Phosphatidylinositol 3-kinase/Akt signaling mediates interleukin-32alpha induction in human pancreatic periacinar myofibroblasts

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by the induction of nuclear factor (NF)-kappaB activation. We studied IL-32alpha expression in human pancreatic periacinar myofibroblasts, which play important roles in the regulation of extracellular matrix metabolism and inflammatory responses in the pancreas. IL-32alpha protein expression was evaluated by Western blot analyses, and IL-32alpha mRNA expression was analyzed by Northern blot and real-time PCR analyses. IL-32alpha mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1beta, IFN-gamma, and TNF-alpha. IL-1beta, IFN-gamma, and TNF-alpha enhanced intracellular accumulation of IL-32alpha protein, but IL-32alpha was not detected in supernatants. Each cytokine dose and time dependently induced IL-32alpha mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1beta-, IFN-gamma-, and TNF-alpha-induced IL-32alpha mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blot. Furthermore, LY294002 suppressed both IL-1beta- and TNF-alpha-induced NF-kappaB activation and IL-1beta-, TNF-alpha-, and IFN-gamma-induced activated protein-1 (AP-1) activation. Blockade of NF-kappaB and AP-1 activation by an adenovirus expressing a stable mutant form of IkappaBalpha and a dominant negative mutant of c-Jun markedly suppressed IL-1beta-, IFN-gamma-, and/or TNF-alpha-induced IL-32alpha mRNA expression. Human pancreatic periacinar myofibroblasts expressed IL-32alpha in response to IL-1beta, TNF-alpha, and IFN-gamma. IL-32alpha mRNA expression is dependent on interactions between the phosphatidylinositol 3-kinase/Akt-pathway and the NF-kappaB/AP-1 system.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes

Background information. Mercurials inhibit AQPs (aquaporins), and site‐directed mutagenesis has identified Cys¹⁸⁹ as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury‐insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys¹⁸⁹ of AQP1. Indeed, the osmotic water permeability (P_f) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl₂. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His‐tagged rAPR4 (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni²⁺‐nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. Results. The water permeability of AQP4 proteoliposomes with or without HgCl₂ was measured with a stopped‐flow apparatus. Surprisingly, the P_f of AQP4 proteoliposomes was significantly decreased by 5 μM HgCl₂ within 30 s, and this effect was completely reversed by 2‐mercaptoethanol. The dose‐ and time‐dependent inhibitory effects of Hg²⁺ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site‐directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys¹⁷⁸, which is located at loop D facing the intracellular side, is a target responding to Hg²⁺. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the P_f of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105)

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).     

Detection of Legionella pneumophila serogroup 1 antigen in respiratory samples using an immunochromatographic membrane test

The immunochromatographic membrane test (ICT) efficacy of Legionella antigen detection (Binax Now Legionella®) was evaluated using respiratory samples, including bronchial washings (44 cases) and sputum (128 cases), from suspected Legionella pneumonia patients. The ICT results using respiratory samples agreed well with isolation of L. pneumophila SG1 and ICT using urines.     

Elucidation of the role of activin in organogenesis using a multiple organ induction system with amphibian and mouse undifferentiated cells in vitro

Studies performed over the last century have clarified the mechanisms of organ and tissue formation. Mesoderm formation is one of the most important events in early body pattern determination during embryogenesis. In 1988, we found that activin A has mesoderm-inducing activity. As activin A could induce dorsal mesoderm formation, unlike fibroblast growth factor and bone morphogenetic protein, this factor was thought to be the molecular entity of the Spemann-Mangold organizer. Subsequently, the mechanisms of early embryogenesis have been clarified using molecular biological techniques, resulting in the identification of many genes that are involved in organ and tissue development. This finding that activin A could induce dorsal mesoderm formation spurred research into the application of agents that induce organs and tissues in vitro . In this regard, we have shown that many organ types can be induced by activin A in vitro . Moreover, we have found that other types of organs can be induced by changing the conditions of treatment. To date, more than 20 different types of tissues and organs have been successfully induced from Xenopus undifferentiated cells in vitro . In recent years, we have applied these protocols to mouse embryonic stem cells, and we have successfully induced several tissues, such as the pancreas and cardiomyocytes. We are also investigating how the pluripotency of undifferentiated stem cells is regulated. In this review, we summarize the current knowledge regarding activin as a mesoderm-inducing factor and its application for the induction of tissues and organs from undifferentiated cells. Moreover, we provide some examples of in vitro tissue differentiation from mouse embryonic stem cells, which may prove useful in regenerative medicine.     

Structure-based analysis reveals hydration changes induced by arginine hydrochloride

Arginine hydrochloride has been used to suppress protein aggregation during refolding and in various other applications. We investigated the structure of hen egg-white lysozyme (HEL) and solvent molecules in arginine hydrochloride solution by X-ray crystallography. Neither the backbone nor side-chain structure of HEL was altered by the presence of arginine hydrochloride. In addition, no stably bound arginine molecules were observed. The number of hydration water molecules, however, changed with the arginine hydrochloride concentration. We suggest that arginine hydrochloride suppresses protein aggregation by altering the hydration structure and the transient binding of arginine molecules that could not be observed.