Hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues in subcritical water

The hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues with different glycosidic bonds, 1-kestose, d-melezitose, d-raffinose, and lactosucrose, in subcritical water were conducted over the temperature range of 150-230 degrees C and at a constant pressure of 10 MPa. The hydrolysis of trisaccharides in subcritical water proceeded consecutively, i.e., one cleavage of the two bonds antedated the other. The preceding cleavage was not expressed by the first-order kinetics, but by the kinetics considering the concentration of the acidic compounds, which were produced by the degradation of the constituent monosaccharides. The hydrolysis of the constituent disaccharides, except sucrose composed of the alpha-Glc-(1-->2)-beta-Fru bond, obeyed first-order kinetics. All of the rate constants of the hydrolytic kinetics were determined, and the values were found to depend on the type of bond.     

Zinc is required for Fc epsilon RI-mediated mast cell activation

Zinc (Zn) is an essential nutrient, and its deficiency causes growth retardation, immunodeficiency, and neuronal degeneration. However, the precise roles and molecular mechanism(s) of Zn function in immune response have not been clarified. Mast cells (MCs) are granulated cells that play a pivotal role in allergic reactions and inflammation. The granules of MCs contain various chemical mediators and inflammatory cytokines that are released upon FcepsilonRI cross-linking. In this study, we report that Zn is essential for MC activation both in vitro and in vivo. We showed that a Zn chelator, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine, inhibited in vivo allergic reactions such as PCA and PSA. Consistent with this, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine significantly inhibited the FcepsilonRI-induced degranulation and cytokine production. We found that Zn was required for FcepsilonRI-induced translocation of granules to the plasma membrane, a process that we have shown to be important for MC degranulation. In addition, we showed that Zn was essential for plasma membrane translocation of protein kinase C and subsequent nuclear translocation of NF-kappaB, leading to cytokine production, such as IL-6 and TNF-alpha. These results revealed that Zn was involved in multiple steps of FcepsilonRI-induced MC activation and required for degranulation and cytokine production.     

Porphyromonas gingivalis FimA Fimbriae: Fimbrial Assembly by fimA Alone in the fim Gene Cluster and Differential Antigenicity among fimA Genotypes

The periodontal pathogen Porphyromonas gingivalis colonizes largely through FimA fimbriae, composed of polymerized FimA encoded by fimA. fimA exists as a single copy within the fim gene cluster (fim cluster), which consists of seven genes: fimX, pgmA and fimA-E. Using an expression vector, fimA alone was inserted into a mutant from which the whole fim cluster was deleted, and the resultant complement exhibited a fimbrial structure. Thus, the genes of the fim cluster other than fimA were not essential for the assembly of FimA fimbriae, although they were reported to influence FimA protein expression. It is known that there are various genotypes for fimA, and it was indicated that the genotype was related to the morphological features of FimA fimbriae, especially the length, and to the pathogenicity of the bacterium. We next complemented the fim cluster-deletion mutant with fimA genes cloned from P. gingivalis strains including genotypes I to V. All genotypes showed a long fimbrial structure, indicating that FimA itself had nothing to do with regulation of the fimbrial length. In FimA fimbriae purified from the complemented strains, types I, II, and III showed slightly higher thermostability than types IV and V. Antisera of mice immunized with each purified fimbria principally recognized the polymeric, structural conformation of the fimbriae, and showed low cross-reactivity among genotypes, indicating that FimA fimbriae of each genotype were antigenically different. Additionally, the activity of a macrophage cell line stimulated with the purified fimbriae was much lower than that induced by Escherichia coli lipopolysaccharide.     

