In the immune system, mast cells are a key cell type in the pathogenesis of immunoglobulin E (IgE)-dependent hypersensitivity reactions. Engagement of the high-affinity IgE receptors by multivalent antigens initiates the downstream activation of signal-transducing enzymes and evokes degranulation and cytokine production via an increase in the intracellular Ca2+ concentration. In addition, mast cells also play a prominent role in non-IgE-mediated hypersensitivity reactions. Mast cells are closely apposed to nerves in vivo and are likely to be regulated functionally by nerves. However, the molecular mechanisms for mast cell activation in an IgE-dependent and -independent manner have not been fully clarified. Confocal laser scanning microscopy has played an essential role in cell biology by allowing visualization of specific intracellular signaling molecules with high spatiotemporal resolution in living cells. We have studied intracellular movements of Ca2+ using a specific fluorescent probe and several types of signaling molecules using derivatives of green fluorescent protein in a living single mast cell using a microscopic strategy. We here describe our imaging analysis of the calcium signals to the nucleus, the movement of secretory granules in the degranulation process, and the nucleocytoplasmic shuttling of mitogen-activated protein kinase in mast cells. Further, we demonstrate that direct communication between mast cells and nerves occurs. These findings provide useful information from a new perspective to understand the molecular mechanisms of allergic reaction and inflammation.
Megakaryocytes are the hematopoietic precursors of platelets, which play an essential role in thrombosis and hemostasis. Platelet factor 4 (PF4) is expressed exclusively in megakaryocytes and platelets and serves as a lineage-specific marker of megakaryocytic differentiation. We previously characterized a number of upstream enhancer and repressor elements and demonstrated that GATA-1 and ETS-1 are important for PF4 gene expression. Recently, we have determined the novel regulatory element termed “TME” in the PF4 promoter and identified a group of binding proteins from megakaryocytic HEL cells. Here we review the function of these proteins in PF4 gene expression and discuss megakaryocyte-specific gene expression and megakaryocytepoiesis.
Highly stereoselective reactions have been developed using lone pairs of oxygen atoms, in other words, the nucleophilic ability of oxygen atoms. They consist of five types of reactions: 1) asymmetric synthesis using acetals, 2) organic reactions via Beckmann fragmentation intermediates, 3) organic reactions via dication equivalents, 4) rearrangement of 2,3-epoxy-1-alcohol derivatives, and 5) organic reactions using orthoesters. In this review, asymmetric syntheses using acetals are described in detail. They are divided into four categories: 1) diastereoselective 1,2-addition(alkylation, reduction) to the α-keto acetals and their derivatives, 2) asymmetric carbon-carbon bond formation by a combination of Beckmann fragmentation and asymmetric synthesis using C2-symmetric chiral acetals, 3) asymmetric synthesis via chiral oxonium ions prepared by intramolecular haloetherification of chiral ene acetals having C2-symmetric hydrobenzoin, and 4) asymmetric desymmetrization of meso-1,2-, meso-1,3-, and meso-1,4-diols via chiral oxonium ions prepared by intramolecular haloetherification of chiral ene acetals derived from optically pure norbornen aldehyde derivatives, and their applications to asymmetric syntheses of biologically active natural products.
Eosinophils are one of the cells that play a critical role in the pathogenesis of allergic diseases. The increase in the number of eosinophils in such diseases is regulated by interleukin-5 (IL-5). The author have prepared recombinant rat IL-5 using a baculovirus expression system and examined its biological activities in rat eosinophils. It was demonstrated that recombinant rat IL-5 prolongs the survival of mature eosinophils and differentiates immature eosinophils into mature eosinophils, suggesting that rat IL-5 is a factor for eosinophilia in rats. Recombinant rat eosinophil-associated ribonuclease (Ear)-1 and Ear-2 were also prepared. Eosinophil granule proteins are thought to cause tissue damage due to their cytotoxic activity, but using recombinant rat Ear-1 and Ear-2, it was found that rat Ear-1 and Ear-2 have strong RNase A activity and bactericidal activity, suggesting that these proteins play critical roles in host defense. Finally, the important role of acetylation of histones was clarified in the differentiation of HL-60 clone 15 cells into eosinophils using the histone deacetylase inhibitors sodium n-butyrate, apicidin, and trichostatin A. These findings would be useful for further investigations of the role of eosinophils in allergic inflammation.
