Yoshihisa Hagihara

Mapping the core of the |[beta]|2-microglobulin amyloid fibril by H/D exchange

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of β2-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid β-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded β-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.

Association of zinc ion release and oxidative stress induced by intratracheal instillation of ZnO nanoparticles to rat lung

Zinc oxide (ZnO) nanoparticles are one of the important industrial nanoparticles. The production of ZnO nanoparticles is increasing every year. On the other hand, it is known that ZnO nanoparticles have strong cytotoxicity. In vitro studies using culture cells revealed that ZnO nanoparticles induce severe oxidative stress. However, the in vivo influence of ZnO nanoparticles is still unclear. In the present study, rat lung was exposed to ZnO nanoparticles by intratracheal instillation, and the influences of ZnO nanoparticles to the lung in the acute phase, particularly oxidative stress, were examined. Additionally, in vitro cellular influences of ZnO nanoparticles were examined using lung carcinoma A549 cells and compared to in vivo examinations. The ZnO nanoparticles used in this study released zinc ion in both dispersions. In the in vivo examinations, ZnO dispersion induced strong oxidative stress in the lung in the acute phase. The oxidative stress induced by the ZnO nanoparticles was stronger than that of a ZnCl(2) solution. Intratracheal instillation of ZnO nanoparticles induced an increase of lipid peroxide, HO-1 and alpha-tocopherol in the lung. The ZnO nanoparticles also induced strong oxidative stress and cell death in culture cells. Intracellular zinc level and reactive oxygen species were increased. These results suggest that ZnO nanoparticles induce oxidative stress in the lung in the acute phase. Intracellular ROS level had a high correlation with intracellular Zn(2+) level. ZnO nanoparticles will stay in the lung and continually release zinc ion, and thus stronger oxidative stress is induced.

The role of disulfide bond in the amyloidogenic state of β2‐microglobulin studied by heteronuclear NMR

β2‐Microglobulin (β2‐m) is a major component of dialysis‐related amyloid fibrils. Although recombinant β2‐m forms needle‐like fibrils by in vitro extension reaction at pH 2.5, reduced β2‐m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of β2‐m in the acid‐denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. {1H}‐15N NOE at the regions between the two cysteine residues (Cys25–Cys80) revealed a marked difference in the pico‐ and nanosecond time scale dynamics between that the acid‐denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, ΔCα, ΔCO, and ΔHα, and 3JHNHα coupling constants indicated that both the oxidized and reduced β2‐m at pH 2.5 have marginal α‐helical propensity at regions close to the C‐terminal cysteine, although it is a β‐sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of β2‐m, and that the marginal helical propensity at the C‐terminal regions might play a role in modifying this potential.

α-Tocopherol suppresses lipid peroxidation and behavioral and cognitive impairments in the Ts65Dn mouse model of Down syndrome

It is widely accepted that oxidative stress is involved in the pathogenesis of Down syndrome, but the effectiveness of antioxidant treatment remains inconclusive. We tested whether chronic administration of α-tocopherol ameliorates the cognitive deficits exhibited by Ts65Dn mice, a mouse model of Down syndrome. α-Tocopherol was administered to pregnant Ts65Dn females, from the day of conception throughout the pregnancy, and to pups over their entire lifetime, from birth to the end of the behavioral testing period. Cognitive deficits were confirmed for Ts65Dn mice fed a control diet, revealing reduced anxiety or regardlessness in the elevated-plus maze task test and spatial learning deficits in the Morris water maze test. However, supplementation with α-tocopherol attenuated both cognitive impairments. In addition, we found that levels of 8-iso-prostaglandin F(2α) in brain tissue and hydroxyoctadecadienoic acid and 7-hydroxycholesterol in the plasma of Ts65Dn mice were higher than those of control mice. Supplementation with α-tocopherol decreased levels of lipid peroxidation products in Ts65Dn mice. Furthermore, we found out that α-tocopherol improved hypocellularity in the hippocampal dentate gyrus of Ts65Dn mice. These results imply that α-tocopherol supplementation from an early stage may be an effective treatment for the cognitive deficits associated with Down syndrome.

Association of the physical and chemical properties and the cytotoxicity of metal oxide nanoparticles: metal ion release, adsorption ability and specific surface area

Association of cellular influences and physical and chemical properties were examined for 24 kinds of industrial metal oxide nanoparticles: ZnO, CuO, NiO, Sb(2)O(3), CoO, MoO(3), Y(2)O(3), MgO, Gd(2)O(3), SnO(2), WO(3), ZrO(2), Fe(2)O(3), TiO(2), CeO(2), Al(2)O(3), Bi(2)O(3), La(2)O(3), ITO, and cobalt blue pigments. We prepared a stable medium dispersion for each nanoparticle and examined the influence on cell viability and oxidative stress together with physical and chemical characterizations. ZnO, CuO, NiO, MgO, and WO(3) showed a large amount of metal ion release in the culture medium. The cellular influences of these soluble nanoparticles were larger than insoluble nanoparticles. TiO(2), SnO(2), and CeO(2) nanoparticles showed strong protein adsorption ability; however, cellular influences of these nanoparticles were small. The primary particle size and the specific surface area seemed unrelated to cellular influences. Cellular influences of metal oxide nanoparticles depended on the kind and concentrations of released metals in the solution. For insoluble nanoparticles, the adsorption property was involved in cellular influences. The primary particle size and specific surface area of metal oxide nanoparticles did not affect directly cellular influences. In conclusion the most important cytotoxic factor of metal oxide nanoparticles was metal ion release.

