Poly-L-arginine-Induced internalization of tight junction proteins increases the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules.

We investigated whether poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 monolayer to hydrophilic macromolecules and clarified the disposition of tight junction (TJ) proteins. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran (FD-4) permeation were determined after treatment with PLA. TJ proteins were visualized using immunofluorescence microscopy after PLA exposure and depletion, and their expression levels were determined. The barrier function of TJs was also evaluated by measuring the alterations in the TEER and in the localization of TJ proteins. PLA induced an increase in hydrophilic macromolecule, FD-4, permeation through Caco-2 cell monolayers and a decrease in the TEER in a concentration-dependent manner, without any significant impact on the cell viability. This increased paracellular permeability induced by PLA was found to be internalized of claudin-4, ZO-1, tricellulin and mainly occludin from cell-cell junction to the subcellular space. ZO-1 appeared to play an important role in the reconstitution of TJ strand structures following PLA depletion. These results indicate that the PLA led to the internalization of TJ proteins to the subcellular space, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

Photocatalytic degradation of chlorinated ethanes in the gas phase on the porous TiO(2) pellets: effect of surface acidity.

The photocatalytic degradation of chlorinated ethanes was studied in a tubular photoreactor packed with TiO(2) pellets prepared by sol-gel method. The steady-state condition was not obtained, but the deterioration in the photocatalytic activity was observed during the irradiation. Effects of mole fractions of water vapor, O(2), and C(2)H(5)Cl or C(2)H(4)Cl(2) and reaction temperature on the photodegradation of C(2)H(5)Cl or C(2)H(4)Cl(2) were examined, and these data were compared with those obtained by the photodegradation of chlorinated ethylenes. On the basis of the products detected with and without oxygen in the reactant's gas stream, we proposed the degradation mechanism. Measurement of diffuse reflectance infrared Fourier transform spectroscopy of pyridine adsorbed on the catalysts showed that decrease in the conversion for the photodegradation of C(2)H(5)Cl was attributable to the formation of Brønsted acid sites. Comparison of the data obtained with the TiO(2) and the sulfated TiO(2) (SO(4)(2-)/TiO(2)) pellets indicated that the photodegradation of C(2)H(5)Cl was suppressed by the presence of the Brønsted sites, but that of trichloroethylene was not affected. Such a difference is attributable to the adsorption process of these reactants on the acid sites on the catalyst surface.

Flavin adenine dinucleotide-dependent 4-phospho-D-erythronate dehydrogenase is responsible for the 4-phosphohydroxy-L-threonine pathway in vitamin B6 biosynthesis in Sinorhizobium meliloti.

The vitamin B6 biosynthetic pathway in Sinorhizobium meliloti is similar to that in Escherichia coli K-12; in both organisms this pathway includes condensation of two intermediates, 1-deoxy-D-xylulose 5-phosphate and 4-phosphohydroxy-L-threonine (4PHT). Here, we report cloning of a gene designated pdxR that functionally corresponds to the pdxB gene of E. coli and encodes a dye-linked flavin adenine dinucleotide-dependent 4-phospho-D-erythronate (4PE) dehydrogenase. This enzyme catalyzes the oxidation of 4PE to 3-hydroxy-4-phosphohydroxy-alpha-ketobutyrate and is clearly different in terms of cofactor requirements from the pdxB gene product of E. coli, which is known to be an NAD-dependent enzyme. Previously, we revealed that in S. meliloti IFO 14782, 4PHT is synthesized from 4-hydroxy-l-threonine and that this synthesis starts with glycolaldehyde and glycine. However, in this study, we identified a second 4PHT pathway in S. meliloti that originates exclusively from glycolaldehyde (the major pathway). Based on the involvement of 4PE in the 4PHT pathway, the incorporation of different samples of 13C-labeled glycolaldehyde into pyridoxine molecules was examined using 13C nuclear magnetic resonance spectroscopy. On the basis of the spectral analyses, the synthesis of 4PHT from glycolaldehyde was hypothesized to involve the following steps: glycolaldehyde is sequentially metabolized to D-erythrulose, D-erythrulose 4-phosphate, and D-erythrose 4-phosphate by transketolase, kinase, and isomerase, respectively; and D-erythrose 4-phosphate is then converted to 4PHT by the conventional three-step pathway elucidated in E. coli, although the mechanism of action of the enzymes catalyzing the first two steps is different.

Purification and characterization of pyridoxine 5'-phosphate phosphatase from Sinorhizobium meliloti.

Here we report the purification and biochemical characterization of a pyridoxine 5'-phosphate phosphatase involved in the biosynthesis of pyridoxine in Sinorhizobium meliloti. The phosphatase was localized in the cytoplasm and purified to electrophoretic homogeneity by a combination of EDTA/lysozyme treatment and five chromatography steps. Gel-filtration chromatography with Sephacryl S-200 and SDS/PAGE demonstrated that the protein was a monomer with a molecular size of approximately 29 kDa. The protein required divalent metal ions for pyridoxine 5'-phosphate phosphatase activity, and specifically catalyzed the removal of Pi from pyridoxine and pyridoxal 5'-phosphates at physiological pH (about 7.5). It was inactive on pyridoxamine 5'-phosphate and other physiologically important phosphorylated compounds. The enzyme had the same Michaelis constant (K(m)) of 385 muM for pyridoxine and pyridoxal 5'-phosphates, but its specific constant [maximum velocity (V(max))/K(m)] was nearly 2.5 times higher for the former than for the latter.

Efficacy and safety of milnacipran in the treatment of generalized anxiety disorder: an open study.

OBJECTIVE The aim of this open study was to assess the efficacy and safety of the serotonin-noradrenaline reuptake-inhibiting antidepressant, milnacipran, for the treatment of generalised anxiety disorder. METHODS Twelve patients, treated with milnacipran at doses from 30 to 150 mg/day for 8 weeks, were evaluated at baseline and after 1, 2, 3, 4, 6 and 8 weeks of treatment using the Hamilton Anxiety Rating (HAM-A) scale and a self-rating visual analogue scale. RESULTS Two patients dropped out early in the trial because of adverse effects. The 10 patients who completed the trial showed a marked improvement in anxiety symptoms with HAM-A scores being reduced from 21.6±6.5 (mean±SD) to 1.5±1.8 after 8 weeks. All of the patients who completed the 8 weeks of treatment had a reduction of at least 70% in their HAM-A score. Adverse effects, principally nausea, were mild and occurred early in the study, regressing as the study progressed. CONCLUSION These results suggest that milnacipran is effective and well tolerated in patients suffering from generalised anxiety disorder. A full-scale double-blind placebo trial is clearly warranted.

Biosynthesis of vitamin B6 in Rhizobium: in vitro synthesis of pyridoxine from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine.

Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD+, NADP+, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD+ for formation of pyridoxine 5'-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.