Diacylglycerol Kinase η Activity in Cells Using Protein Myristoylation and Cellular Phosphatidic Acid Sensor

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PtdOH) and regulates the balance between two lipid second messengers: diacylglycerol and PtdOH. Several lines of evidence suggest that the η isozyme of DGK is involved in the pathogenesis of bipolar disorder. However, the detailed molecular mechanisms regulating the pathophysiological functions remain unclear. One reason is that it is difficult to detect the cellular activity of DGKη. To overcome this difficulty, we utilized protein myristoylation and a cellular PtdOH sensor, the N-terminal region of α-synuclein (α-Syn-N). Although DGKη expressed in COS-7 cells was broadly distributed in the cytoplasm, myristoylated (Myr)-AcGFP-DGKη and Myr-AcGFP-DGKη-KD (inactive (kinase-dead) mutant) were substantially localized in the plasma membrane. Moreover, DsRed monomer-α-Syn-N significantly colocalized with Myr-AcGFP-DGKη but not Myr-AcGFP-DGKη-KD at the plasma membrane. When COS-7 cells were osmotically shocked, all DGKη constructs were exclusively translocated to osmotic shock-responsive granules (OSRG). DsRed monomer-α-Syn-N markedly colocalized with only Myr-AcGFP-DGKη at OSRG and exhibited a higher signal/background ratio (3.4) than Myr-AcGFP-DGKη at the plasma membrane in unstimulated COS-7 cells (2.5), indicating that α-Syn-N more effectively detects Myr-AcGFP-DGKη activity in OSRG. Therefore, these results demonstrated that the combination of myristoylation and the PtdOH sensor effectively detects DGKη activity in cells and that this method is convenient to examine the molecular functions of DGKη. Moreover, this method will be useful for the development of drugs targeting DGKη. Furthermore, the combination of myristoylation (intensive accumulation in membranes) and α-Syn-N can be applicable to assays for various cytosolic PtdOH-generating enzymes.     

Elevation of Chemosensitivity of Lung Adenocarcinoma A549 Spheroid Cells by Claudin-2 Knockdown through Activation of Glucose Transport and Inhibition of Nrf2 Signal

Claudin-2 (CLDN2), a tight junctional protein, is involved in the chemoresistance in a three-dimensional spheroid culture model of human lung adenocarcinoma A549 cells. However, the mechanism has not been fully clarified. We found that the knockdown of CLDN2 expression by siRNA in the spheroid reduces the expression of glucose transporters and metabolic enzymes. In a two-dimensional culture model, the expression of these proteins was increased by glucose deprivation or fasentin, an inhibitor of glucose transporter. In addition, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant enzymes including heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and a glutamate–cysteine ligase modifier subunit were increased by fasentin. The fluorescence intensities of JC-1, a probe of mitochondrial membrane potential, and MitoROS 580, a probe of mitochondrial superoxide production, were increased by fasentin. These results suggest that mitochondrial production of reactive oxygen species is increased by glucose deficiency. The knockdown of CLDN2 enhanced the flux of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), a fluorescent deoxyglucose derivative, in a transwell assay, and the accumulation of glucose and 2-NBDG in spheroid cells. The expression of Nrf2 was decreased by CLDN2 knockdown, which was inhibited by fasentin and sulforaphane, a typical Nrf2 activator, in spheroid cells. The sensitivity of spheroid cells to doxorubicin, an anthracycline antitumor antibiotic, was enhanced by CLDN2 knockdown, which was inhibited by fasentin and sulforaphane. We suggest that CLDN2 induces chemoresistance in spheroid cells mediated through the inhibition of glucose transport and activation of the Nrf2 signal.     

