Biomedical subjects

Kiichi Sato

Publications and source records attributed to Kiichi Sato.

At least 19 recordsLinked to original sources

PubMed2007

Construction of a dimethyl sulfoxide sensor based on dimethyl sulfoxide reductase immobilized on a au film electrode.

A novel dimethyl sulfoxide (DMSO) sensor using DMSO reductase and film electrodes was constructed. The Au and Ag electrodes were fabricated on slide glass by vacuum deposition and the application of a photolithographic technique. The micro-chamber (4 x 50 x 1 mm, volume 200 microl) was fabricated on a poly(dimethylsiloxane) (PDMS) polymer. The Pt electrode was implanted in a PDMS polymer. DMSO reductase was immobilized on a Au film electrode with bovine serum albumin (BSA)-glutaraldehyde. This sensor could determine DMSO in an unpurged aqueous solution with glucose oxidase (GOD) and catalase (CAT) for oxygen removal. The DMSO sensor showed a linear response within 1 mM DMSO with a correlation coefficient of 0.999. The detection limit was 200 microM (3sigma), and the sensitivity was 23.8 mA M(-1) cm(-2). The relative standard deviations at each concentration were within 3.6%.

Calibration↗

PubMed2006

Existence of autocrine loop between interleukin-6 and transforming growth factor-beta1 in activated rat pancreatic stellate cells.

Interleukin (IL)-6 is a proinflammatory cytokine assumed to participate in pancreatic fibrosis by activating pancreatic stellate cells (PSCs). Autocrine TGF-beta1 is to central in PSC functional regulation. In this study, we examined IL-6 secretion from culture-activated rat PSCs and its regulatory mechanism. Activated PSCs express and secrete IL-6. When anti-TGF-beta1 neutralizing antibody was added in the culture medium, IL-6 secretion from activated PSCs was inhibited, whereas exogenous TGF-beta1 added in the culture medium enhanced IL-6 expression and secretion by PSCs in a dose dependent manner. Infection of PSCs with an adenovirus expressing dominant-negative Smad2/3 attenuated basal and TGF-beta1-stimulated IL-6 expression and secretion of PSCs. We also demonstrated the reciprocal effect of PSCs-secreted IL-6 on autocrine TGF-beta1. Anti-IL-6 neutralizing antibody inhibited TGF-beta1 secretion from PSCs. Preincubation of cells with 10 nM PD98059, an extracellular signal-regulated kinase (ERK)-dependent pathway inhibitor, attenuated IL-6-enhanced TGF-beta1 expression and secretion of PSCs. In addition, IL-6 activated ERK in PSCs. These data indicate the existence of autocrine loop between IL-6 and TGF-beta1 through ERK- and Smad2/3-dependent pathways in activated PSCs.

Animals↗

PubMed2006

Cyclooxygenase-2 is required for activated pancreatic stellate cells to respond to proinflammatory cytokines.

Cyclooxygenase-2 (COX-2) mediates various inflammatory responses and is expressed in pancreatic tissue from patients with chronic pancreatitis. To examine the role of COX-2 in chronic pancreatitis, we investigated its participation in regulating functions of pancreatic stellate cells (PSCs), using isolated rat PSCs. COX-2 was expressed in culture-activated PSCs but not in freshly isolated quiescent PSCs. TGF-beta1, IL-1beta, and IL-6 enhanced COX-2 expression in activated PSCs, concomitantly increasing the expression of alpha-smooth muscle actin (alpha-SMA), a parameter of PSC activation. The COX-2 inhibitor NS-398 blocked culture activation of freshly isolated quiescent PSCs. NS-398 also inhibited the enhancement of alpha-SMA expression by TGF-beta1, IL-1beta, and IL-6 in activated PSCs. These data indicate that COX-2 is required for the initiation and promotion of PSC activation. We further investigated the mechanism by which cytokines enhance COX-2 expression in PSCs. Adenovirus-mediated expression of dominant negative Smad2/3 inhibited the increase in expression of COX-2, alpha-SMA, and collagen-1 mediated by TGF-beta1 in activated PSCs. Moreover, dominant negative Smad2/3 expression attenuated the expression of COX-2 and alpha-SMA enhanced by IL-1beta and IL-6. Anti-TGF-beta neutralizing antibody also attenuated the increase in COX-2 and alpha-SMA expression caused by IL-1beta and IL-6. IL-6 as well as IL-1beta enhanced TGF-beta1 secretion from PSCs. These data indicate that Smad2/3-dependent pathway plays a central role in COX-2 induction by TGF-beta1, IL-1beta, and IL-6. Furthermore, IL-1beta and IL-6 promote PSC activation by enhancing COX-2 expression indirectly through Smad2/3-dependent pathway by increasing TGF-beta1 secretion from PSCs.

Actins↗

PubMed2006

Sonic hedgehog stimulates the proliferation of rat gastric mucosal cells through ERK activation by elevating intracellular calcium concentration.

