TMX, a human transmembrane oxidoreductase of the thioredoxin family: the possible role in disulfide-linked protein folding in the endoplasmic reticulum

Various proteins sharing thioredoxin (Trx)-like active site sequences (Cys-Xxx-Xxx-Cys) have been found and classified in the Trx superfamily. Among them, transmembrane Trx-related protein (TMX) was recently identified as a novel protein possessing an atypical active site sequence, Cys-Pro-Ala-Cys. In the present study, we describe the properties of this membranous Trx-related molecule. Endogenous TMX was detected as a protein of approximately 30 kDa with a cleavable signal peptide. TMX was enriched in membrane fractions and exhibited a similar subcellular distribution with calnexin localized in the endoplasmic reticulum (ER). The examination of membrane topology of TMX suggested that the N-terminal region containing the Trx-like domain was present in the ER lumen, where protein disulfide isomerase (PDI) was found to assist protein folding. Recombinant TMX showed PDI-like activity to refold scrambled RNase. These results indicate the possibility that TMX can modify certain molecules with its oxidoreductase activity and be involved in the redox regulation in the ER.     

Flavonoid-induced reduction of ENaC expression in the kidney of Dahl salt-sensitive hypertensive rat

Flavonoid, a plant extract, exhibits various biological actions. Dietary flavonoid intake is reported to reduce an elevated blood pressure, however the mechanism is unknown. The epithelial Na+ channel (ENaC) in the kidney plays a key role in the regulation of blood pressure by contributing to the Na+ reabsorption in renal tubules. Thus, we investigated the effect of quercetin, a flavonoid, on ENaC mRNA expression in the kidney of hypertensive Dahl salt-sensitive rats. Dahl salt-sensitive rats of 8 weeks were acclimated for 1 week in a metabolic cage and were subsequently kept for 4 weeks under four different conditions: (1) normal salt diet (0.3% NaCl), (2) normal salt diet with quercetin (10 mg/kg/day), (3) high-salt diet (8% NaCl), and (4) high-salt diet with quercetin. Quercetin diminished the alphaENaC mRNA expression in the kidney associated with reduction of the systolic blood pressure elevated by high-salt diet, suggesting that one of the mechanisms of the flavonoid's antihypertensive effect on salt-sensitive hypertension would be mediated through downregulation of ENaC expression in the kidney.     

Flavin reductase coupling with two monooxygenases involved in dibenzothiophene desulfurization: purification and characterization from a non-desulfurizing bacterium,Paenibacillus polymyxa A-1

The dibenzothiophene (DBT) desulfurizing bacterium metabolizes DBT to form 2-hydroxybiphenyl without breaking the carbon skeleton. Of the DBT desulfurization enzymes, DszC and DszA catalyze monooxygenation reactions, both requiring flavin reductase. We searched for non-DBT-desulfurizing microorganisms producing a flavin reductase that couples more efficiently with DszC than that produced by the DBT desulfurizing bacterium Rhodococcus erythropolis D-1, and found Paenibacillus polymyxa A-1 to be a promising strain. The enzyme was purified to complete homogeneity. K(m) values for FMN and NADH were 2.1 microM and 0.57 mM, respectively. Flavin compounds were good substrates, some nitroaromatic compounds were also active, and regarding the electron donor, the activity for NADPH was about 1.5 times that for NADH. In the coupling assay with DszC, only FMN or riboflavin acted as the electron acceptor. The coupling reactions of P. polymyxa A-1 flavin reductase with DszC and DszA proceeded more efficiently (3.5- and 5-fold, respectively) than those of R. erythropolis D-1 flavin reductase when identical enzyme activities of each flavin reductase were added to the reaction mixture. The result of the coupling reaction suggested that, in the microbial DBT desulfurization, flavin reductase from the non-DBT-desulfurizing bacterium was superior to that from the DBT-desulfurizing bacterium.     

Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension

Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. The prostacyclin synthase (PGIS) gene is a candidate gene for cardiovascular disease. The purpose of this study was to locate possible mutations in the PGIS gene related to hypertension and cerebral infarction. Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we discovered a T to C transition at the +2 position of the splicing donor site of intron 9 in patients with essential hypertension (EH). In vitro expression analysis of an allelic minigene consisting of exons 8-10 revealed that the nucleotide transition causes skipping of exon 9. This in turn alters the translational reading frame of exon 10 and introduces a premature stop codon (TGA). A three-dimensional model shows that the splice site mutation produces a truncated protein with a deletion in the heme-binding region. This splice site mutation was found in only one subject in 200 EH patients and 200 healthy controls. Analysis of the patient's family members revealed the mutation in two of the three siblings. The urinary excretion of prostacyclin metabolites in subjects with the mutation was significantly decreased. All subjects displaying the splice site mutation in the PGIS gene were hypertensive. In this study, we report a novel splicing mutation in the PGIS gene, which is associated with hypertension in a family. It is thought that this mechanism may involve in the pathophysiology of their hypertension.     

Comparison of bacteriological, genetic and pathological characters between Escherichia coli O115a,c:K(B) and Citrobacter rodentium.

Murine pathogenic Escherichia coli O115a,c:K(B) (MPEC) is the causative agent of mouse megaenteron, the pathology of which resembles that of transmissible murine colonic hyperplasia caused by Citrobacter rodentium. We compared their genetic and pathological features to reveal the relationship between these two bacteria. To evaluate the genetic distances, 16S rDNA genes were sequenced and biochemical reactions were tested. Mouse strain susceptibility tests, using CF1 MPEC-susceptible germfree mice and BALB/cA(Jic) resistant mice were performed. MPEC strains and C. rodentium showed more than 99.6% identity by comparison of 16S rDNA gene sequences. All results from biochemical reactions and the mouse strain susceptibility tests were identical. It is proposed that MPEC should be reclassified as C. rodentium.     

Elimination of Pasteurella pneumotropica from a contaminated mouse colony by oral administration of Enrofloxacin.

Enrofloxacin, a fluoroquinolone bactericidal antibiotic, was administered in an attempt to eradicate Pasteurella pneumotropica (P. pneumotropica) from a contaminated mouse colony. Contaminated mice, maintained within 4 animal rooms, were administered Enrofloxacin in drinking water at a daily dosage of 25.5 mg/kg for 2 weeks. Following one week of Enrofloxacin treatment, mice were selected randomly from each room and examined for P. pneumotropica. This procedure was repeated two or three times until all mice examined tested negative for the Pasteurella strain. With the exception of one room, treated mice consistently tested negative for P. pneumotropica for up to 45 weeks following completion of Enrofloxacin treatment. Thus, oral administration of Enrofloxacin significantly eliminated P. pneumotropica from a contaminated mouse colony.     

The interaction of sodium dodecyl sulfate with cucumber green mottle mosaic virus protein and tobacco mosaic virus protein

The binding of sodium dodecyl sulfate to coat protein subunits of cucumber green mottle mosaic virus and tobacco mosaic virus was studied by equilibrium dialysis. The amount of dodecyl sulfate bound to the cucumber virus protein in 0.1 m phosphate buffer (pH 7.2) was found to be 1.55 g/g, which was the same value as that obtained with the tobacco virus protein. The presence of 8 m urea markedly decreased the degree of binding of dodecyl sulfate to the proteins. The amount of binding to the cucumber virus protein was reduced to 0.56 g/g, and that to the tobacco virus protein decreased to 0.8 g/g. The net charges of both proteins were negative at neutral pH and the amount of negative charge of the cucumber virus protein, obtained from the potentiometric titration curves, was larger than that of the tobacco virus protein, either in the native state or in the denatured state. In dodecyl sulfate/polyacrylamide gel electrophoresis the cucumber virus protein migrated faster than the tobacco virus protein. On the other hand, in the presence of 8 m urea, the electrophoretic migration rate of the cucumber virus protein was equal to that of the tobacco virus protein. Sedimentation equilibrium experiments in 6 m guanidinium chloride gave molecular weights of 17,700 and 17,200 for the tobacco mosaic virus and the cucumber virus proteins, respectively. These results suggest that the effective negative charge density of the cucumber virus protein-dodecyl sulfate complex is higher than that of the tobacco virus proteindodecyl sulfate complex in 0.1% dodecyl sulfate solution. The conformation of both proteins was investigated by circular dichroism measurements. Both proteins have a slightly higher degree of α-helix content in dodecyl sulfate solution than in the native state. The addition of 8 m urea to both proteins while in this solution induced a change in conformation to one having a much smaller degree of ordered structure, although the change in the cucumber virus protein was more intense than that in the tobacco virus protein.     

