Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.     

IL-4-transfected tumor cell vaccines activate tumor-infiltrating dendritic cells and promote type-1 immunity

We previously demonstrated that IL-4 gene-transfected glioma cell vaccines induce effective therapeutic immunity in preclinical glioma models, and have initiated phase I trials of these vaccines in patients with malignant gliomas. To gain additional mechanistic insight into the efficacy of this approach, we have treated mice bearing the MCA205 (H-2(b)) or CMS-4 (H-2(d)) sarcomas. IL-12/23 p40(-/-) and IFN-gamma(-/-) mice, which were able to reject the initial inoculation of IL-4 expressing tumors, failed to mount a sustained systemic response against parental (nontransfected) tumor cells. Paracrine production of IL-4 in vaccine sites promoted the accumulation and maturation of IL-12p70-secreting tumor-infiltrating dendritic cells (TIDCs). Adoptive transfer of TIDCs isolated from vaccinated wild-type, but not IL-12/23 p40(-/-), mice were capable of promoting tumor-specific CTL responses in syngeneic recipient animals. Interestingly, both STAT4(-/-) and STAT6(-/-) mice failed to reject IL-4-transfected tumors in concert with the reduced capacity of TIDCs to produce IL-12p70 and to promote specific antitumor CTL reactivity. These results suggest that vaccines consisting of tumor cells engineered to produce the type 2 cytokine, IL-4, critically depend on type 1 immunity for their observed therapeutic efficacy.     

X-ray diffraction from a left ventricular wall of rat heart

We studied x-ray diffraction from the left ventricular wall of an excised, perfused whole heart of a rat using x rays from the third-generation synchrotron radiation facility, SPring-8. With the beam at right angles to the long axis of the left ventricle, well-oriented, strong equatorial reflections were observed from the epicardium surface. The reflections became vertically split arcs when the beam passed through myocardium deeper in the wall, and rings were observed when the beam passed into the inner myocardium of the wall. These diffraction patterns were explained by employing a layered-spiral model of the arrangement of muscle fibers in the heart. In a quiescent heart with an expanded left ventricle, the muscle fibers at the epicardium surface were found to have a (1,0) lattice spacing smaller than in the rest of the wall. The intensity ratio of the (1,0) and (1,1) equatorial reflections decreased on contraction with a similar time course in all parts of the wall. The results show that it is possible to assign the origin of reflections in a diffraction diagram from a whole heart. This study offers a basis for interpretation of x-ray diffraction from a beating heart under physiologically and pathologically different conditions.     

Receptor-operated Ca2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini

We investigated signal transduction between receptor-operated Ca(2+) influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca(2+) ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca(2+)](o), loading of the intracellular Ca(2+) chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca(2+) or Mn(2+) entry induced by CCK and carbachol, it did not affect intracellular Ca(2+) release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of G(q) protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize G(q)-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca(2+)](o)-dependent pancreatic exocytosis.     

Survey on cellular phone usage on students in Thailand

The number of cellular phone subscribers is increasing every year and there have been reports of health disorders related to the high-frequency radio waves. This paper considers the dependence of Thai university and high school students on cellular phones. A survey form (cellular phone dependence questionnaire: CPDQ) was distributed to 181 female and 177 male Thai university students and to 240 female and 140 male Thai high school students. The surveys were collected, Cronbach alpha coefficient was calculated, and a factor analysis was performed using the principal factor method and varimax rotation. The total scores were 16.54 to 20.04 and the Cronbach's alpha coefficients were 0.808 to 0.930. According to a factor analysis of 20 scored items, 4 factors were extracted for both male and female high school students, and the cumulative correlation coefficients of the male and female groups were 64.85% and 62.70%, respectively. Five factors were extracted for male university students and 6 factors were extracted for female university students, and the cumulative correlation coefficients were 58.08% and 57.91%, respectively. The W value results of the Shapiro-Wilk W-test for male university students, female university students, male high school students and female high school students were 0.969, 0.984, 0.964, and 0.913 respectively, thus verifying the normality of the score distributions.The total scores for the Thai university students were higher than the scores for the Thai high school students. The factor analysis of female high school students confirmed a large difference compared to male university students, male high school students, and Japanese female university students. (The Japanese students were surveyed in an earlier study by Toda et al.). Also, the CPDQ total score was high, which indicated a strong tendency toward dependence.     

Cystic Fibrosis Transmembrane Conductance Regulator Interacts with Multiple Immunoglobulin Domains of Filamin A

Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa·partner complexes as templates, we generated in silico models of IgFLNa·CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated (∼160 nm) strands, whereas CFTR is compact (6∼8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa·partner complexes, the FLNa-monomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.     

Improved catalytic and stereoselective glycosylation with glycosyl N-trichloroacetylcarbamate: application to various 1-hydroxy sugars

Efficient catalytic and stereoselective glycosylation was achieved by activating a glycosyl N-trichloroacetylcarbamate with a catalytic amount of Lewis acid in the presence of a glycosyl acceptor and 5 ? molecular sieves. Catalytic one-pot dehydrative glycosylation of a 1-hydroxy carbohydrate was achieved stereoselectively by reaction with trichloroacetyl isocyanate, followed by activation with a catalytic amount of activators.