Conformational differences among metarhodopsin I, metarhodopsin II, and opsin probed by wide-angle X-ray scattering

Among the photoproducts of vertebrate rhodopsin, only metarhodopsin II (Meta-II) preferentially adopts the active structure in which transmembrane helices are rearranged. Light-induced helical rearrangement of rhodopsin in membrane-embedded form was directly monitored by wide-angle X-ray scattering (WAXS) using nanodiscs. The change in the WAXS curve for the formation of Meta-II was characterized by a peak at 0.2 Å⁻¹ and a valley at 0.6 Å⁻¹, which were not observed in metarhodopsin I and opsin. However, acid-induced active opsin (Opsin*) showed a 0.2 Å⁻¹ peak, but no 0.6 Å⁻¹ valley. Analyses using the model structures based on the crystal structures of dark state and Meta-II suggest that the outward movement of helix VI occurred in Opsin*. However, the displaced helices III and V in Meta-II resulting from the disruption of cytoplasmic ionic lock were restored in Opsin*, which is likely to destabilize the G-protein-activating structure of opsin

Polar-Nonpolar Interfaces of Normal Bicontinuous Cubic Phases in Nonionic Surfactant/Water Systems Are Parallel to the Gyroid Surface

We investigated the structures of normal (type I) bicontinuous cubic phases in hexa-, hepta-, and octaethylene glycol dodecyl ether/water mixtures by small-angle X-ray crystallography of single-crystal domains. Reconstructed electron densities showed that the hydrophilic chains with high electron density are confined to a film centered on the surface of the Gyroid (a triply periodic minimal surface), while hydrophobic chains with low electron density are distributed within the pair of interwoven labyrinths carved out by the Gyroid. Further, the local minimum within the high electron density region, due to bulk water, coincides precisely with the Gyroid. This minimum is less pronounced in mixtures with longer ethylene glycol chains, consistent with their decreased water content. Our analysis clearly shows that the polar-nonpolar interfaces are parallel to the Gyroid surface in all mixtures. The repulsive hydration or overlapping force between the pair of facing monolayers of ethylene glycol chains on either side of the Gyroid surface is the likely origin of the parallel interfaces.This work was supported by JSPS KAKENHI Grant Numbers 15K05243 and 18K03557. Part of this research was based on a Cooperative Research Project of the Research Institute of Electronics, Shizuoka University. The synchrotron radiation experiments were performed using BL40B2 at SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal Nos. 2016A1174, 2016B1339, 2017A1352)

Luminal acidification of diverse organelles by V-ATPase in animal cells

Eukaryotic cells contain organelles bounded by a single membrane in the cytoplasm. These organelles have differentiated to carry out various functions in the pathways of endocytosis and exocytosis. Their lumina are acidic, with pH ranging from 4.5 to 6.5. This article describes recent studies on these animal cell organelles focusing on (1) the primary proton pump (vacuolar-type H+-ATPase) and (2) the functions of the organelle luminal acidity. We also discuss similarities and differences between vacuolar-type H+-ATPase and F-type ATPase. Our own studies and interests are emphasized

Hyperammonemia in a carbamoyl-phosphate synthetase 1 deficiency recipient after living-donor liver transplantation from a carrier donor: a case report

Carbamoyl-phosphate synthetase 1 (CPS1) deficiency is an autosomal recessive congenital urea cycle disorder (UCD) characterized by hyperammonemia. The recipients of liver transplantation (LT) for UCD are often children, and the potential donors are often the parents. Hereditary congenital diseases involving UCD entail the possibility of both parents being genetically heterozygous. Herein, we describe the case of a 12-year-old girl with CPS1 deficiency receiving a liver transplant (soon after birth) from her father, who had a heterozygous CPS1 mutation. She was referred to our hospital with respiratory distress after contracting two infections (respiratory syncytial virus and human metapneumovirus) within a short period, both of which presented with hyperammonemia. Medication for hyperammonemia quickly lowered the ammonia levels. The hyperammonemia was thought to be caused by the heterozygous mutation in the donor liver; moreover, it is likely that the low enzyme activity in the patient’s liver was increased due to the infections. This is the first study to report hyperammonemia in a CPS1 deficiency patient due to an infection after LT. Thus, patients with CPS1 deficiency should be aware of the development of hyperammonemia after LT

