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Abstract: The structure of heavy hyperhydrogen 6 Λ H is studied within the framework of a four-body cluster model. Interactions among the constituent subunits are determined so as to reproduce reasonably well the observed low energy properties of the and tnn subsystems. As long as we reproduce the energy and width of 5H within the error bar, the ground state of 6 Λ H is obtained as a resonant state. [Copyright &y& Elsevier]

Abstract: The success of Sazanov''s group in determining the X-ray structure of the whole bacterial complex I is a great contribution to the progress of complex I research. In this mini-review of 35years'' history of my laboratory and collaborators, we characterized the function of protein-associated semiquinone molecules in the proton-pumping mechanism in complex I (NADH-quinone oxidoreductase). We have constructed most of the frame work of our hypothesis, utilizing EPR techniques before the X-ray structures of complex I were reported by Sazanov''s and Brandt''s groups. One of the semiquinones (SQNf) is extremely sensitive to a proton motive force imposed on the energy-transducing membrane, while the other (SQNs) is insensitive. Their sensitivity to rotenone inhibition also differs. These differences were exploited using tightly coupled bovine heart submitochondrial particles with a high respiratory control ratio (>8). We determined the distance between SQNf and iron–sulfur cluster N2 on the basis of their direct spin–spin interaction. We are extending this line of work using reconstituted bovine heart complex I proteoliposomes which shows a respiratory control ratio >5. Two frontier research groups support our view point based on their mutagenesis studies. High frequency (33.9GHz; Q-band) EPR experiments appear to favor our two-semiquinone model. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). [Copyright &y& Elsevier]

Abstract: Considerable disagreement still exists concerning the superoxide generation sites in the purified bovine heart NADH-ubiquinone oxidoreductase (complex I). Majority of investigators agree that superoxide is generated at the flavin site. Here we present a new hypothesis that the generation of superoxide reflects a dynamic balance between the flavosemiquinone (semiflavin or SF) and the semiquinone (SQ), like a “tug-of-war” through electrons. All preparations of bovine heart complex I, which have been isolated at Yoshikawa''s laboratory, have one protein-bound endogenous ubiquinone per complex I (Shinzawa-Itoh et al., Biochemistry, 49 (2010) 487–492). Using these preparations, we measured (i) EPR signals of the SF, the SQ and iron–sulfur cluster N2 simultaneously with cryogenic EPR and (ii) superoxide production with both the room temperature spin-trapping technique and the partially acetylated cytochrome c method. Our experimental evidence was (1) without added decylubiquinone (DBQ), no catalytic oxidation of NADH occurs. The NADH addition produced mostly SF and it generated superoxide as reported by Kussmaul and Hirst (PNAS, 103 (2006) 7607–7612). (2) During catalytic electron transfer from NADH to DBQ, the superoxide generation site was mostly shifted to the SQ. (3) A quinone-pocket binding inhibitor (rotenone or piericidin A) inhibits the catalytic formation of the SQ, and it enhances the formation of SF and increases the overall superoxide generation. This suggests that if electron transfer was inhibited under pathological conditions, superoxide generation from the SF would be increased. [Copyright &y& Elsevier]

We analyzed the mechanism of oxygen-atom-pair chemisorption on a tungsten (0 0 1) surface by performing theoretical cluster-model calculations based on the density functional theory. In the analysis, we supposed that p(4×1) structures of oxygen-covered tungsten (0 0 1) surface above coverage of a fourth monolayer come from chains of quasi-compounds of WO2. Calculated electronic structures and vibrational modes normal to the surface of the quasi-compounds of WO2 agrees well with the experimental results. The model clusters for the quasi-compound are however more unstable than ones for single-site chemisorption of oxygen at fourfold hollow and bridge site from comparing their adsorption energies. In order to explain the driving force behind the quasi-compound reaction, we analyzed electrovalent chemisorption energies and electronic dipole moments of the model clusters for oxygen chemisorption on the tungsten (0 0 1) surface and then we propose that the driving force is due to the dipole repulsion between surface polarization induced by oxygen chemisorption and the polarization that occurs between the incoming oxygen molecule and the surface. [Copyright &y& Elsevier]

The efficacy of S-allylcysteine (SAC) as a free radical scavenger was studied using rat brain ischemia models. In a middle cerebral artery occlusion model, preischemic administration of SAC had the following effects: it improved motor performance and memory impairment and reduced water content and the infarct size. In a transient global ischemia model, the time course of free radical (alkoxyl radical) formation as studied by electron paramagnetic resonance (EPR) spectroscopy and α-phenyI-N-tert-butylnitrone (PBN) was biphasic; the first peak occurred at 5 min and the second at 20 min after reperfusion. Although SAC did not attenuate the first peak, it did affect the second peak, which is related to lipid peroxidation. The lipid peroxidation as estimated by thiobarbituric acid reactive substances (TBARS) increased significantly at 20 min after reperfusion. SAC decreased TBARS to the levels found without ischemia. These results suggest that SAC could have beneficial effects in brain ischemia and that the major protective mechanism may be the inhibition of free radical-mediated lipid peroxidation. [ABSTRACT FROM AUTHOR]