Sonic Hedgehog (Shh), a member of hedgehog peptides family, is expressed in gastric gland epithelium. To elucidate Shh function to gastric mucosal cells, we examined the effect of Shh on the proliferation of a rat normal gastric mucosal cell line, RGM-1. RGM-1 cells express essential components of Shh receptor system, patched-1, and smoothened. Shh enhanced DNA synthesis in RGM-1 cells and elevated intracellular calcium concentration ([Ca2+]i). In addition, Shh as well as calcium ionophore A32187 rapidly activated ERK. However, Shh failed to activate ERK under calcium-free culture condition. Pretreatment of cells with PD98059 attenuated the DNA synthesis promoted by Shh. Moreover, when cells were pretreated with cyclopamine, Shh could not elevate [Ca2+]i, activate ERK or promote DNA synthesis. On the other hand, although Shh induced Gli-1 nuclear accumulation in RGM-1 cells, Shh activated ERK even in cells pretreated with actinomycin D. These results indicate that Shh promotes the proliferation of RGM-1 cells through an intracellular calcium- and ERK-dependent but transcription-independent pathway via Patched/Smoothened receptor system.

Animals↗

PubMed2006

Microbioassay system for an anti-cancer agent test using animal cells on a microfluidic gradient mixer.

We developed a novel microbioassay system equipped with a gradient mixer of two solutions, and we applied the microfluidic system to an anti-cancer agent test using living animal cells on a microchip. A microchannel for the gradient mixing of two solutions and eight other microchannels for cell assay were fabricated on a poly(dimethylsiloxane) substrate using a soft-lithography method. The functions necessary for this bioassay, i.e., cell culturing, chemical stimulation, cell staining, and fluorescence determination, were integrated into the microfluidic chip. Eight gradient concentrations of the fluorescein solution, ranging from 1 to 98 microg/ml, were archived at 0.1 microl/min on a microchip. A stomach cancer cell line was cultured, and a cell viability assay was conducted using 5-Fluorouracil as an anti-cancer agent on the microchip. Cell viability changed according to the estimated concentration of the agent solution. With the microbioassay system, an anti-cancer agent test was conducted using living cells simultaneously in eight individual channels with the gradient concentration of the agent on a microchip.

Animals↗

PubMed2005

Autocrine loop between TGF-beta1 and IL-1beta through Smad3- and ERK-dependent pathways in rat pancreatic stellate cells.

Pancreatic stellate cells (PSCs) are activated during pancreatitis and promote pancreatic fibrosis by producing and secreting ECMs such as collagen and fibronectin. IL-1beta has been assumed to participate in pancreatic fibrosis by activating PSCs. Activated PSCs secrete various cytokines that regulate PSC function. In this study, we have examined IL-1beta secretion from culture-activated PSCs as well as its regulatory mechanism. RT-PCR and ELISA have demonstrated that PSCs express IL-1beta mRNA and secrete IL-1beta peptide. Inhibition of TGF-beta(1) activity secreted from PSCs by TGF-beta(1)-neutralizing antibody attenuated IL-1beta secretion from PSCs. Exogenous TGF-beta(1) increased IL-1beta expression and secretion by PSCs in a dose-dependent manner. Adenovirus-mediated expression of dominant-negative (dn)Smad2/3 expression reduced both basal and TGF-beta(1)-stimulated IL-1beta expression and secretion by PSCs. Coexpression of Smad3 with dnSmad2/3 restored IL-1beta expression and secretion by PSCs, which were attenuated by dnSmad2/3 expression. In contrast, coexpression of Smad2 with dnSmad2/3 did not alter them. Furthermore, inhibition of IL-1beta activity secreted from PSCs by IL-1beta-neutralizing antibody attenuated TGF-beta(1) secretion from PSCs. Exogenous IL-1beta enhanced TGF-beta(1) expression and secretion by PSCs. IL-1beta activated ERK, and PD-98059, a MEK1 inhibitor, blocked IL-1beta enhancement of TGF-beta(1) expression and secretion by PSCs. We propose that an autocrine loop exists between TGF-beta(1) and IL-1beta in activated PSCs through Smad3- and ERK-dependent pathways.

Animals↗

PubMed2005

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway.

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.

Adenoviridae↗

PubMed2005

Role of trehalose synthesis pathways in salt tolerance mechanism of Rhodobacter sphaeroides f. sp. denitrificans IL106.

The photosynthetic bacterium Rhodobacter sphaeroides (R. sphaeroides) f. sp. denitrificans IL106 accumulates trehalose as the major organic osmoprotectant in response to a salt stress. An analysis of the R. sphaeroides 2.4.1 genome sequence revealed the presence of five different genes encoding enzymes belonging to three putative trehalose biosynthesis pathways (OtsA-OtsB, TreY-TreZ, and TreS). The function of the different pathways of trehalose was studied by characterizing strains defective in individual trehalose biosynthetic routes. A phenotypic comparison revealed that trehalose synthesis in R. sphaeroides f. sp. denitrificans IL106 is mediated mainly by the OtsA-OtsB pathway and, to some extent, by the TreY-TreZ pathway. Strains with the simultaneous inactivation of these two pathways were completely unable to synthesize trehalose. On the other hand, treS mutants showed an increase in the trehalose level. These results suggest that treS plays a role in trehalose degradation. In addition, treS was found to be important in reducing trehalose after osmotic stress was removed. In this report, we show that the strains that accumulate the most trehalose adapt to salt stress earlier. This is the first report of an organism using multiple pathways to synthesize trehalose solely for use as a compatible solute against salt stress.

