Dendrimer-immobilized culture surface as a tool to evaluate formation of cellular cytoskeleton of anchorage-dependent cells

Anchorage-dependent cultivation of human epithelial and keratinocyte cells was carried out on surfaces modified with synthesized dendrimers. Notable elongation of the epithelial cells was recognized on the culture surface immobilized with a dendrimer having D-glucose as a functional ligand, but not when a dendrimer having L-glucose was used or when the dendrimer was ligand-free. This morphological change was attributable to a temporary grasping of the cells at the D-glucose moiety via a glucose transporter-mediated mechanism present in the cell membrane. Following visualization of the actin filaments of the cells, it was considered that the cellular elongation on the D-glucose-bound dendrimer surface reflected the degree of formation of the cellular cytoskeleton. The cellular roundness was calculated by means of image analysis of the individual cells and employed as a parameter to evaluate the formation of the cellular cytoskeleton. In the culture of keratinocytes on the D-glucose-bound dendrimer surface, it was demonstrated that the decrease in the ratio of elongated cells (i.e., cells with a low roundness value) was correlated with the deterioration in the growth potential associated with cellular senescence.     

Host-dependent activation of IS1203v excision in Shiga toxin-producing Escherichia coli

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.     

Cloning and characterization of a β-N-acetylglucosaminidase (BmFDL) from silkworm Bombyx mori

In insects, β-N-acetylglucosaminidase (GlcNAcase) participates in critical physiological processes such as fertilization, metamorphosis, and glycoconjugate degradation. Insects produce glycoproteins carrying paucimannosidic-type N-glycans, the terminal GlcNAc residue of which is cleaved by a GlcNAc-linkage specific GlcNAcase, also known as the fused lobes (FDL) protein. To obtain information on the structure of GlcNAcases and insight into their contribution to physiological processes, we cloned Bombyx mori FDL (BmFDL) from silkworm larvae. The full-length cDNA (1.9 kb) encoded a protein of 633 amino acids with 42% amino acid sequence identity to Drosophila melanogaster FDL (DmFDL). Recombinant BmFDL cleaved only β-1,2-linked GlcNAc residues from the α-1,3 branch of biantennary N-glycan. This substrate specificity was similar to that of DmFDL. Microsomal FDL activity was inhibited by anti-BmFDL antibodies. Taken together, our results suggest that BmFDL is a N-glycan-processing GlcNAcase in B. mori.     

Preparation of samples for proficiency testing of pesticide residue analysis in processed foods

To conduct proficiency testing for the analysis of pesticide residues in processed foods, fortified samples of retort curry and pancake were examined. In the case of retort curry, heating and mixing were necessary at the time of preparation to provide a homogenous analytical sample. A mixture of 4 carbamates and 11 organophosphorus pesticides was spiked and 14 of them showed consistent results in the samples. In the case of pancake, 10 kinds of pesticides were added to the pastry. The prepared pastry was them cooked. The relative concentrations of most of the pesticides in the pancake were not affected and all the pesticides showed consistent results in the samples. These results showed that the two tested samples were suitable for proficiency testing.     

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.     

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.