極限環境生物由来ヘム蛋白質の環境適応機構の解明

内容の要約広島大学(Hiroshima University)博士(農学)Doctor of Agriculturedoctora

Difference in NaCl tolerance of membrane-bound 5′-nucleotidases purified from deep-sea and brackish water Shewanella species

Shewanella species are widely distributed in sea, brackish, and fresh water areas, growing psychrophilically or mesophilically, and piezophilically or piezo-sensitively. Here, membrane-bound 5′-nucleotidases (NTases) from deep-sea Shewanella violacea and brackish water Shewanella amazonensis were examined from the aspect of NaCl tolerance in order to gain an insight into protein stability against salt. Both NTases were single polypeptides with molecular masses of ~59 kDa, as determined on mass spectroscopy. They similarly required 10 mM MgCl2 for their activities, and they exhibited the same pH dependency and substrate specificity for 5′-nucleotides. However, S. violacea 5′-nucleotidase (SVNTase) was active enough in the presence of 2.5 M NaCl, whereas S. amazonensis 5′-nucleotidase (SANTase) exhibited significantly reduced activity with the same concentration of the salt. Although SVNTase and SANTase exhibited high sequence identity (69.7%), differences in the ratio of acidic to basic amino acid residues and the number of potential salt bridges maybe being responsible for the difference in the protein stability against salt. 5′-Nucleotidases from these Shewanella species will provide useful information regarding NaCl tolerance, which may be fundamental for understanding bacterial adaptation to growth environments.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan [No. 26240045], a grant from the Japan Society for the Promotion of Science [No. 25-1446], and The Salt Science Research Foundation [No. 1655]

Structural and functional insights into thermally stable cytochrome c' from a thermophile

Thermophilic Hydrogenophilus thermoluteolus cytochrome c0 (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c0 (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 A ° . In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit–subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit–subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit–subunit interactions with conservation of the ligand-binding ability.This work was performed under the Cooperative Research Program of the “Network Joint Research Center for Materials and Devices”

Cardiovascular and respiratory effects of the degree of head-down angle during robot-assisted laparoscopic radical prostatectomy

Background: Robot-assisted laparoscopic radical prostatectomy (RALP) requires a steep Trendelenburg position and CO2 pneumoperitoneum for several hours to secure the surgical visual field. The present study was performed to investigate the influence of each angle of Trendelenburg position during RALP on cardiovascular and respiratory homeostasis. Methods: Forty-seven ASA physical status 1 and 2 patients underwent open retropubic radical prostatectomy (RRP) or RALP. Patients receiving RALP were randomized to undergo the operation in the 20°, 25° or 30° Trendelenburg position. Heart rate (HR), mean arterial pressure (MAP), respiratory rate (RR), end-tidal CO2 pressure (PetCO2), tidal volume (Vt), peak inspiratory pressure (PIP) and dynamic compliance (Cdyn) were recorded during the operation. Results: Angle of head-down tilt was significantly correlated with MAP, PIP and Cdyn, but not with HR, RR or PetCO2. MAP decreased gradually over time in each group in the Trendelenburg position with pneumoperitoneum. As the angle of head-down tilt became stronger, MAP, RR, PetCO2 and PIP tended to increase and Cdyn tended to decrease. Conclusions: This study demonstrated that the degree of the head-down angle at RALP affected the cardiovascular and respiratory parameters. Pneumoperitoneum with head-down position in RALP influenced the cardiovascular and respiratory system to a greater extent than RRP, and these effects were stronger with deeper head-down angle. © 2013 John Wiley & Sons, Ltd

Luxury Perfusion Syndrome Confirmed by Sequential Studies of Regional Cerebral Blood Flow and Volume after Extracranial to Intracranial Bypass Surgery: Case Report

Abstract We report a case of luxury perfusion syndrome with temporary neurological deterioration after extracranial to intracranial bypass surgery. A preoperative computed tomographic scan showed no detectable infarct, and the measurement of regional cerebral blood flow showed severe depression of ipsilateral hemispheric perfusion. The patient developed temporary neurological deterioration after bypass surgery, with no recognizable pathological signs on postoperative computed tomographic and angiographic studies. Regional cerebral blood flow and volume were more elevated during the period of neurological deterioration than after the subsequent recovery. This strongly suggests that excessive blood flow directed into chronically ischemic brain through a graft may induce a luxury perfusion syndrome resulting in neurological deterioration

Conferment of CO-Controlled Dimer–Monomer Transition Property to Thermostable Cytochrome c′ by Mutation in the Subunit–Subunit Interface

Cytochrome c′ (CP) is a gas-binding homo-dimeric heme protein. Mesophilic Allochromatium vinosum CP (AVCP) and thermophilic Hydrogenophilus thermoluteolus CP (PHCP) have high sequence and structure similarities. AVCP is known to exhibit a dimer–monomer transition upon CO binding/dissociation, whereas detailed CO-binding properties of PHCP remain unrevealed. Here, we found that the CO-binding affinity of wild-type PHCP is lower than that of AVCP, and the PHCP dimer does not dissociate to monomers under CO-saturated reduced conditions. The CO-binding affinity of PHCP increased by mutations in the subunit–subunit interface (F11T, T18F, or F71D). The T18F, F71D, and T18F/F71D PHCP variants exhibited similar dimer–monomer transitions upon CO binding/dissociation to that of AVCP, although the F11T variant did not. The simulated structures of the PHCP variants revealed that the T18F and F71D mutations caused rearrangement in the subunit–subunit interface, whereas the F11T mutation did not, indicating that the effective dimer–monomer transitions upon CO binding/dissociation are induced by the rearrangement in the subunit–subunit interface. The present results indicate that subunit–subunit interface mutation of oligomeric proteins is a useful approach in the adjustment of protein stability and ligand binding affinity, leading to a change in the quaternary structure