Anisotropic elastic constants and thermal expansivities in monocrystal CrB2, TiB2, and ZrB2

The elastic constants and thermal expansivities in monocrystals of three transition-metal diborides with the AlB₂ structure, CrB₂, TiB₂, and ZrB₂, have been investigated in the temperature ranges from 300 to 1373 K and from 300 to 1073 K. The anisotropic parameters deduced from the measured elastic constants and thermal expansivities indicate that of the three diborides, the anisotropy is the most and least significant in CrB₂ and ZrB₂, respectively. The factors determining the significance in anisotropy in atomic bonding in AlB₂-type diborides are analyzed by an approach similar to the valence-force-field method and are discussed in terms of the deformation of the electronic charge around the metal atoms occurring to fit themselves in the (0 0 0 1) basal plane.     

Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413–511 of human ORC subunit 1, hORC1^413–511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1^413–511. Here, we investigated the interaction of hORC1^413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1^413–511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1^413–511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1^413–511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.     

Protective Role of Proton-Sensing TDAG8 in Lipopolysaccharide-Induced Acute Lung Injury

Acute lung injury is characterized by the infiltration of neutrophils into lungs and the subsequent impairment of lung function. Here we explored the role of TDAG8 in lung injury induced by lipopolysaccharide (LPS) administrated intratracheally. In this model, cytokines and chemokines released from resident macrophages are shown to cause neutrophilic inflammation in the lungs. We found that LPS treatment increased TDAG8 expression in the lungs and confirmed its expression in resident macrophages in bronchoalveolar lavage (BAL) fluids. LPS administration remarkably increased neutrophil accumulation without appreciable change in the resident macrophages, which was associated with increased penetration of blood proteins into BAL fluids, interstitial accumulation of inflammatory cells, and damage of the alveolar architecture. The LPS-induced neutrophil accumulation and the associated lung damage were enhanced in TDAG8-deficient mice as compared with those in wild-type mice. LPS also increased several mRNA and protein expressions of inflammatory cytokines and chemokines in the lungs or BAL fluids. Among these inflammatory mediators, mRNA and protein expression of KC (also known as CXCL1), a chemokine of neutrophils, were significantly enhanced by TDAG8 deficiency. We conclude that TDAG8 is a negative regulator for lung neutrophilic inflammation and injury, in part, through the inhibition of chemokine production.     

Enhancing dependency pair method using strong computability in simply-typed term rewriting

We enhance the dependency pair method in order to prove termination using recursive structure analysis in simply-typed term rewriting systems, which is one of the computational models of functional programs. The primary advantage of our method is that one can exclude higher-order variables which are difficult to analyze theoretically, from recursive structure analysis. The key idea of our method is to analyze recursive structure from the viewpoint of strong computability. This property was introduced for proving termination in typed λ-calculus, and is a stronger condition than the property of termination. The difficulty in incorporating this concept into recursive structure analysis is that because it is defined inductively over type structure, it is not closed under the subterm relation. This breaks the correspondence between strong computability and recursive structure. In order to guarantee the correspondence, we propose plain function-passing as a restriction, which is satisfied by many non-artificial functional programs.     

Characterization of mechanical stress distributions in a cross section of cucumber fruits: bisector reference line represents tissue anatomy

This study sought to statistically analyze the difference of planar stress distribution in a cross section of cucumber cultivars. The method dealt with statistically comparable data, incorporating both the stress magnitude and their positional information, and was developed to reduce complexity without losing anatomical information. Utilizing the trigonal symmetry of cross‐sectioned cucumbers, a reference line bisecting the cross section was introduced on the rupture frame for referencing the relative locations of stress‐sensing points to the underlying anatomical tissue distribution. The stress–location curves on the reference line statistically differed among the cultivars, especially at the center of the cross section. The corresponding tissue for the center of the curve was the seed cavity, so the cultivar differences arise from mainly the mechanical differences in this tissue. The stress–strain analysis of definite tissues revealed flesh stiffness to exceed the seed cavity stiffness, but both differed from the average stiffness of the whole cross section in all cultivars. Copyright © 2004 Society of Chemical Industry     