A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs

Behavior of vascular resistance undergoing various pressure insufflation and perfusion on decellularized lungs

Bioengineering of functional lung tissue by using whole lung scaffolds has been proposed as a potential alternative for patients awaiting lung transplant. Previous studies have demonstrated that vascular resistance (Rv) could be altered to optimize the process of obtaining suitable lung scaffolds. Therefore, this work was aimed at determining how lung inflation (tracheal pressure) and perfusion (pulmonary arterial pressure) affect vascular resistance. This study was carried out using the lungs excised from 5 healthy male Sprague-Dawley rats. The trachea was cannulated and connected to a continuous positive airway pressure (CPAP) device to provide a tracheal pressure ranging from 0 to 15 cmH(2)O. The pulmonary artery was cannulated and connected to a controlled perfusion system with continuous pressure (gravimetric level) ranging from 5 to 30 cmH(2)O. Effective Rv was calculated by ratio of pulmonary artery pressure (P-PA) by pulmonary artery flow (V'(PA)). Rv in the decellularized lungs scaffolds decreased at increasing V'(PA), stabilizing at a pulmonary arterial pressure greater than 20 cmH(2)O. On the other hand, CPAP had no influence on vascular resistance in the lung scaffolds after being subjected to pulmonary artery pressure of 5 cmH(2)O. In conclusion, compared to positive airway pressure, arterial lung pressure markedly influences the mechanics of vascular resistance in decellularized lungs. (C) 2016 Elsevier Ltd. All rights reserved

High-capacitance supercapacitors using nitrogen-decorated porous carbon derived from novolac resin containing peptide linkage

We fabricated nitrogen-decorated porous carbon exhibiting high capacitance per unit volume and unit weight via chemical activation of novolac resin containing peptide linkage. The porosity and the amount of nitrogen atoms were controlled by changing the molecular weight of novolac resin, the added amount of potassium hydroxide, or both. After chemical activation, positively charged nitrogen atoms (i.e., pyridine/pyrrole) at 400.3 eV in photoemission spectra contributed to both a shift in the point of zero charge toward negative potential and the generation of pseudocapacitance. Suitably developed pores and the positively charged nitrogen atoms make nitrogen-decorated novolac resin-derived porous carbon a promising material for electrodes in high-performance supercapacitors.ArticleELECTROCHIMICA ACTA. 55(20):5624-5628 (2010)journal articl

Concentration of mitochondrial DNA mutations by cytoplasmic transfer from platelets to cultured mouse cells

Accumulation of mutations in mitochondrial DNA (mtDNA) is thought to be responsible for mitochondrial, and other, diseases and biological phenomena, such as diabetes, cancer, neurodegenerative diseases, and aging. Mouse models may elucidate the relationship between mutations in mtDNA and these abnormalities. However, because of the difficulty of mtDNA manipulation, generation of mouse models has not sufficiently progressed to enable such studies. To overcome this difficulty and to establish a source of diverse mtDNA mutations, we here generated cultured mouse cells containing mtDNA derived from an mtDNA mutator mouse that accumulates random mtDNA mutations with age. Mutation analysis of the obtained transmitochondrial cytoplasmic hybrid cells (cybrids) revealed that the cells harbored diverse mtDNA mutations occurring at a higher frequency than in mouse tissues, and exhibited severe respiration defects that would be lethal in tissues or organs. Abnormal respiratory complex formation and high stress on the mitochondrial protein quality control system appeared to be involved in these severe respiration defects. The mutation rates of the majority of highly accumulated mutations converged to either approximately 5%, 10%, or 40%, suggesting that these mutations are linked on the respective mtDNA molecules, and mtDNA in cybrid cells likely consisted of mtDNA molecules clonally expanded from the small population of introduced mtDNAs. Thus, the linked mutations in these cybrid cells cannot be evaluated individually. In addition, mtDNA mutations homologous to confirmed pathogenic mutations in human were rarely observed in our generated cybrids. However, the transmitochondrial cybrids constitute a useful tool for concentrating pathogenic mtDNA mutations and as a source of diverse mtDNA mutations to elucidate the relationship between mtDNA mutations and diseases