Family M42 aminopeptidase from the syntrophic bacterium Symbiobacterium thermophilum: characterization using recombinant protein

The chromosomal DNA of the syntrophic thermophile Symbiobacterium thermophilum contains open reading frames of the genes encoding family M42 aminopeptidases, Pep1079, Pep1080, and Pep1081. To characterize these peptidases, the genes were cloned into Escherichia coli and overexpressed. Our experiments using the recombinant proteins confirmed that Pep1079, Pep1080, and Pep1081 are components of arginyl or lysinyl aminopeptidases that require Co2+ for enzymatic activity. Coexistence of Pep1079 and Pep1080 is necessary for expressing high peptidase activity. Pep1081 enhances the activity of Pep1079 and Pep1080.     

Influence of different feeding systems on the growth performance and muscle development of Japanese Black steers

This study investigated the growth performance and gene expression for muscle development between grass hay-fed (GH) and concentrate-fed (CT) steers. Daily gain and energy intake during the fattening period of the GH group were lower than those of the CT group. Analysis of C/EBPα, PPARγ2, myosin heavy chain (MHC), and myostatin gene expressions was performed by real-time PCR. Expressions of C/EBPα and myostatin in semitendinosus and longissimus lumborum (LL) muscles were higher in the CT group than in the GH group at the end of fattening. In LL muscle, MHC expression at the end of fattening was greater in the GH group than in the CT group. These results suggest that regulation of adipogenesis and myogenesis by the expression of genes involved in muscle development might have occurred in the skeletal muscle of the GH group by the feeding of grass hay and/or because of the low energy intakes.     

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.     

Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease Tl

Hydrophobic effects on binding of ribonuclease Tl to guaninebases of several ribonucleotides have been proved by mutatinga hydrophobic residue at the recognition site and by measuringthe effect on binding. Mutation of a hydrophobic surface residueto a more hydrophobic residue (Tyr45 – Trp) enhances thebinding to ribonucleotides, including mononucleotide inhibitorand product, and a synthetic substrate-analog trinudeotide aswell as the binding to dinucleotide substrates and RNA. Enhancementson binding to non-substrate ribonucleotides by the mutationhave been observed with free energy changes ranging from –2.2 to – 3 .9 kJ/mol. These changes are in good agreementwith that of substrate binding, –2.3 kJ/mol, which iscalculated from Michaelis constants obtained from kinetic studies.It is shown, by comparing the observed and calculated changesin binding free energy with differences in the observed transferfree energy changes of the amino acid side chains from organicsolvents to water, that the enhancement observed on guaninebinding comes from the difference in the hydrophobic effectsof the side chains of tyrosine and tryptophan. Furthermore,a linear relationship between nucleolytic activities and hydrophobicityof the residues (Ala, Phe, Tyr, Trp) at position 45 is observed.The mutation could not change substantially the base specificityof RNase Tl, which exhibits a prime requirement for guaninebases of substrates.     

D.s.c. investigation of the states of water in poly(vinyl alcohol-co-itaconic acid) membranes

The state of water in water-swollen poly(vinyl alcohol-co-itaconic acid) membranes, having various water contents from 0.31 to 0.83, was investigated by d.s.c. measurements and compared with those in water-swollen poly(vinyl alcohol) membranes. The amount of freezing water in the membranes was estimated by use of a relationship between the phase transition temperature and the enthalpy of the crystallization of supercooled water. The melting temperature of the water in the membranes immersed in urea and NaCl 0–2 mol l^?1 aqueous solutions was also determined by d.s.c. analysis. The present study proposes a method for estimating the solubilities of urea and NaCl in both of the freezing and the non-freezing water using the melting point depression of the freezing water in the membranes immersed in the solute solution.     

Radical polymerization of (phenylethynyl)styrenes and characterization of poly(phenylethynyl)styrenes as a thermally curable material

Summary The radical polymerizations of 2-, 3-, and 4-(phenylethynyl)styrenes (1a–c) and the copolymerizations of 1a–c (M₁) with styrene (M₂) were carried out using AIBN as the initiator in toluene at 60°C. The number-average molecular weights (M _ns) were extremely low for poly(2-phenylethynylstyrene) (2a) and poly[(phenylethynyl)styrene-co-styrene] (3a), and increased in the order of 2a, 3a << 2b, 3b < 2c, 3c. Monomer reactivity ratios were determined as r ₁= 1.80 and r ₂= 0.51 for 1a, r ₁= 1.72 and r ₂= 0.53 for 1b, and r ₁= 3.17 and r ₂= 0.24 for 1c. Polymers 2a–c and 3a–c underwent an exothermic reaction at elevated temperature to form organic solvent-insoluble polymers. Although the decomposition of 2a was observed from 200°C, 2b and 2c exhibited a high heat-resistance property in both nitrogen and air atmospheres, in particular, 2b showed no significant weight loss below 450°C. Received: 28 January 1998/Accepted: 5 March 1998     

Fine Patterning and Characterization of Gel Films Derived from Methyltriethoxysilane and Tetraethoxysilane

Pregrooves of 1.6 µm pitch for optical data storage have been embossed successfully by pressing a stamper against x CH₃Si(OC₂H₅)₃(100 - x )Si(OC₂H₅)₄-derived gel films (60 ≤ x ≤ 100 mol%) on glass-disk substrates of 130 mm diameter. When x is <40 mol%, the resultant films are too hard to emboss patterns uniformly. The shrinkage of the patterns is ∼4% for all the films when 60 lessthan equal to x lessthan equal to 100 mol%, even after heat treatment at 350°C, so that the nearly net negative shape of the stamper is preserved. The methyl groups in the films decompose at temperatures from 500° to 600°C.