platinum was varied from 0 to 100%. The CP-Au/Pt were alloy-structured. They were well dispersed in water. The average diameter of platinum nanoparticles (CP-Pt) was 4.7 ± 1.5 nm. Hydrogen peroxide (H2O2) was quenched by CP-Au/Pt consisting of more than 50% platinum whereas superoxide anion radical () was quenched by any CP-Au/Pt. The CP-Au/Pt quenched these two reactive oxygen species in dose-dependent manners. The CP-Pt is the strongest quencher. The CP-Pt decomposed H2O2 and consequently generated O2 like catalase. The CP-Pt actually quenched which was verified by a superoxide dismutase (SOD) assay kit. This quenching activity against persisted like SOD. Taken together, CP-Pt may be a SOD/catalase mimetic which is useful for medical treatment of oxidative stress diseases.

Effects of a potent antioxidant, platinum nanoparticle, on the lifespan of Caenorhabditis elegans

We have shown that platinum nanoparticles (nano-Pt) are a superoxide dismutase (SOD)/catalase mimetic. Various data have shown extension of the Caenorhabditis elegans lifespan by antioxidant treatment. The present study was designed to elucidate the survival benefit conferred by nano-Pt, as compared to the well-known SOD/catalase mimetic EUK-8. At 0.5mM, nano-Pt significantly extended the lifespan of wild-type N2 nematodes and at 0.25 and 0.5mM, nano-Pt recovered the shortened lifespan of the mev-1(kn1) mutant, which is due to excessive oxidative stress. In both instances, EUK-8 at 0.05, 0.5, and 5mM did not extend nematode lifespan. Even when 0.4M paraquat was loaded exogenously, nano-Pt (0.1 and 0.5mM) and EUK-8 (0.5 and 5mM) were effective in rescuing worms. Moreover, 0.5mM nano-Pt significantly reduced the accumulation of lipofuscin and ROS induced by paraquat. We measured the in vitro dose-dependent quenching of O(2)(-) and H(2)O(2), indicating that nano-Pt is a more potent SOD/catalase mimetic than EUK-8. Nano-Pt prolonged the worm lifespan, regardless of thermotolerance or dietary restriction. Taken together, nano-Pt has interesting anti-ageing properties.

Two Mechanistically and Temporally Distinct NF-κB Activation Pathways in IL-1 Signaling

The cooperative activation of NF-κB by two distinct pathways that diverge at TRAF6 critically contributes to the IL-1–dependent inflammatory response. A One-Two Punch Members of the interleukin-1 (IL-1) family of cytokines stimulate proinflammatory responses through their activation of the transcription factors nuclear factor κB (NF-κB) and activating protein 1 (AP-1). The binding of IL-1 to its receptor complex triggers the activation of the E3 ubiquitin ligase and scaffold protein TRAF6 and its association with the mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) TAK1, which leads to the activation of NF-κB. Another MAP3K, MEKK3, is also involved in IL-1–mediated activation of NF-κB, but how TAK1 and MEKK3 might physically or functionally interact has been unclear. Yamazaki et al. showed that early IL-1 signaling induced the formation of a transient complex of TRAF6, TAK1, and MEKK3. Formation of this complex, which was dependent on TRAF6-mediated ubiquitination of TAK1, led to NF-κB activation. In a later phase of IL-1 signaling, TRAF6 activated NF-κB in a MEKK3-dependent, TAK1-independent manner. Together, these two pathways resulted in the prolonged activation of NF-κB required to trigger an effective proinflammatory response. The cytokine interleukin-1 (IL-1) mediates immune and inflammatory responses by activating the transcription factor nuclear factor κB (NF-κB). Although transforming growth factor–β–activated kinase 1 (TAK1) and mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3) are both crucial for IL-1–dependent activation of NF-κB, their potential functional and physical interactions remain unclear. Here, we showed that TAK1-mediated activation of NF-κB required the transient formation of a signaling complex that included tumor necrosis factor receptor–associated factor 6 (TRAF6), MEKK3, and TAK1. Site-specific, lysine 63–linked polyubiquitination of TAK1 at lysine 209, likely catalyzed by TRAF6 and Ubc13, was required for the formation of this complex. After TAK1-mediated activation of NF-κB, TRAF6 subsequently activated NF-κB through MEKK3 independently of TAK1, thereby establishing continuous activation of NF-κB, which was required for the production of sufficient cytokines. Therefore, we propose that the cooperative activation of NF-κB by two mechanistically and temporally distinct MEKK3-dependent pathways that diverge at TRAF6 critically contributes to immune and inflammatory systems.

