Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.     

Structural properties of mammalian mitochondrial ADP/ATP carriers: identification of possible amino acids that determine functional differences in its isoforms

The structural properties of isoforms of the mitochondrial ADP/ATP carrier expressed in mammals were characterized in order to understand their possible functional differences. To accomplish this, the cDNA clone of the bovine type 2 isoform was isolated and characterized. We also extensively explored the rat type 3 isoform, but it was not detected. We next compared the amino acid sequences of the ten ADP/ATP carriers, which are expressed in mammals. As a result, amino acids at positions 45, 147 and 164 were found to show strict isoform dependency regardless of species differences. Thus, they are expected to determine functional differences in the isoforms of the ADP/ATP carrier.     

Liquid chromatographic-mass spectrometric determination of haloperidol and its metabolites in human plasma and urine

Haloperidol and its two metabolites, reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP) in human plasma and urine were analyzed by HPLC-MS using a new polymer column (MSpak GF-310), which enabled direct injection of crude biological samples without pretreatment. Recoveries of haloperidol and reduced haloperidol spiked into plasma were 64.4-76.1% and 46.8-50.2%, respectively; those for urine were 87.3-99.4% and 94.2-98.5%, respectively; those of CPHP for both samples were not less than 92.7%. The regression equations for haloperidol, reduced haloperidol and CPHP showed good linearity in the ranges of 10-800, 15-800 and 400-800 ng/ml, respectively, for both plasma and urine. Their detection limits were 5, 10 and 300 ng/ml, respectively, for both samples. Thus, the present method was sensitive enough for detection and determination of high therapeutic and toxic levels for haloperidol and its metabolites present in biological samples.     

Proteome approaches to characterize seed storage proteins related to ditelocentric chromosomes in common wheat (Triticum aestivum L.)

Changes in protein composition of wheat endosperm proteome were investigated in 39 ditelocentric chromosome lines of common wheat (Triticum aestivum L.) cv. Chinese Spring. Two-dimensional gel electrophoresis followed by Coomassie Brilliant Blue staining has resolved a total of 105 protein spots in a gel. Quantitative image analysis of protein spots was performed by PDQuest. Variations in protein spots between the euploid and the 39 ditelocentric lines were evaluated by spot number, appearance, disappearance and intensity. A specific spot present in all gels was taken as an internal standard, and the intensity of all other spots was calculated as the ratio of the internal standard. Out of the 1755 major spots detected in 39 ditelocentric lines, 1372 (78%) spots were found variable in different spot parameters: 147 (11%) disappeared, 978 (71%) up-regulated and 247 (18%) down-regulated. Correlation studies in changes in protein intensities among 24 protein spots across the ditelocentric lines were performed. High correlations in changes of protein intensities were observed among the proteins encoded by genes located in the homoeologous arms. Locations of structural genes controlling 26 spots were identified in 10 chromosomal arms. Multiple regulators of the same protein located at various chromosomal arms were also noticed. Identification of structural genes for most of the proteins was found difficult due to multiple regulators encoding the same protein. Two novel subunits (1B(Z,) 1BDz), the structure of which are very similar to the high molecular weight glutenin subunit 12, were identified, and the chromosome arm locations of these subunits were assigned.     

Removal of mucus for ultrastructural observation of the surface of human gastric epithelium using pronase

Background. Helicobacter pylori adhering to the human gastric epithelium causes gastric diseases such as ulcer, carcinoma and lymphoma. It is thus important to observe in detail both the surface of the epithelial cells and the H. pylori that adhered to it for the elucidation of H. pylori‐induced diseases by scanning electron microscopy (SEM). Since the thick mucus layer blocks the observation of the cell surface and the bacteria, it is generally eliminated during the processing for SEM by roughly mechanical methods, but these treatments also demolish the ultrastructure of the cells. We studied the nonmechanical method for removal of mucus layer of gastric epithelium using pronase. Materials and Methods. To determine the optimal concentration of pronase, mucin was used as a substrate for inhibition of the viscosity. Pronase was added in 2% mucin at the concentration of 10, 50, 100, 500, 1000, 2000 or 5000 unit/ml and the flowing time of the mixture was measured. Based on the digestion experiment, biopsied specimens from 24 patients with dyspepsic symptoms were fixed in glutaraldehyde and then washed in rolling with different concentration of pronase. After the pretreatment by pronase, the specimens were treated according to the standard process for SEM. Results. We succeeded in removing the mucus layer on the surface of epithelial cells from the biopsied specimens fixed in glutaraldehyde by rinsing with 2000 unit/ml pronase for 24 hours. Conclusions. Using our digestive method without destroying the ultrastructure, the earliest stage which H. pylori has adhered onto the human gastric epithelium can be observed for the investigation of H. pylori‐induced gastric disorders by SEM.     

Methacholine-induced cGMP production is regulated by nitric oxide generation in rabbit submandibular gland cells

Guanosine 3′,5′-monophosphate (cGMP) is an intracellular messenger in various kinds of cell. We investigated the regulation of cGMP production by nitric oxide (NO) in rabbit submandibular gland cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose- and time-dependent manner, but the α-agonist phenylephrine, substance P and the β-agonist isoproterenol failed to evoke cGMP production. In fura-2-loaded cells, methacholine induced an increase in intracellular Ca²⁺ ([Ca²⁺]_i) in a concentration-dependent manner, which was similar to that for cGMP production. When the external Ca²⁺ was chelated with EGTA, methacholine failed to induce cGMP production. Ca²⁺ ionophore A23187 and thapsigargin, which induce the increase in [Ca²⁺]_i without activation of Ca²⁺-mobilizing receptors, mimicked the effect of methacholine. cGMP production induced by methacholine, A23187 and thapsigargin was clearly inhibited by N^G-nitro- -arginine methylester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS). S-Nitroso-N-acetyl- -penicillamine (SNAP), a NO donor, induced cGMP formation. In the lysate of rabbit submandibular gland cells, Ca²⁺-regulated nitric oxide synthase activity was detected. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is regulated by NO generation via the increase in [Ca²⁺]_i.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

A specific peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand,pioglitazone, ameliorates gastric mucosal damage induced by ischemia and reperfusion in rats

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. The aim of the present study was to investigate the effects of pioglitazone on ischemia-reperfusion (I/R)-induced gastric mucosal injury in rats. Gastric ischemia was induced for 30 min by applying a small vascular clamp to the celiac artery and reperfusion was produced by removal of the clamp in male Sprague-Dawley rats treated with and without pioglitazone. Pioglitazone was given to the rats intraperitoneally 2 h before the vascular clamping. The area of gastric mucosal erosion (erosion index) significantly increased from mean basal levels after 60 min of reperfusion. This erosion index was significantly inhibited by pretreatment with pioglitazone in a dose-dependent manner. The concentration of thiobarbituric acid reactive substances (TBARS) and myeloperoxidase (MPO) activity in the gastric mucosa were both significantly increased after I/R, and pretreatment with pioglitazone significantly reduced these increases. The contents of both mucosal TNF-alpha and CINC-2beta in the I/R group were significantly increased compared with the levels in the sham-operated group. These increases in TNF-alpha and CINC-2beta were significantly inhibited by pretreatment with pioglitazone at a dose of 10 mg/kg. The results of the present study indicate that pioglitazone inhibited lipid peroxidation and reduced development of the gastric mucosal inflammation induced by I/R in rats. This investigation suggests that pioglitazone has potential as a new therapeutic agent for reperfusion injury.