Adherence protects the binding sites of Helicobacter pylori urease from acid-induced damage

Colonization by Helicobacter pylori partly depends on acid-dependent adherence by urease to gastric mucin. To further verify the relevance of urease adherence to colonization, the influence of acidity on the binding sites of H. pylori urease was investigated. When enzyme-based in vitro ligand capture assays were used, the effect of acidity on the binding site of H. pylori urease was determined against a backdrop medium consisting of acidic buffers simulating the luminal side of gastric mucus. A high degree of stability was exhibited by adherent urease, suggesting a pivotal role by the denatured enzyme in the persistence of the bacterium within the acidified compartment of gastric mucus.     

Contents of sulfur amino acids, and cystathionine beta-synthase and gamma-lyase activities in various tissues from agkistroden blomhoffi (mamushi)

The concentrations of sulfur-containing amino acids, taurine, cystathionine, methionine and cystine, as well as cystathionine beta-synthase and gamma-lyase activities in various tissues of Agkistrodon blomhoffi (mamushi) were measured. The concentration of taurine in examined tissues was greater than the concentration of other sulfur-containing amino acids. The concentration of cystathionine in various tissues was also much higher than those of methionine and cystine, but the concentration of cystathionine in the brain was lower than that of methionine. In all tissues examined in this study, cystathionine beta-synthase activity was much higher than that of cystathionine gamma-lyase. The ratios of cystathionine beta-synthase to gamma-lyase activities in various tissues were 5.6 to approximately 85.6. The concentration of sulfur-containing amino acids in muscle and skin divided into eight portions of the body were also determined. The concentrations of methionine and cystine in each portion of muscle and skin were almost the same, but the concentrations of taurine and cystathionine in each portion of the body were varied.     

Autocrine/Paracrine secretion of IL-6 family cytokines causes angiotensin II-induced delayed STAT3 activation

We recently reported that angiotensin II (AngII) biphasically activates the JAK/STAT pathway and induces delayed phosphorylation of STAT3 in the late stage (120 min) in cardiomyocytes. This study was designed to determine the mechanism of delayed phosphorylation of STAT3. Conditioned medium prepared from AngII-stimulated cardiomyocytes could reproduce the tyrosine phosphorylation of STAT3 at 5 min. This delayed phosphorylation was almost completely inhibited by anti-gp130 blocking antibody RX435, but not by TAK044 (ET-A/B-R antagonist), prazosin, or propranolol. AngII induced phosphorylation of gp130 in the late stage, which was temporally in parallel with the delayed phosphorylation of STAT3. AngII augmented IL-6, CT-1, and LIF mRNA expression at 30-60 min, but not CNTF expression. AngII increased IL-6 protein levels by 3-fold in the conditioned media at 2 h compared with the control. These findings indicated that AngII-induced delayed activation of STAT3 is caused by autocrine/paracrine secreted IL-6 family cytokines.     

Dose-dependent biphasic accumulation of TP53 protein in normal human embryo cells after X irradiation

The effects of various doses of X radiation on the kinetics of accumulation of TP53 protein (formerly known as p53) were examined in normal human embryo cells. We found that the rate of accumulation of TP53 protein was biphasic at X-ray doses between 1 and 4 Gy, while monophasic accumulation was observed after X irradiation with doses higher than 6 Gy. The first phase of accumulation was detected within 1 h after irradiation, and a second phase of accumulation was detected between 6 and 12 h after irradiation. The induction of CDKN1A (formerly known as p21(WAF1/CIP1)) and MDM2 proteins was also biphasic after doses of 4 Gy or less, while monophasic accumulation was observed after 6 Gy or higher. We found that the proteasome inhibitor ALLN increased the constitutive level of TP53 protein, and no change was observed in the TP53 level after X irradiation when cells were treated with ALLN. These results indicate that the dose-dependent accumulation of TP53 is due to an inhibition of TP53 degradation, and that the induction of MDM2 might be responsible in part for the different kinetics of accumulation of TP53.     

Hypotaurine disproportionation reaction: Identification of products

Summary Hypotaurine, concentrated under reduced pressure in HCl solution, partially (30–40%) degrades into taurine (about 30%), 2-aminoethyl-2-aminoethanethiolsulfonate (about 10%) and ethanolamine. The degradation products were identified using LC/APCI-MS, NMR, amino acid analysis and various chromatographics. The identities were confirmed by comparing the HPLS, MS and NMR characteristics of authentic compounds. One of the degradation processes during concentration of HCl solution of hypotaurine is therefore a disproportionation reaction which can interfere with the experimental results, when studying hypotaurine in biological systems.Abbreviations LC/APCI-MS liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry - TLC thin layer chromatography - DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition

We have analysed the function of a gene of Bacillus subtilis , the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23°C but not at 37°C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23°C and in the conditional null mutant grown in low concentrations of IPTG at 37°C. In addition, immunofluorescence microscopy showed that subcellular localization of the Spo0J partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.     

Rho and Rab Small G Proteins Coordinately Reorganize Stress Fibers and Focal Adhesions in MDCK Cells

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.     

Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and −10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and ±2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at −80°C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.