Molecular biology of oxygen tolerance in lactic acid bacteria: Functions of NADH oxidases and Dpr in oxidative stress

Lactic acid bacteria including Streptococcus mutans lack cytochromes and heme-containing proteins. Most lactic acid bacteria also lack catalase. However, they can grow in the presence of air. In view of the defense against oxygen toxicity, the lack of catalase in lactic acid bacteria is not always consistent with its aerotolerance. Mechanisms, by which lactic acid bacteria establish their growth in air, are therefore an active area of investigation. We identified two kinds of NADH oxidase genes, nox-1 and nox-2 for H2O2-forming NADH oxidase (Nox-1) and H2O-forming NADH oxidase (Nox-2), respectively, in S. mutans and found that Nox-1 is homologous with flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase (AhpR), consisting of AhpF and AhpC. We also identified ahpC which is homologous with ahpC of S. typhimurium, upstream of nox-1 in S. mutans. In the first and second parts of this article, we will refer to the role of Nox-1 which acts together with AhpC as bi-component peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohol and/or H2O. We will also refer to the role of Nox-2 in carbohydrate metabolism of S. mutans in its aerobic life. Nox-2 was found to be involved in regenerating NAD+, which is required for glycolysis in S. mutans. While studying nox-1 and ahpC double deletion mutant of S. mutans, we found that the mutant still showed the same level peroxide tolerance as did the wild-type strain. The finding suggested the existence of another antioxidant system in addition of Nox-1 and AhpC in S. mutans. We identified a new gene, dpr (for Dps-like Peroxide Resistance gene) and its product, Dpr, as an iron-binding protein which is responsible for oxygen tolerance in S. mutans. In the third part of this article, we will refer to the current status of knowledge of molecular cloning of dpr, the characteristics of dpr-disruption mutants, and a mechanism by which Dpr confers aerotolerance to S. mutans.     

Interaction of modified tail-anchored proteins with liposomes: effect of extensions of hydrophilic segment at the COOH-terminus of holo-cytochromes b₅

A group of membrane proteins having a single COOH-terminal hydrophobic domain capable of post-translational insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b(5) (Hcytb5) into ER membranes, we produced and purified various membrane-bound forms of Hcytb5 with their heme b-bound, in which various truncated forms of NH(2)-terminal bovine opsin sequence were appended at the COOH-terminus of the native form. We analyzed the integration of the TA-domains of these forms onto protein-free liposomes. The integration occurred efficiently even in the presence of a small amount of sodium cholate and, once incorporated, such proteoliposomes were very stable. The mode of the integration was further analyzed by treatment of the proteoliposomes with trypsin either on the extravesicular side or on the luminal side. LC-MS analyses of the trypsin digests obtained from the proteoliposomes indicated that most of the C-terminal hydrophilic segment of the native Hcytb5 were exposed towards the lumen of the vesicles and, further, a significant part of the population of the extended C-terminal hydrophilic segments of the modified Hcytb5 were exposed in the lumen as well, suggesting efficient translocation ability of the TA-domain without any assistance from other protein factors. Present results opened a route for the use of the C-terminal TA-domain as a convenient tool for the transport of proteins as well as short peptides into artificial liposomes.     

Analysis of locality of early-stage maturation in confluent state of human retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells were cultured on the laminin-coated and plain surfaces. The measurement of local nucleus density in non-stratified region, which correlated with formation of tight junction, is the indicator of the maturation, and the parameters can be applied to the evaluation of the early-stage maturation of RPE cells in culture.     

Package Design of Ready-to-Drink Coffee Beverages Based on Food <Emphasis Type="Italic">Kansei</Emphasis> Model—Effects of Straw and Cognition Terms on Consumer’s Pleasantness

To design a consumer-oriented package that complements the taste and aroma of ready-to-drink chilled-cup coffee beverages by using the food kansei model, the effects of the diameter and the color of drinking straws as well as the cognition terms of coffee on consumer sensory characteristics and preferences were investigated. Variance and factor analyses of the sensory scores for chilled-cup coffee with milk and sugar using straws of different diameters, as rated by consumer panelists, extracted two perceived factors (F1, contribution ratio 36.5%, and F2, 28.6%). A two-dimensional plot of the average F1 and F2 scores of 123 panelists showed that the perceived characteristics of the same taste and aroma varied according to the straw diameter. An image investigation of different straw colors and another sensory evaluation using “black,” “brown,” and “green” straws were also performed. A principal component analysis of the image data revealed that the sensory characteristics of coffee with milk and sugar were imaged from the straw color. The second evaluation suggested that the images of straw colors affected the sensory characteristics. In addition, cluster and multiple-comparison analyses of Internet research data from consumers regarding the cognition terms for coffee exhibited three clusters representing the cognitive characteristics of terms by consumers and by developers and the differences of attractiveness degree on the cognition terms due to the consumers’ personal attributes. These studies provide useful information for the development of packages of chilled-cup coffee beverages.     

Characterization on the Conformation of Glucoamylase from Rhizopus niveus and Rhizopus delemar

Using the pH-jump (neutral to above 11) for Tyr-OH ionization as a probe, two glucoamylases from Rhizopus niveus and Rhizopus delemar were found to be caused the change in conformation, which is in a kinetically single step. The pH titration at pHs below 12 was carried out on a multidimensional correlation spectrophotometer. Correlations among the spectrophotometric properties: fluorescence, absorption and circular dichroism are almost identical for the two enzymes. These results suggest that conformational characteristics of the niveus enzyme is almost the same as that of the delemar one.     

