Akiko Hamamoto

New water disinfection system using UVA light-emitting diodes

Aim:  To evaluate the ability of high‐energy ultraviolet A (UVA) light‐emitting diode (LED) to inactivate bacteria in water and investigate the inactivating mechanism of UVA irradiation.

Vibrio parahaemolyticus Infection Induces Modulation of IL-8 Secretion Through Dual Pathway via VP1680 in Caco-2 Cells

BACKGROUND Vibrio parahaemolyticus causes acute gastroenteritis and inflammations in humans. A variety of pathogenic bacteria can stimulate mitogen-activated protein kinases (MAPKs) in host cells. Phosphorylation of MAPKs leads to production of interleukin (IL)- 8 and subsequently causes inflammations. Thus, MAPK cascades were strong candidates for the main signaling pathway of V. parahaemolyticus-induced acute inflammation. METHODS To determine whether the signaling pathway on V. parahaemolyticus infection induces inflammation, we analyzed the secretion level of IL-8 and phosphorylation of MAPKs by use of intestinal epithelial Caco-2 cells. RESULTS V. parahaemolyticus infection of Caco-2 cells activated extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK signal pathways, leading to IL-8 secretion, whereas MAPK inhibitors, UO126 or SB203580, suppressed IL-8 secretion. A strain carrying a deletion of VP1680, a type three secretion system 1 (T3SS1) effector protein, failed to activate phosphorylation of ERK1/2 and p38 MAPK and secretion of IL-8. ERK1/2 pathway inhibitor, UO126, failed IL-8 promoter activity, whereas p38 MAPK inhibitor, SB203580, decreased the stabilization of IL-8 messenger RNA following V. parahaemolyticus infection. CONCLUSIONS We showed that V. parahaemolyticus infection of Caco-2 cells results in the secretion of IL-8, and that VP1680 plays a pivotal role in manipulating host cell signaling and is responsible for triggering IL-8 secretion.

Insulin Activates ATP-Sensitive Potassium Channels via Phosphatidylinositol 3-Kinase in Cultured Vascular Smooth Muscle Cells

The effects of insulin on the vasculature are significant because insulin resistance is associated with hypertension. To increase the understanding of the effects of insulin on the vasculature, we analyzed changes in potassium ion transport in cultured vascular smooth muscle cells (VSMCs). Using the potential-sensitive fluorescence dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)], we found that insulin induced membrane hyperpolarization after 2 min in A10 cells. Insulin-induced hyperpolarization was suppressed by glibenclamide, an ATP-sensitive potassium (KATP) channel blocker. Using a cell-attached patch clamp experiment, the KATP channel was activated by insulin in both A10 cells and isolated VSMCs from rat aortas, indicating that insulin causes membrane hyperpolarization via KATP channel activation. These effects were not dependent on intracellular ATP concentration, but wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, significantly suppressed insulin-induced KATP channel activation. In addition, insulin enhanced phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and protein kinase B (Akt) after 2 min. These data suggest that KATP channel activation by insulin is mediated by PI3-K. Furthermore, using a nitric oxide synthase (NOS) inhibitor, we found that NOS might play an important role downstream of PI3-K in insulin-induced KATP channel activation. This study may contribute to our understanding of mechanisms of insulin resistance-associated hypertension.

Haemolysin produced by Vibrio mimicus activates two Cl– secretory pathways in cultured intestinal-like Caco-2 cells

Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco‐2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH‐induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA‐AM or by Rp‐adenosin 3′,5′‐cyclic monophosphorothioate triethylammonium salt (Rp‐cAMPS). The Cl– channel inhibitors 4,4′‐disothiocyanatostibene‐2,2′‐disulfonic acid (DIDS), glybenclamide, and 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) suppressed the VMH‐induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco‐2 cells to secrete Cl– by activating both Ca2+‐dependent and cAMP‐dependent Cl– secretion mechanisms. VMH forms ion‐permeable pores in the lipid bilayer that are non‐selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH‐induced Cl– secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+‐dependent and cAMP‐dependent Cl– secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.

Differences in stress response after UVC or UVA irradiation in Vibrio parahaemolyticus.