Platinum nanoparticles have an activity similar to mitochondrial NADH:ubiquinone oxidoreductase

This study was designed to examine if platinum nanoparticles have an activity similar to mitochondrial complex I, NADH:ubiquinone oxidoreductase. Platinum nanoparticles were prepared by a citrate reduction of H(2)PtCl(6) and protected by citrate itself and pectin (CP-Pt). Time- and dose-dependent decreases in NADH and a time-dependent increase in NAD(+) were observed in the presence of 50 microM CP-Pt; these observations were made using a spectrophotometric method in which the maximum absorption spectra at 340 and 260 nm were used for NADH and NAD(+), respectively. The required platinum concentration in CP-Pt to achieve a 50% oxidation of NADH for 3h was approximately 20 microM, and this NADH oxidation did not require oxygen as an electron acceptor. We also verified NAD(+) formation using an NAD(+)/NADH quantification kit. The absorption peak shift from 278 to 284 nm of 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (CoQ(1)) was observed by incubating CoQ(1) with CP-Pt in an aqueous buffer. A further analysis with HPLC revealed the reduction of CoQ(1) to CoQ(1)H(2) by CP-Pt. As a whole, platinum nanoparticles have an NADH:ubiquinone oxidoreductase-like activity. This suggests that platinum nanoparticles are a potential medicinal substance for oxidative stress diseases with suppressed mitochondrial complex I.

Apoptosis triggered by phagocytosis-related oxidative stress through FLIPs down-regulation and JNK activation

Tumor necrosis factor‐α (TNF‐α)‐activated neutrophils phagocytose and eliminate bacteria by using such oxidants as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), which is produced from H2O2 by myeloperoxidase (MPO). Thereafter, neutrophils eventually undergo apoptosis to prevent excessive inflammation. However, it is unclear how this process is regulated. Here, we show that cotreatment of TNF‐α‐resistant neutrophilic HL‐60 cells with taurine chloramine (TauCl), a detoxified form of HOCl, and TNF‐α renders them susceptible to apoptosis, mostly by preventing nuclear factor‐κB (NF‐κB) activation. Of several NF‐κB target genes tested, FLICE inhibitory protein short form (FLIPS) was specifically down‐regulated by TauCl. TNF‐α/TauCl cotreatment‐induced apoptosis was largely blocked by stable expression of FLIPS. Cotreatment with TNF‐α and H2O2 promoted apoptotic signaling via MPO activation and subsequent attenuation of FLIPS expression. TNF‐α priming with H2O2 or bacteria caused MPO‐dependent apoptosis in human neutrophils. However, FLIPS knock‐down by siRNA did not affect the viability of cells treated with TNF‐α, implying that TauCl may affect another pathway in TNF‐α‐driven apoptosis. Indeed, oxidization of thioredoxin‐1 (Trx‐1) by TauCl induced the activation of apoptosis signal‐regulating kinase 1 (ASK1) and cJun N‐terminal kinase (JNK), thereby triggering TNF‐α‐mediated apoptosis. Taken together, these results indicate that the antiapoptotic signaling induced by TNF‐α via NF‐κB activation can be altered to promote apoptosis via H2O2‐MPO‐mediated FLIPS down‐regulation and JNK activation.

