Synthesis of norlignans and in vitro inhibitory activity of antigen-induced degranulation

The synthesis and biological evaluation of a series of novel norlignans are described. Norlignans were evaluated for their inhibitory activity on the release of β-hexosaminidase, a marker of degranulation, from RBL-2H3 cells induced by the IgE-antigen complex. The results showed that norlignans 4c and 4e potently inhibited degranulation, with IC(50) values of 18.3 and 17.9 μM, respectively.     

Enzymatic formation of carbohydrate rings catalyzed by single-walled carbon nanotubes

Macrocyclic carbohydrate rings were formed via enzymatic reactions around single-walled carbon nanotubes (SWNTs) as a catalyst. Cyclodextrin glucanotransferase, starch substrate and SWNTs were reacted in buffer solution to yield cyclodextrin (CD) rings wrapped around individual SWNTs. Atomic force microscopy showed the resulting complexes to be rings of 12–50 nm in diameter, which were highly soluble and dispersed in aqueous solution. They were further characterized by Raman and Fourier transform infrared spectroscopy and molecular simulation using density functional theory calculation. In the absence of SWNT, hydrogen bonding between glucose units determines the structure of maltose (the precursor of CD) and produces the curvature along the glucose chain. Wrapping SWNT along the short axis was preferred with curvature in the presence of SWNTs and with the hydrophobic interactions between the SWNTs and CD molecules. This synthetic approach may be useful for the functionalization of carbon nanotubes for development of nanostructures.     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

Phosphorylation is required for alteration of kv1.5 K(+) channel function by the Kvbeta1.3 subunit.

The Kv1.5 K(+) channel is functionally altered by coassembly with the Kvbeta1.3 subunit, which induces fast inactivation and a hyperpolarizing shift in the activation curve. Here we examine kinase regulation of Kv1.5/Kvbeta1.3 interaction after coexpression in human embryonic kidney 293 cells. The protein kinase C inhibitor calphostin C (3 microM) removed the fast inactivation (66 +/- 1.9 versus 11 +/- 0.25%, steady state/peak current) and the beta-induced hyperpolarizing voltage shift in the activation midpoint (V(1/2)) (-21.9 +/- 1.4 versus -4.3 +/- 2.0 mV). Calphostin C had no effect on Kv1.5 alone with respect to inactivation kinetics and V(1/2). Okadaic acid, but not the inactive derivative, blunted both calphostin C effects (V(1/2) = -17.6 +/- 2.2 mV, 38 +/- 1.8% inactivation), consistent with dephosphorylation being required for calphostin C action. Calphostin C also removed the fast inactivation (57 +/- 2.6 versus 16 +/- 0.6%) and the shift in V(1/2) (-22.1 +/- 1.4 versus -2.1 +/- 2.0 mV) conferred onto Kv1.5 by the Kvbeta1.2 subunit, which shares only C terminus sequence identity with Kvbeta1. 3. In contrast, modulation of Kv1.5 by the Kvbeta2.1 subunit was unaffected by calphostin C. These data suggest that Kvbeta1.2 and Kvbeta1.3 subunit modification of Kv1.5 inactivation and voltage sensitivity require phosphorylation by protein kinase C or a related kinase.     

Proteoglycan isolated from Phellinus linteus inhibits tumor growth through mechanisms leading to an activation of CD11c+CD8+ DC and type I helper T cell-dominant immune state

Dendritic cells (DC) are known to not only induce the activation of T cells, but are also associated with the polarization of T cells. This study investigated whether or not proteoglycan (PG) isolated from Phellinus linteus induces the phenotypic and functional maturation of CD11c+ DC in vitro and in vivo. PG was found to induce the phenotypic and functional maturation of bone marrow-derived DC via Toll-like receptors (TLR) 2 and 4 in vitro. Administration of PG in vivo strongly inhibited the MCA-102 tumor growth and increase in vivo. The ratio of CD8+ DC to CD8- DC increased, and PG enhanced IL-12 and IFN-gamma production, and expression of surface molecules including major histocompatibility complexes (MHC) classes I, MHC II, CD80, and CD86 in MCA-102-challenged mice. PG also caused a marked increase in the production of Th (helper T cells)-1 cytokine (IFN-gamma) and a decrease in the production of Th-2 cytokine (IL-4) by splenic cells and inguinal lymph node cells in MCA-102 tumor-bearing mice. Furthermore, PG stimulated the proliferation of CD4+ and CD8+ T cells. In addition, a combination of PG and tumor lysate-pulsed DC inhibited completely the growth of MCA-102 cells in tumor-bearing mice. These results indicate that the administration of PG inhibited the tumor growth through a mechanism leading to a Th-1 dominant immune state and the activation of CD11cCD8+ DC.     

Enhancement of in situ hybridization to detect bovine herpesvirus 4 in paraffin sections

In situ hybridization (ISH) protocols including different pre-treatment regimes were developed and compared for their effects on detecting bovine herpesvirus 4 (BoHV-4) in formalin fixed, paraffin embedded tissue. Results were compared for the hybridization and background signal intensities, and cellular morphology. We found that optimum results were obtained using enzyme treatment-thermal cycling as a pre-treatment of ISH. The results showed that the combination of protease and thermal cycling would be recommended as a means of supplementing in situ hybridization methods, especially when using long-term formalin fixed, paraffin-embedded tissue.     

O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic beta cells

O-GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues with O-GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O-GlcNAc. However, the nature and significance of the Akt1 O-GlcNAc modification is unknown. The relationship of O-GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine beta-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O-GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O-GlcNAcase revealed an inverse correlation between O-GlcNAc modification and Ser473 phosphorylation of Akt1. MALDI-TOF/TOF mass spectrometry analysis of Akt1 immunoprecipitates from glucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified with O-GlcNAc. Furthermore, in vitro O-GlcNAc-modification analysis of wildtype and mutant Akt1 revealed that S473 was targeted by recombinant OGT. A S473A Akt1 mutant demonstrated reduced basal and glucosamine-induced Akt1 O-GlcNAc modification compared with wildtype Akt1. Furthermore, wildtype Akt1, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Akt1 Ser473 may undergo both phosphorylation and O-GlcNAc modification, and the balance between these may regulate murine beta-pancreatic cell fate.