Functional Characterization of the GH10 and GH11 Xylanases from Streptomyces olivaceoviridis E-86 Provide Insights into the Advantage of GH11 Xylanase in Catalyzing Biomass Degradation

We functionally characterized the GH10 xylanase (SoXyn10A) and the GH11 xylanase (SoXyn11B) derived from the actinomycete Streptomyces olivaceoviridis E-86. Each enzyme exhibited differences in the produced reducing power upon degradation of xylan substrates. SoXyn10A produced higher reducing power than SoXyn11B. Gel filtration of the hydrolysates generated by both enzymes revealed that the original substrate was completely decomposed. Enzyme mixtures of SoXyn10A and SoXyn11B produced the same level of reducing power as SoXyn10A alone. These observations were in good agreement with the composition of the hydrolysis products. The hydrolysis products derived from the incubation of soluble birchwood xylan with a mixture of SoXyn10A and SoXyn11B produced the same products as SoXyn10A alone with similar compositions. Furthermore, the addition of SoXyn10A following SoXyn11B-mediated digestion of xylan produced the same products as SoXyn10A alone with similar compositions. Thus, it was hypothesized that SoXyn10A could degrade xylans to a smaller size than SoXyn11B. In contrast to the soluble xylans as the substrate, the produced reducing power generated by both enzymes was not significantly different when pretreated milled bagasses were used as substrates. Quantification of the pentose content in the milled bagasse residues after the enzyme digestions revealed that SoXyn11B hydrolyzed xylans in pretreated milled bagasses much more efficiently than SoXyn10A. These data suggested that the GH10 xylanases can degrade soluble xylans smaller than the GH11 xylanases. However, the GH11 xylanases may be more efficient at catalyzing xylan degradation in natural environments (e.g. biomass) where xylans interact with celluloses and lignins.     

Temperature-swing adsorption of proteins in water using N-isopropylacrylamide copolymer gel particles

The adsorption and desorption behaviors of bovine serum albumin (BSA) in water for temperature-responsive polymer gel particles have been investigated by the temperature-swing operation between 298 and 313 K, where the cationic N-isopropylacrylamide (NIPA) gels copolymerized with vinylbenzyl trimethylammonium chloride (VBTA) or 2-(dimethylamino)ethyl methacrylate (DMAEMA) were used. The NIPA-VBTA and the NIPA-DMAEMA copolymer gels adsorbed BSA while the NIPA homopolymer gel hardly adsorbed BSA, indicating that the copolymer gels adsorb BSA through the electrostatic attraction between the positively charged groups in the gels and the negatively charged BSA. The adsorption amounts for the NIPA-DMAEMA gels were smaller than those for the NIPA-VBTA gels. This may be because almost every VBTA group, which is a quaternary ammonium salt, can be positively charged in water, while only some of the tertiary amine DMAEMA groups are protonated in water. Moreover, it was found that both the copolymer gels with a large mesh size of the polymer network repeatedly adsorbed BSA at 298 K and desorbed some of pre-adsorbed BSA at 313 K by the temperature-swing operation. This BSA desorption may result from the decrease of the number of the positively charged groups accessible to BSA due to the shrinking of the constituent polymer chains.     

Electrodialysis plant at Hatsushima

A commercial multistage batch system electrodialysis plant for desalination of brackish groundwater was installed at Hatsushima, Atami City in April 1973. The plant has three stages each of which is operated at a constant voltage. Each stage has a dialyzer which is provided with 150 pairs of ion exchange membranes having an effective area of 1 m² each. This plant is used for drinking water supply and has a capacity of 200 m³/day when the raw water has a saline concentration of 6,000 ppm TDS. The operation was very stable and easy, and it was possible to produce water of constant quality at all times regardless of fluctuations in the raw water concentration. The dialyzer was chemically cleaned for removal of deposits on membranes thereby reducing the maintenance cost by 62% without adversely influencing ion exchange membranes.