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 °C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.

Cellular responses induced by cerium oxide nanoparticles: induction of intracellular calcium level and oxidative stress on culture cells

Cerium oxide (CeO(2)) is an important metal oxide used for industrial products. Many investigations about the cellular influence of CeO(2) nanoparticles have been done, but results are contradictory. It has been reported that CeO(2) nanoparticles have an anti-oxidative effect in cells, but it has also been reported that CeO(2) nanoparticles induce oxidative stress. We investigated the potential influence on cells and the mechanisms induced by CeO(2) nanoparticles in vitro. We prepared a stable CeO(2) culture medium dispersion. Cellular responses in CeO(2) medium-exposed cells were examined. Cellular uptake of CeO(2) nanoparticles was observed. After 24-h exposure, a high concentration of CeO(2) nanoparticles (∼200 mg/ml) induced an increase in the intracellular level of reactive oxygen species (ROS); a low concentration of CeO(2) nanoparticles induced a decrease in the intracellular ROS level. On the other hand, exposure of CeO(2) nanoparticle for 24 h had little influence on the cell viability. Exposure of CeO(2) nanoparticles increased the intracellular Ca(2+) concentration and also Calpain was activated. These results suggest that CeO(2) nanoparticles have a potential to induce intracellular oxidative stress and increase the intracellular Ca(2+) level, but these influences are small.

Assessment of antioxidant capacity for scavenging free radicals in vitro: A rational basis and practical application

With increasing evidence showing the involvement of oxidative stress induced by free radicals in the development of various diseases, the role of radical-scavenging antioxidants has received much attention. Although many randomized controlled clinical trials do not support the beneficial effects of indiscriminate supplementation of antioxidants, more recent studies suggest that antioxidants such as vitamin E may be effective for prevention and treatment of some diseases when given to the right subjects at the right time. Many studies on the antioxidant capacity assessed by various available methods showed inconsistent results and the assessment of antioxidant capacity has been the subject of extensive studies and arguments. This study was performed to elucidate the basic chemistry required for the development of a reliable method for the assessment of antioxidant capacity for radical scavenging in vitro. In this study, the capacity of α-tocopherol and its related compounds, ascorbic acid, and uric acid for scavenging radicals was assessed from their effects on the rate of decay of hydrophilic and lipophilic probes with various reactivities toward free radicals induced by hydrophilic and lipophilic radicals in homogeneous solution and heterogeneous micelle systems. Fluorescein, pyranine, and pyrogallol red were used as hydrophilic probes, and BODIPY and N,N-diphenyl-p-phenylenediamine were used as lipophilic probes. We show that the rate and amount of radical scavenging by antioxidants, termed the antioxidant radical absorbance capacity, could be assessed by an appropriate combination of radical initiator and probe. This method was applied to the assessment of radical-scavenging capacity of human plasma, wine, and green tea powder.

Trifluoroethanol-induced conformational transition of hen egg-white lysozyme studied by small-angle X-ray scattering

The trifluoroethanol (TFE)‐induced conformational transition of hen lysozyme was studied with the combined use of far‐UV circular dichroism (CD) and small‐angle X‐ray scattering. At pH 2.0 and 20°C, the addition of TFE to the native lysozyme induced a cooperative transition to an intermediate state with an increased helical content (TFE state). Small‐angle X‐ray scattering measurements indicated that the TFE state has a radius of gyration which is 20% larger than that of the native state and assumes a chain‐like conformation with some remaining globularity. The TFE‐induced transition curves obtained by CD and the small‐angle X‐ray scattering measurements agreed well, consistent with a two‐state transition mechanism. A singular value decomposition analysis of Kratky plots of the small‐angle X‐ray scattering profiles indicated that two basic scattering functions reproduce the observed spectra, further confirming the validity of a two‐state approximation.

Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion

Although the membrane fusion of spermatozoon and egg cells is the central event of fertilization, the underlying molecular mechanism remains virtually unknown. Gene disruption studies have showed that IZUMO1 on spermatozoon and CD9 on oocyte are essential transmembrane proteins in sperm-egg fusion. In this study, we dissected IZUMO1 protein to determine the domains that were required for the function of sperm-egg fusion. We found that a fragment of the N terminus (Asp5 to Leu113) interacts with fertilization inhibitory antibodies. It also binds to the egg surface and effectively inhibits fusion in vitro. We named this fragment ‘IZUMO1 putative functional fragment (IZUMO1PFF)’. Surprisingly, IZUMO1PPF still maintains binding ability on the egg surface of Cd9-/- eggs. A series of biophysical measurements using circular dichroism, sedimentation equilibrium and small angle X-ray scattering revealed that IZUMO1PFF is composed of an N-terminal unfolded structure and a C-terminal ellipsoidal helix dimer. Egg binding and fusion inhibition were not observed in the IZUMO1PFF derivative, which was incapable of helix formation. These findings suggest that the formation of a helical dimer at the N-terminal region of IZUMO1 is required for its function. Cos-7 cells expressing the whole IZUMO1 molecule bound to eggs, and IZUMO1 accumulated at the interface between the two cells, but fusion was not observed. These observations suggest that IZUMO1 alone cannot promote sperm-egg membrane fusion, but it works as a factor that is related to the cellular surface interaction, such as the tethering of the membranes by a helical region corresponding to IZUMO1PFF-core.