Evaluation of the Amounts of Sennosides A and B in Rhubarb-containing Kampo Medicines to Create a Ranking of Kampo Medicines for Appropriate Selection of Laxatives.

OBJECTIVES: To evaluate 20 Kampo medicines, which comprised 6 formulas, Otsujito, Junchoto, Tokakujokito, Bofutsushosan, Mashiningan, and Keishikashakuyakudaioto, from 7 brands, to create a ranking of Kampo medicines for appropriate selection of laxatives. METHODS: The amounts of sennosides A and B, the important components showing laxative effects contained in Kampo medicines, were analysed using High Performance Liquid Chromatography. RESULTS: We found that the amounts of sennosides A and B were different among brands, even when they had the same formula. Furthermore, the amounts of sennosides differed when the same amounts of rhubarb were used. CONCLUSIONS: These results suggest that the differences in amounts of sennosides are caused by the quality of the rhubarb used. Kampo medicines containing laxatives other than rhubarb, including disodium sulphate and hemp seed, had synergistic laxative effects. Thus, in the future, it may be possible to adjust laxative potency of Kampo medicines through further clinical tests.

Application of the cellular oxidation biosensor to Toxicity Identification Evaluations for high-throughput toxicity assessment of river water.

Toxicity Identification Evaluation (TIE) is a useful method for the classification and identification of toxicants in a composite environment water sample. However, its extension to a larger sample size has been restrained owing to the limited throughput of toxicity bioassays. Here we reported the development of a high-throughput method of TIE Phase I. This newly developed method was assisted by the fluorescence-based cellular oxidation (CO) biosensor fabricated with roGFP2-expressing bacterial cells in 96-well microplate format. The assessment of four river water samples from Langat river basin by this new method demonstrated that the contaminant composition of the four samples can be classified into two distinct groups. The entire toxicity assay consisted of 2338 tests was completed within 12 h with a fluorescence microplate reader. Concurrently, the sample volume for each assay was reduced to 50 µL, which is 600 to 4700 times lesser to compare with conventional bioassays. These imply that the throughput of the CO biosensor-assisted TIE Phase I is now feasible for constructing a large-scale toxicity monitoring system, which would cover a whole watershed scale.

A bacterial biosensor for oxidative stress using the constitutively expressed redox-sensitive protein roGFP2.

A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd(2+), Cu(2+), Pb(2+), Zn(2+) and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10(-7) (arsenite), 1.0 × 10(-4) (naphthalene), 1.0 × 10(-4) (Cu(2+)), 3.8 × 10(-4) (H(2)O(2)), 1.0 × 10(-3) (Cd(2+)), 1.0 × 10(-3) (Zn(2+)), 1.0 × 10(-2) (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb(2+)), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.

[Two cases of nonrecurrent inferior laryngeal nerve--different branching levels from vagus nerve].

We observed two cases of nonrecurrent inferior laryngeal nerve (NRILN). Case 1, a 71 year old man was diagnosed as having papillary carcinoma. NRILN was found during his operation. It directly branched from the right cervical trunk of the vagus nerve at the level of the cricoid cartilage and then entered the larynx after running behind the thyroid gland. Case 2, a 64 year old woman was diagnosed as having primary hyperparathyroidism. In this patient, the NRILN branched at the level of the inferior pole of the thyroid gland, rose up beside the tracheal wall and entered the larynx. In both patients, preoperative CT scan and postoperative MR angiography revealed the aberrant right subclavian artery. A postoperative barium swallow test showed the compression of the esophagus by this anomalous artery in case 1. Although it is possible to predict the presence of NRILN by preoperative imaging tests, the branching level from the vagus nerve is unpredictable. Surgery must be performed with this point in mind, if the presence of NRILN is suspected.

Expression and regulation of gap junctions in rat cholangiocytes.