Fabrication of CaCO3-Coated Vesicles by Biomineralization and Their Application as Carriers of Drug Delivery Systems

We fabricated CaCO₃-coated vesicles as drug carriers that release their cargo under a weakly acidic condition. We designed and synthesized a peptide lipid containing the Val-His-Val-Glu-Val-Ser sequence as the hydrophilic part, and with two palmitoyl groups at the N-terminal as the anchor groups of the lipid bilayer membrane. Vesicles embedded with the peptide lipids were prepared. The CaCO₃ coating of the vesicle surface was performed by the mineralization induced by the embedded peptide lipid. The peptide lipid produced the mineral source, CO₃²⁻, for CaCO₃ mineralization through the hydrolysis of urea. We investigated the structure of the obtained CaCO₃-coated vesicles using transmission electron microscopy (TEM). The vesicles retained the spherical shapes, even in vacuo. Furthermore, the vesicles had inner spaces that acted as the drug cargo, as observed by the TEM tomographic analysis. The thickness of the CaCO₃ shell was estimated as ca. 20 nm. CaCO₃-coated vesicles containing hydrophobic or hydrophilic drugs were prepared, and the drug release properties were examined under various pH conditions. The mineralized CaCO₃ shell of the vesicle surface was dissolved under a weakly acidic condition, pH 6.0, such as in the neighborhood of cancer tissues. The degradation of the CaCO₃ shell induced an effective release of the drugs. Such behavior suggests potential of the CaCO₃-coated vesicles as carriers for cancer therapies.     

Mechano-electrochemistry of a passive surface using an in situ micro-indentation test

Depassivation–repassivation of iron surfaces in boric–borate solutions were investigated by using the micro-indentation test. A pair of current peaks due to repair of the passive film following rupture of the film were observed during a series of indenter drives, i.e., loading and unloading of the indenter. The shape of the current peak depended on environmental conditions (conductivity and pH of the solution) and substrate conditions (mechanical processing history, alloyed element) as well as indentation conditions (repetition, maximum depth, and maximum load). Plastic deformation of the surface was accompanied by surface depassivation, while no depassivation occurred during the elastic deformation, indicating that the passive film on iron has a ductile property. The solution conditions did not affect the scale of depassivation but affected the rate of repassivation. Dislocations in the substrate made surface depassivation difficult but enhanced reactivity during the repassivation. The test also revealed that type-312L stainless steel has high corrosion resistance in a concentrated NaCl solution.     

Effect of gamma radiolysis on pit initiation of zircaloy-2 in water containing sea salt

In spent fuel pools at the Fukushima Daiichi Nuclear Power Station (1F), seawater was injected for cooling purposes after the tsunami disaster in March 2011. It is well known that the chloride in the seawater has the potential to cause localized corrosion (e.g., pitting corrosion) in metals. In this study, we evaluated the pitting potentials of zircaloy-2, the material used in the fuel cladding tubes in 1F, as a function of chloride concentration. To accomplish this, we used artificial seawater under gamma-ray irradiation and investigated the effect of radiolysis on pit initiation of zircaloy-2 in water containing sea salt. Changes in the composition of water containing sea salt were analyzed as well, both before and after gamma-ray irradiation. The characteristics of the resultant oxide films formed on zircaloy-2 were evaluated by X-ray photoelectron spectroscopy and electrochemical impedance spectroscopy. The experimental results showed that the pitting potential under irradiation was slightly higher than that under conditions in which no radiation was present, and that the pitting potential decreased with increasing chloride concentration in the presence as well as the absence of radiation. Solution analyses for water containing sea salt showed that hydrogen peroxide was generated by irradiation. The oxide film was composed of zirconium oxide and was made thicker during the irradiation. The higher pitting potential could thus be explained by the capacity of hydrogen peroxide to oxidize the surface and enhance oxide film formation. Under gamma-ray irradiation, the zircaloy-2 surface with an oxide film formed by radiolysis products was found to be resistant to pitting in the presence of chloride.     

Production of Carbon Nanotube by Ethylene Decomposition over Silica-Coated Metal Catalysts

Formation of carbon nanofibers (CNFs) and carbon nanotubes (CNTs) through the decomposition of ethylene at 973 K was achieved using various metal catalysts covered with silica layers. CNFs of various diameters were formed by ethylene decomposition over a Co metal catalyst supported on the outer surface of the silica. In contrast, silica-coated Co catalysts formed CNTs with uniform diameters by ethylene decomposition. Silica-coated Ni/SiO₂ and Pt/carbon black also formed CNTs with uniform diameters, while CNFs and CNTs with various diameters were formed over Ni/SiO₂ and Pt/carbon black without a silica coating. These results indicate that silica layers that envelop metal particles prevent sintering of the metal particles during ethylene decomposition. This results in the preferential formation of CNTs with a uniform diameter.     

Measurement of particle concentration in powder coating process using capacitance computed tomography and wavelet analysis

Capacitance computed tomography techniques were used to visualize particles movement in the draft tube of a spouted fluidized bed for the coating process of drug production. A total of 512 frames images of the particle concentration distribution were obtained at 10-millisecond intervals over a coating time of 5 min using a capacitance computed tomography system. The three-dimensional capacitance CT images (time and two-dimensional space images) were decomposed to wavelet time and space levels to extract the dominant particle distribution feature using three-dimensional discrete wavelet multiresolution at different coating times. As a result, the time and space dominant particle distribution with a specific frequency level can be visualized.