Identification of a Taurine Transport Inhibitory Substance in Sesame Seeds

An ethanol extract from sesame seeds inhibited the taurine uptake in human intestinal epithelial Caco-2 cells. The uptake of such α-amino acids as leucine and glutamic acid was not inhibited by the extract, indicating that this inhibition is specific to the taurine uptake. The unknown inhibitor in the sesame extract was purified by reversed-phase HPLC by monitoring the inhibitory effect on taurine uptake. The isolated substance was identified as lysophosphatidylcholine, linoleoyl (Lyso-PC), by NMR and MS analysis. Lyso-PC inhibited the taurine uptake in a dose-dependent manner with an IC50 value of approximately 200 μM. Although Lyso-PC is known to be a surface active and cell lytic compound, neither damage nor loss of integrity of the Caco-2 cell monolayer was apparent after treating with 200 μM Lyso-PC. Inhibition was observed by incubating cells with Lyso-PC for only 1 min prior to the uptake experiments. These results suggest the direct effect of Lyso-PC on the cell membrane to be the main mechanism for this inhibition. Lyso-PC may play a role in the regulation of certain intestinal transporters.

ARD1 binding to RIP1 mediates doxorubicin-induced NF-κB activation.

NF-κB is activated by several cellular stresses. Of these, the TNFα-induced activation pathway has been examined in detail. It was recently reported that receptor-interacting protein 1 (RIP1) is involved in DNA damage-induced NF-κB activation by forming a complex with the p53 interacting death domain protein (PIDD) and NF-κB essential modulator (NEMO) in the nucleus, although the underlying mechanism of this interaction has yet to be clarified. This study shows that siRNA knock-down of arrest-defective 1 protein (ARD1) abrogated doxorubicin- but not TNFα-induced activation. Conversely, the over-expression of ARD1 greatly enhanced NF-κB activation induced by doxorubicin. Immunoprecipitation experiments revealed that ARD1 interacted with RIP1 via the acetyltransferase domain. Furthermore, the over-expression of several domain-deleted ARD1 constructs demonstrated that the N-terminal and acetyltransferase domains of ARD1 were required for doxorubicin-induced NF-κB activation. Treatment of deacetylase inhibitor, trichostatin A, significantly increased doxorubicin-induced NF-κB activation in the presence of ARD1 but not acetyltransferase-defective ARD1 mutant. Moreover, N-terminal domain-deleted ARD1 could not be localized in the nucleus in response to doxorubicin treatment. These data indicate that the interaction between ARD1 and RIP1 plays an important role in the DNA damage-induced NF-κB activation, and that the acetyltransferase activity of ARD1 and its localization in to the nucleus are involved in such stress response.

Taurine Is Involved in Oxidation of IκBα at Met45

The neutrophil is a phagocyte and functions to kill invading microorganisms in the defense of the host1. Phagocytosis rapidly turns on the respiratory burst, resulting in release of superoxide anion radical (O2.-)in the phagosomes as illustrated in Fig. 1. Superoxide anion radical is formed from (O2) oxygen by one-electron reduction that is catalyzed by NADPH oxidase assembled for activation at the phagosomal membrane. Cytosolic NADPH is the electron donor for this reduction. Spontaneous dismutation converts O2.- to hydrogen peroxide (H202) in phagosomes: 2O2.- + 2H+ → 02 + H202. Myeloperoxidase (MPO) also released into phagosomes catalyzes the production of hypochlorous acid (HClO) from H202and chloride ion (Cl-). The final product, HClO, is a strong oxidant that most effectively kills microorganisms. However, reactive oxygen species (ROS) such as, H202and HClO cause damage to surrounding tissues as observed in inflammation. If HClO infiltrates into neutrophils themselves, it impairs their proteins, lipids and genes, eventually leading to their cell death. Because taurine, the most abundant free amino acid in the neutrophil, has a high reactivity with HClO, taurine chloramine (TauCl) is scarcely synthesized when taurine encounters HClO. The cytotoxicity of TauCl is much less than that of HClO and hence the biological function of taurine has been thought to be antioxidation and detoxification in the neutrophil2.