Mutagenic specificity in DNA base sequence by irradiation of health lamp light (UV-B) in Escherichia coli.

A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene, was irradiated with health lamp (HL) light containing UV-B. Plasmid mutations were scored by transforming an indicator strain of Escherichia coli carrying a suppressive blue amber mutation in the beta-galactosidase gene. Plasmid survival was also measured by transforming activity of the indicator strain. The majority of mutations induced by HL light were GC-AT transitions (69%) and the rest were transversions (31%). Some hot-spots in the mutations were observed by sequencing the suppressor gene. Mutagenic specificity in DNA base sequences induced by HL in E. coli agrees well with previous reports about 254-nm or 313-nm light effects on mammalian cells. This agreement may depend on the substitution of the inserted base instead of a G residue at the opposite site of a damaged C residue from conformational change of DNA structure in both bacterial and mammalian cells.     

Involvement of phosphoinositide 3-kinase and its association with pp60src in cholecystokinin-stimulated pancreatic acinar cells

Phosphoinositide 3-kinase (PI3K) is a lipid kinase which phosphorylates the D3 position of the phosphoinositide derivatives and is known to be activated by a host of protein tyrosine kinases. PI3K has been demonstrated to play an important role in mitogenesis and cell transformation in several cell systems. However, the functional roles of PI3K in pancreatic acinar cells remain to be determined. The objective of this study was to identify and characterize the PI3K pathway and its relation to other non-receptor protein tyrosine kinases in mediating signal transduction of pancreatic acinar cells. Intact acini isolated from the rat pancreas were incubated with or without cholecystokinin octapeptide (CCK-8). A Triton X-100-soluble and 10000 rpm supernatant of the cell sonicates was used for immunoprecipitation and Western immunoblotting. When a monoclonal anti-phosphotyrosine antibody (clone 4G10) was used, two major tyrosine-phosphorylated bands were observed at the location of p85 and p60. CCK (10 pM and 10 nM) significantly enhanced the tyrosine phosphorylation of these two bands. Furthermore, when a monoclonal anti-PI3K antibody (clone UB93-3) which recognizes the N-terminal SH2 domain of the p85 regulatory subunit of PI3K was used, CCK (10 pM-10 nM) dose-dependently increased the amount of the immunodetectable PI3K band with a peak occurring at 5 min. The increase in the immunodetectable PI3K band elicited by CCK did not require the presence of extracellular Ca2+. The pp60src inhibitor, herbimycin A (6 microM), and the PI3K inhibitor, wortmannin (6 microM), both decreased intensities of the PI3K band elicited by CCK. Herbimycin A abolished phosphotransferase activities of the Src kinase following stimulation with CCK, whereas wortmannin had no effect, suggesting that Src is an upstream regulator of PI3K. Wortmannin (3-6 microM) abolished CCK-stimulated pancreatic amylase secretion. Immunoprecipitation studies using an anti-Src antibody (clone CD11) or PI3K antibody in conjunction with the anti-phosphotyrosine antibody showed that, in response to CCK, tyrosine phosphorylations of Src and PI3K were enhanced at the location of p60 and p85, respectively. Src was co-immunoprecipitated with PI3K following stimulation with CCK, suggesting that pp60src forms an immunocomplex with PI3K via the N-SH2 domain of the p85 regulatory subunit. Thus PI3K and its association with Src appear to be involved in mediating CCK-stimulated pancreatic exocytosis.