Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca²⁺ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca²⁺ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inhibitory effect of src homology (SH) 2/SH3 fragments of phospholipase C-gamma on the catalytic activity of phospholipase C isoforms. Identification of a novel phospholipase C inhibitor region.

In order to study the regulatory mechanisms of phospholipase C-gamma (PLC-gamma) via the intrinsic SH2/SH3 region (Z region), two recombinant Z proteins, rP45Z and rP38Z, derived from rat PLC-gamma 1 and PLC-gamma 2, respectively, were purified from the inclusion bodies of Escherichia coli. We examined their direct effects on phosphoinositide hydrolysis induced by four different PLC isoforms purified from bovine brain and thymus, and found that both of these Z proteins suppress the enzyme activity of all four PLC isoforms in a dose-dependent manner. This suppressive effect is very potent and stoichiometric. The kinetics studies indicate that the suppression is non-competitive. This suppression is eliminated by treatment with proteases but is not affected by heat treatment at 95 degrees C for 15 min, indicating that the primary structure might be important for the action of Z proteins. Comparative studies suggested that two Z proteins but not Src and phosphatidylinositol 3-kinase possess, adjacent to their SH2 and SH3 motifs, a phospholipase C inhibitor (PCI) region that strongly suppresses their phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity. A series of synthetic peptides identical with the sequence of the proposed PCI region, including an octamer, YRKMRLRY, inhibited PIP2 hydrolysis induced by four different phospholipase C isoforms. These results demonstrate that both types of phospholipase C-gamma contain the PCI sequence which is responsible for the inhibition of PIP2 hydrolysis, indicating that phospholipase C-gamma is a self-regulating enzyme.     

Identification of another actin-related protein (Arp) 2/3 complex binding site in neural Wiskott-Aldrich syndrome protein (N-WASP) that complements actin polymerization induced by the Arp2/3 complex activating (VCA) domain of N-WASP.

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic (VCA) or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. We focused on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduced actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP led to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP was much smaller than that of wild-type N-WASP. Partial deletion of acidic residues did not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion.     

Imaging mass spectroscopy delineates the thinned and thickened walls of intracranial aneurysms

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The wall thickness of intracranial aneurysms (IAs) is heterogeneous. Although thinning of the IA wall is thought to contribute to IA rupture, the underlying mechanism remains poorly understood. Recently, imaging mass spectroscopy (IMS) has been used to reveal the distribution of phospholipids in vascular diseases. To investigate the feature of phospholipid composition of IA walls, we conducted IMS in a rat model of experimentally induced IA.

Isolation of a cDNA encoding human lysophosphatidic acid phosphatase that is involved in the regulation of mitochondrial lipid biosynthesis.

In this study, we isolated cDNA encoding lysophosphatidic acid (LPA) phosphatase (LPAP). The amino acid sequence deduced from the cDNA encoding LPAP had 421 residues including a putative signal peptide and was homologous to acid phosphatase, especially at the active site. Human LPAP had 28.5% amino acid identity to human prostatic acid phosphatase. Northern blot analysis showed a ubiquitous expression of LPAP, which was marked in kidney, heart, small intestine, muscle, and liver. Human chromosome map obtained by fluorescence in situ hybridazation showed that the gene for LPAP was localized to chromosome 1 q21. The mutant in which histidine was replaced with alanine at the active site and the putative signal peptide-deleted LPAP had no LPA phosphatase activity. In addition, the putative signal peptide-deleted LPAP showed no mitochondrial localization. The site of intracellular localization of endogenous LPAP was also mitochondria in MDCK cells and differentiated C2C12 cells. The LPAP homologous phosphatase, human prostatic acid phosphatase, also has LPA phosphatase activity. LPAP-stable transfected NIH 3T3 cells showed less phosphatidic acid, phosphatidylglycerol, and cardiolipin. These results suggested that LPAP regulates lipid metabolism in mitochondria via the hydrolysis of LPA to monoacylglycerol.     

Local lymphogenic migration pathway in normal mouse spleen

Although the immunological and hemodynamical significance of the spleen is of great importance, few reports detail the lymphatic vessels in this organ. We have used an immunohistochemical three-dimensional imaging technique to characterize lymphatic vessels in the normal mouse spleen and have successfully demonstrated their spatial relationship to the blood vascular system for the first time. Lymphatic markers, such as LYVE-1, VEGFR-3, and podoplanin, show different staining patterns depending on their location in the spleen. LYVE-1-positive lymphatic vessels run reverse to the arterial blood flow along the central arteries in the white pulp and trabecular arteries and exit the spleen from the hilum. These lymphatic vessels are surrounded by type IV collagen, indicating that they are collecting lymphatic vessels rather than lymphatic capillaries. Podoplanin is expressed not only in lymphatic vessels, but also in stromal cells in the white pulp. These podoplanin-positive cells form fine meshworks surrounding the lymphatic vessels and central arteries. Following intravenous transplantation of lymphocytes positive for green fluorescent protein (GFP⁺) into normal recipient mice, donor cells appear in the meshworks within 1 h and accumulate in the lymphatic vessels within 6 h after injection. The GFP⁺ cells further accumulate in a draining celiac lymph node through the efferent lymphatic vessels from the hilum. These meshworks might therefore act as an extravascular lymphatic pathway and, together with ordinary lymphatic vessels, play a primary role in the cell traffic of the spleen, additional to the blood circulatory system.     

Innovative treatment system for digester liquor using anammox process

This study demonstrated that partial nitritation using nitrifying activated sludge entrapped in a polyethylene glycol (PEG) gel carrier, as a pretreatment to anammox process, could be successfully applied to digester liquor of biogas plant at a nitrogen loading rate of 3.0 kg-N/m³/d. The nitritation process produced an effluent with a NO₂–N/NH₄–N ratio between 1.0 and 1.4, which was found to be suitable for the subsequent anammox process. A high SS concentration (2000–3000 mg/l) in the digester liquor did not affect partial nitritation treatment performances. Effluent from this partial nitritation reactor was successfully treated in the anammox reactor using anammox sludge entrapped in the PEG gel carrier with T-N removal rates of greater than 4.0 kg-N/m³/d. Influent BOD and SS contents did not inhibit anammox activity of the anammox gel carrier. The combination of partial nitritation and anammox reactors using PEG entrapped nitrifying and anammox bacteria was shown to be effective for the removal of high concentration ammonium in the digester liquor of a biogas plant.     

Potent small molecule mouse CD22-inhibitors: Exploring the interaction of the residue at C-2 of sialic acid scaffold

Our previous study revealed that compound 1 (9-(4′-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcα2-6GalOMP) has the most promising affinity for mCD22. Replacing the subterminal galactose residue of 1 with benzyl or biphenylmethyl as aglycone led to 38- and 20-fold higher potency, respectively. This discovery represents a new direction in inhibitor design suitable for pharmaceutical development.