To understand the folding process of α-helical membrane proteins in a lipid bilayer environment, the mechanisms of membrane partitioning and self-association of the helix should be elucidated. Considering the inhomogeneity of biological membranes composed of various lipids, not only the amino acid sequence of the transmembrane helices but also the composition of the lipid bilayers are determinants for folding and intramembrane distribution of membrane proteins thorough the balance between helix-helix, helix-lipid, and lipid-lipid interactions. Thermodynamic study using model transmembrane helices is fascinating to measure these complex interactions experimentally. The effects of lipid composition on membrane partitioning and self-association of an inert model transmembrane helix, (AALALAA)3, were examined. Partitioning of the helix into phosphoethanolamine-containing bilayers, gel-phase bilayers, or liquid ordered-phase bilayers significantly decreased, presumably by decreasing the fluidity in the hydrophobic region of the bilayer. It was found that the difference in the length of the hydrophobic regions between helix and lipid bilayers is energetically unfavorable, and partitioning into thicker and thinner membranes were weakened by increasing enthalpic and entropic terms, respectively. In contrast, stronger helix associations driven by the decrease in enthalpy were observed with increasing membrane thickness. These results demonstrate that the surrounding lipids are also important factors determining the behavior of transmembrane helices.
A rapid, sensitive, and specific reverse-phase high-performance liquid chromatography (HPLC) method was developed to quantitate ibuprofen (IBU) and its prodrug, ibuprofen eugenol ester (IEE), simultaneously in rat plasma. IBU, IEE, and the internal standard glycryrrhetic acid (GA) were detected by UV absorption at 230 nm. Extraction recoveries for all compounds ranged between 82.6% and 96.2%. Retention times of IBU, IEE, and GA were 5.62, 15.98, and 18.05 min, respectively. Calibration plots were linear over the range of 0.64 to 64 μg/ml for IBU, and 0.16 to 80 μg/ml for IEE. The limit of quantitation was 0.64 μg/ml for IBU and 0.16 μg/ml for IEE. The intra- and interday variations were less than 10% and accuracy was greater than 90%. The results showed that the established method is reproducible and sensitive and applicable to plasma samples collected from rats administered IBU and IEE intravenously.
Limited use of generics in Japan has been justified on the basis of problematic quality, distribution and information. Of these three problem areas, the state of provision of information in particular has never been objectively evaluated. We therefore sought to evaluate information according to its necessity and importance to medical practice. To establish criteria for evaluation, we weighted 36 separate pieces of drug information found in package inserts and interview forms according to necessity and importance, based on the results of a survey of nationwide medical institutions with DI offices. We then used the evaluation criteria to evaluate currently available drugs with 20 or more products per formulation. We evaluated 14 formulations (324 products). Generic drugs were found to have 25.3±18.7 to 46.1±14.2% (Mean±S.D.) the information of brand name drugs when products were compared for quantity of information by formulation. However, comparison according to manufacturer returned a larger range of variation at 14.4±8.6 to 64.3±14.2% (Mean±S.D.). These data reveal that manufacturer differences play a large role in the provision of drug information. Drug information was also compared separately by category for both brand name and generic drugs. Generic drugs were found to have insufficient information on clinical data, pharmacokinetics, safety, side effects, and nonclinical tests. Brand name drugs also scored low points for information on pharmacokinetics. It is imperative that both brand name and generic drugs provide more information on pharmacokinetics.