Construction of a genetic linkage map based on amplified fragment length polymorphism markers and development of sequence-tagged site markers for marker-assisted selection of the sporeless trait in the oyster mushroom (Pleurotus eryngii)

A large number of spores from fruiting bodies can lead to allergic reactions and other problems during the cultivation of edible mushrooms, including Pleurotus eryngii (DC.) Quél. A cultivar harboring a sporulation-deficient (sporeless) mutation would be useful for preventing these problems, but traditional breeding requires extensive time and labor. In this study, using a sporeless P. eryngii strain, we constructed a genetic linkage map to introduce a molecular breeding program like marker-assisted selection. Based on the segregation of 294 amplified fragment length polymorphism markers, two mating type factors, and the sporeless trait, the linkage map consisted of 11 linkage groups with a total length of 837.2 centimorgans (cM). The gene region responsible for the sporeless trait was located in linkage group IX with 32 amplified fragment length polymorphism markers and the B mating type factor. We also identified eight markers closely linked (within 1.2 cM) to the sporeless locus using bulked-segregant analysis-based amplified fragment length polymorphism. One such amplified fragment length polymorphism marker was converted into two sequence-tagged site markers, SD488-I and SD488-II. Using 14 wild isolates, sequence-tagged site analysis indicated the potential usefulness of the combination of two sequence-tagged site markers in cross-breeding of the sporeless strain. It also suggested that a map constructed for P. eryngii has adequate accuracy for marker-assisted selection.     

Anti-oxidant activity of acyl ascorbates in intestinal epithelial cells

The anti-oxidant activity of acyl ascorbates, with acyl chain lengths of 10 to 18, was measured in an intestinal epithelial cell line. All the acyl ascorbates exhibited the activity for the oxidization induced by three different types of oxidants, at levels higher than ascorbic acid. Myristoyl ascorbate was the most effective and showed activity at 3×10^–12 to 3×10^–6 M. Incubation with myristoyl ascorbate for more than 3 h was needed to sufficiently suppress the oxidization of the intestinal cell monolayers.     

Relationship between structure and permeability of dipeptide derivatives containing tryptophan and related compounds across human intestinal epithelial (Caco-2) cells

The permeability of dipeptide derivatives containing tryptophans and indole derivatives through Caco-2 cells was used as an in vitro intestinal absorption model in order to clarify structural factors which influence their intestinal epithelial permeation and metabolism. Most peptide derivatives were hydrolysed not only by the cytosolic enzymes in Caco-2 cells during permeation but also by enzymes released to the apical solution before cell permeation. The N-terminal blocked dipeptides were more resistant to hydrolases expressed in the Caco-2 cells and indole derivatives were not entirely degraded. Based on compound concentration dependency and comparison of permeability coefficients in apical-to-basolateral and basolateral-to-apical directions, the main absorption mechanism of compounds were determined. Compounds were then classified into three groups; (1) passively transported compounds, (2) actively transported compounds and (3) compounds excreted by P-glycoprotein.     

Relationships between structure and high-throughput screening permeability of peptide derivatives and related compounds with artificial membranes: application to prediction of Caco-2 cell permeability

To evaluate absorption of compounds across the membrane via a transcellular route, the permeability of peptide derivatives and related compounds was measured by the parallel artificial membrane permeation assay (PAMPA). The permeability coefficients by PAMPA were analyzed quantitatively using classical QSAR and Volsurf approaches with the physicochemical parameters. The results from both approaches showed that hydrogen bonding ability of molecules in addition to hydrophobicity at a particular pH were significant in determining variations in PAMPA permeability coefficients. The relationship between Caco-2 cell permeability and artificial lipid membrane permeability was then determined. The compounds were sorted according to their absorption pathway in the plot of the Caco-2 cell and PAMPA permeability coefficients.     

Failure of multinucleated giant cell formation in k562 cells infected with newcastle disease virus and human parainfluenza type 2 virus

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV-2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV-2 is due to disturbance of the viral envelope-cell fusion step or to defect in the cell-cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin-neuraminidase and fusion proteins expressed on the cell surface of NDV-infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus-induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus-induced cell fusion may function on the cell surface of K562 cells.