The SOS response is a global regulatory network for repairing DNA damage induced by various environmental stresses such as UV irradiation. The Escherichia coli SOS response has been extensively studied. However, there are no reports on the SOS response in Vibrio parahaemolyticus. In this study, we examined the SOS response in V. parahaemolyticus and compared the differential expression of genes induced by UVC and UVA irradiation. In UVC-exposed wild-type cells, expression of several DNA repair genes was increased. However, expression of these genes was not increased in ΔrecA or lexA mutants. Cell filamentation was observed in wild-type cells, but not in ΔrecA and lexA mutant cells. Sensitivity to UVC was significantly increased in ΔrecA, lexA mutant and Δlon strains compared with wild type. In the case of UVA irradiation, LexA-controlled DNA repair genes were minimally induced and cell filamentation was not observed. Sensitivity to UVA was the same in the mutant and wild-type strains. These findings suggest that there is a RecA-LexA-mediated SOS response in V. parahaemolyticus, and that this response is important to UVC tolerance but does not contribute to UVA tolerance.

Sterilization Using 365 nm UV-LED

There are several methods used for sterilization. In those methods chlorine, heat and UV rays are traditionally used. In recent years, the UV sterilization is taken notice as a sterilization method that the sterilized object does not change in quality and is environment-friendly. In this paper, an UV-LED is focused because it does not contain harmful substance and has longer operating life. The results have showed that complete germicidal effects for E. coli and Vibrio parahaemolyticus by UV-LED exposure of 30 minutes and 10 minutes, respectively. These results suggest that the UV-LED has sterilization effects. Therefore, UV-LED can be used as a sterilization device.

Effects of Ultraviolet LED on Bacteria

Sterilization technology is absolutely essential for our daily life. For example, it is used for water and sewerage system, foods, and medicine. Method that widely used for sterilization are using drug, heating, ultraviolet (UV) radiation, and ozone. Chlorine is used extensively in sterilization because of its easiness and low cost. Using chlorine has some adverse effects such as alteration of quality of the target. Also it is bad for environment discharge water sterilized by drug to rivers and oceans. Conventional method for UV sterilization is using UV lamps. It is used to sterilize workspaces and tools used in biology laboratories and medical facilities. Low pressure mercury-vapor lamps emit 254 nm wavelength of UV which coincides very well with peaks of the germicidal effectiveness curve (i.e., effectiveness for UV absorption by DNA). Low pressure mercury-vapor lamps contains mercury and it has harmful effects to an environment and human body. In this study, we investigate effects of high-intensity ultraviolet light-emitting diode (UV LED) on E. coli and Vibrio parahaemolyticus. Aside from mercury-free, UV LED is low consumption. Exposure time of the UV is varied from 5 to 60 minutes. The wavelength of UV LED used is 365nm, its output is 15mW, and exposure distance between the UV LED and bacteria is 20mm. The result showed that germicidal effects for 100% of E.coli and 100% of Vibrio parahaemolyticus took 30 minutes and 10 minutes, respectively. These results suggest that the UV LED has sterilized effects.

Angiotensin II decreases glucose uptake by downregulation of GLUT1 in the cell membrane of the vascular smooth muscle cell line A10.

Recent evidence suggests a crosstalk between angiotensin II (Ang II) and insulin. However, whether this crosstalk affects glucose uptake, particularly in terms of actin filament involvement, has not yet been studied in vascular smooth muscle cells. Pretreatment of cells with either Ang II or cytochalasin D disarranged actin filaments in a time-dependent manner and inhibited glucose uptake. However, insulin increased actin reorganization and glucose uptake. Membrane fractionation studies showed that Ang II decreased GLUT-1 at the cell membrane, whereas it increased GLUT-1 in the cytoplasm, indicating that Ang II may cause internalization of GLUT-1 via actin disorganization, consequently decreasing glucose uptake. The effects of Ang II on glucose uptake and actin reorganization were blocked by AT1 receptor antagonist, but not by AT2 antagonist. Either P38 or ERK1/2 inhibitors partially reversed the Ang II-inhibited actin reorganization and glucose uptake, suggesting that MAPK signaling pathways could be involved as downstream events in Ang II signaling, and this signaling may interfere with insulin-induced actin reorganization and glucose uptake. These data imply that Ang II induces insulin resistance by decreasing glucose uptake via disarrangement of actin filaments, which provides a novel insight into understanding of insulin resistance by Ang II at the molecular level.