In the circulation of sickle cell anemia patients, a certain population of erythrocytes has an elevated density. These abnormally dense cells are believed to be at the root of the painful crisis and anemia of the patients. We have developed an in vitro method for the preparation of these heavier erythrocytes by a repeated deoxy-oxy cycling of erythrocytes from sickle cell anemia patients. By using this method, we studied whether certain nutritional supplements would inhibit the formation of dense cells in vitro. It was found that aged garlic extract (AGE) as well as its components with antioxidant activity, i.e., S-allylcysteine and Nα-(1-deoxy-D-fructos-l-yl)-Larginine (fructosyl arginine), inhibited the formation of dense cells in vitro. Vitamin C, vitamin E and the spintrapping agents, 5-diethoxyphophoryl-5-methyl-l-pyrroline-N-oxide and α(4-pyridyl-l-oxide)-N-t-butylnitrone were all found to inhibit the formation of dense cells in vitro. These results suggest that, when extremely stretched sickle-shaped cells are formed by the repeated deoxy-oxy cycling, the erythrocyte membrane becomes susceptible to oxidative injury by reactive oxygen species. The protection of the erythrocyte membrane from such an oxidative injury would prevent the membranes from becoming leaky to the calcium ion, thus inhibiting the activation of the calcium-activated potassium efflux channel and the formation of dense cells. We also developed a new ex vivo method of studying the possible efficacy of antioxidants taken orally on the dense cell formation in sickle cell patients. It involved the use of blood plasma taken from a healthy donor (with normal hemoglobin) of AB blood type who had consumed different types of antioxidants orally. By suspending sickle erythrocytes in such plasma and exposing them to the deoxy-oxy cycling, the degree of dense cell formation was determined. The degree of inhibition in vitro by antioxidants taken orally may be related to their efficacy in inhibiting dense cell formation in the patients. On the basis of these in vivo and ex vivo studies, we propose that a cocktail of antioxidants would have beneficial effects in lessening the incidence and severity of crisis and reducing anemia in sickle cell disease. [ABSTRACT FROM AUTHOR]

Abstract: In many energy transducing systems which couple electron and proton transport, for example, bacterial photosynthetic reaction center, cytochrome bc 1-complex (complex III) and E. coli quinol oxidase (cytochrome bo 3 complex), two protein-associated quinone molecules are known to work together. T. Ohnishi and her collaborators reported that two distinct semiquinone species also play important roles in NADH-ubiquinone oxidoreductase (complex I). They were called SQNf (fast relaxing semiquinone) and SQNs (slow relaxing semiquinone). It was proposed that QNf serves as a “direct” proton carrier in the semiquinone-gated proton pump (Ohnishi and Salerno, FEBS Letters 579 (2005) 4555), while QNs works as a converter between one-electron and two-electron transport processes. This communication presents a revised hypothesis in which QNf plays a role in a “direct” redox-driven proton pump, while QNs triggers an “indirect” conformation-driven proton pump. QNf and QNs together serve as (1e−/2e−) converter, for the transfer of reducing equivalent to the Q-pool. [ABSTRACT FROM AUTHOR]

Fetal bovine serum (FBS) is a common component of culture media and usually used for cellular research, as well as recent cell-based medical products. However, due to the high risk of contaminations and the variation from batch to batch, FBS might influence the outcome of research or cellular manufacturing. FBS also contains moral concerns because it harvested from bovine fetuses taken from pregnant cows. In addition, FBS is most expensive part of cell culture. To overcome several problems regarding FBS, we developed a new serum, adult bovine platelet lysate-derived cell culture supplement (NeoSERA), and investigate its usefulness as an alternative to FBS. NeoSERA is produced in Japan, considered to be a negligible bovine spongiform encephalopathy (BSE) risk country. We obtain adult donor bovine blood from animal less than 36 months old according to the regulation of EMA for BSE (EMA/410/01 rev.3). Using apheresis medical devices with closed disposable kits, sterile platelet-rich plasma (PRP) is collected from healthy donor bovine receiving a regular veterinary check. After stimulation and removal of coagulated fibrin by centrifugation, NeoSERA is collected in a completely closed system. To meet the scope of directives that apply to produce medicinal products from the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/743/00) and the United States Department of Agriculture (9CFR§113.450), NeoSERA is finally g-irradiated at a dose of over 30 kGy. NeoSERA completely meets the standard for biological ingredients in Japan (Ministry of Health, Labour and Welfare Notification No. 375 2014) and also obtained the certificate of eligibility for the raw material of regenerative medicine from Pharmaceuticals and Medical Devices Agency (PMDA, No. 0417002, 2017. 4.17). To test whether NeoSERA is useful for the expansion of mesenchymal stem cells (MSCs), a cell culture experiment was performed. 3 to 5 days after NeoSERA treatment, the proliferation of human bone marrow-, adipose tissue-, umbilical cord- and amnion-derived MSCs was significantly increased (p < 0.05) compared to FBS. Our results confirm that safe, less ethical and economical bovine NeoSERA profoundly enhances the proliferation of MSCs for regenerative medicine and several cell lines for vaccine production. [ABSTRACT FROM AUTHOR]