Hepatocytes and other digestive epithelia exchange second messengers and coordinate their functions by communicating through gap junctions. However, little is known about intercellular communication in cholangiocytes. The aim of this study was to examine expression and regulation of gap junctions in cholangiocytes. Connexin expression was determined by confocal immunofluorescence in rat bile ducts and in normal rat cholangiocyte (NRC) cells, a polarized cholangiocyte cell line. Intercellular Ca(2+) signaling was monitored by fluorescent microscopy. Microinjection studies assessed regulation of gap junction permeability in NRC cells and in SKHep1 cells, a liver-derived cell line engineered to express connexin 43. Immunochemistry showed that cholangiocytes from normal rat liver as well as the NRC cells express connexin 43. Localization of apical, basolateral, and tight junction proteins confirmed that NRC cells are well polarized. Apical exposure to ATP induced Ca(2+) oscillations that were coordinated among neighboring NRC cells, and inhibition of gap junction conductance desynchronized the Ca(2+) oscillations. NRC cells transfected with a connexin 43 antisense were significantly less coupled. Transcellular dye spreading was inhibited by activation of protein kinase A or protein kinase C. The same was observed in transfected SKHep1 cells, which expressed only connexin 43. Rat cholangiocytes and NRC cells express connexin 43, which permits synchronization of Ca(2+) signals among cells. Permeability of connexin 43-gap junctions is negatively regulated by protein kinases A and C. In conclusion, cholangiocytes have the capacity for intercellular communication of second messenger signals via gap junctions in a fashion that is under hormonal control.

Prognostic value of plasma vascular endothelial growth factor in patients with colorectal cancer.

BACKGROUND: Vascular endothelial growth factor (VEGF) is thought to be the most powerful angiogenic factor in many malignancies while and several reports have determined serum VEGF to be a prognostic indicator for various malignancies. However, serum VEGF may not be suitable for the measurement of circulating VEGF levels, given that clot formation during the process of collecting sample induces platelet activation and the subsequent release of many cytokines. MATERIALS AND METHODS: Blood samples were obtained prior to surgery from 33 patients with colorectal cancer, and additional samples were obtained 6 months post-operatively from 15 of the 33 patients. Plasma levels of VEGF were assessed using the quantitative sandwich-enzyme immunoassay technique. We investigated the relationships between plasma levels of pre-operative VEGF, prognosis and clinicopathological factors in patients with colorectal cancer. In addition, the relationship between pre-operative and post-operative plasma levels of VEGF obtained at 6 months was assessed. RESULTS: Although not significant, a trend was observed for high plasma levels of VEGF to occur with more advanced colorectal cancer. Plasma levels of VEGF were only significantly increased in patients who had recurrence and a worsening of colorectal cancer. In blood samples obtained from 15 patients both prior to surgery and 6 months post-operatively, post-operative mean plasma VEGF tended to decrease relative to the pre-operative level. CONCLUSION: These results suggest that high plasma levels of VEGF may be observed in more advanced colorectal cancer patients, especially in patients who develop recurrence or a worsening of their condition. Thus, such patients may require additional immunochemotherapy after surgery.

Molecular basis for pacemaker cells in epithelia.

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca(2+) signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V(1a) vasopressin receptor (V(1a)R). Release of inositol 1,4,5-trisphosphate (InsP(3)) in wild type cells increased Ca(2+) in the injected cell but not in neighboring cells, while the Ca(2+) signal spread to neighbors when gap junctions were expressed. Photorelease of caged Ca(2+) rather than InsP(3) resulted in a small increase in Ca(2+) that did not spread to neighbors with or without gap junctions. However, photorelease of Ca(2+) in cells stimulated with low concentrations of vasopressin resulted in a much larger increase in Ca(2+), which spread to neighbors via gap junctions. Cells expressing tagged V(1a)R similarly had increased sensitivity to vasopressin, and could signal to neighbors via gap junctions. Higher concentrations of vasopressin elicited Ca(2+) signals in all cells. In cx32 or cx43 but not in wild type cells, this signaling was synchronized and began in cells expressing the tagged V(1a)R. Thus, intercellular Ca(2+) signals in epithelia are organized by three factors: 1) InsP(3) must be generated in each cell to support a Ca(2+) signal in that cell; 2) gap junctions are necessary to synchronize Ca(2+) signals among cells; and 3) cells with relatively increased expression of hormone receptor will initiate Ca(2+) signals and thus serve as pacemakers for their neighbors. Together, these factors may allow epithelia to act in an integrated, organ-level fashion rather than as a collection of isolated cells.