Adaptation, Physiological↗

PubMed2005

Monitoring of intercellular messengers released from neuron networks cultured in a microchip.

A cellular biochemistry analysis system was integrated on a quartz glass microchip with a microchamber for cell culture followed by a microchannel for detecting with a thermal lens microscope (TLM). Nerve cells from rat hippocampus were successfully cultured to form neural networks in the microchip. An aqueous solution of glutamate, which is known as a neurotransmitter, was introduced to stimulate the cultured neuron to release a retrograde messenger, arachidonate which is considered to be critical for neuronal plasticity, especially for long-term potentiation (LTP). After the introduction, the solution that flowed through the culture chamber was analyzed using the UV-TLM (excitation wavelength, 244 nm). The measured signal intensity was dependent on glutamate solution concentration, and the neurons were considered to release the retrograde messenger according to the glutamate concentration. This system is suitable for time-course monitoring of ultra trace amounts of chemicals released from very small amount of cultured cells.

Animals↗

PubMed2005

Cell culture and life support system for microbioreactor and bioassay.

A microchip-based cell culture system was developed and a primary culture of rat hepatocytes was realized in the system. The microchip was made of glass plates and had a microchannel and a microculture flask inside. The flask inner surface was coated using collagen solution; then FBS and DMEM were added successively. Rat hepatocytes suspended in a medium was introduced into the microchip and incubated at 37 degrees C in a humidified atmosphere with 5% CO(2). Because of the shortage of dissolved oxygen, the cultured cells in the microchip resulted in a significant decrease in viability. To overcome this, a continuous medium flow oxygen and nutrition supplying system was designed and constructed. The system realized good cell growth for at least 4 days. Liver-specific functions, such as the synthesis of albumin and urea from hepatocytes were confirmed.

Albumins↗

PubMed2005

A case of esophageal stricture due to metastatic breast cancer diagnosed by endoscopic mucosal resection.

Metastasis of breast cancer to the esophagus has been reported but is rare. It is often difficult to diagnose metastases of breast cancer to the esophagus because they are often located in the submucosa and covered with normal mucosa. Although several methods have been reported in order to obtain specimens for pathological diagnosis, the adverse effects including bleeding and perforation were considerable problems. We report a case of a patient with esophageal stricture due to metastatic breast cancer to the esophagus. Pathological diagnosis was successfully obtained using endoscopic mucosal resection of the esophagus.

Adenocarcinoma↗

PubMed2005

Microbioassay system for antiallergic drug screening using suspension cells retaining in a poly(dimethylsiloxane) microfluidic device.

This article describes an antiallergic drug-screening system by the detection of histamine released from mast cells (suspension cells) on a multilayer microchip. In this study, the elastmeric material, poly(dimethylsiloxane) (PDMS), was employed to fabricate microchannels and microchambers. The microchip consists of two sections: a histamine-releasing one, which has a cell chamber, and a histamine-derivatizing one. Both were laminated to one microchip. Rat peritoneal mast cells were retained in the cell chamber (1.2 microL) with a filtering system using a cellulose nitrate membrane. This filtering system could easily retain suspension cells without cell damage. Mast cells were viable for a sufficient time to conduct the assay on the cell chamber. The cells were stimulated with a chemical release compound 48/80 (C48/80), and then histamine flowed into the lower layer, where it was derivatized to the fluorescent molecules with o-phthalaldehyde and its fluorescence was detected on the microchip. This flow system could detect the time course of the histamine release, and this microchip system required only 20 min for the assay. By this integrated system, 51 pmol of histamine released from 500 cells was detected, and the number of cells required for the assay was reduced to 1% compared with conventional bulk systems. By comparing the released histamine levels with and without drugs, their effect could be evaluated. The inhibition ratio of C48/80 induced-histamine release using an antiallergic drug, disodium cromoglicate (DSCG), was related to the concentration of DSCG. This flow system was applicable for antiallergy drug screening by rapid measurement of the inhibition of histamine release from a very small amount of mast cells.

Animals↗

PubMed2005

Development of a microchip-based bioassay system using cultured cells.

We developed a novel bioassay system using a glass microchip and cultured cells. A microchamber for cell culture and microchannels for reactions and detection were fabricated on a Pyrex glass substrate by photolithography and wet etching techniques. Cell culture, chemical and enzymatic reactions, and detection were integrated into the microchip. To keep different temperatures locally in three areas of the microchip, we designed and fabricated a temperature control device. Nitric oxide released from macrophage-like cells stimulated by lipopolysaccharide was successfully monitored with the microchip, the temperature control device, and a thermal lens microscope. The total assay time was reduced from 24 to 4 h, and detection limit of NO was improved from 1 x 10(-6) to 7 x 10(-8) M compared with conventional methods. Moreover, the system could monitor a time course of the release, which is difficult to measure by conventional batch methods. We conclude that this system is promising for a rapid bioassay system with very small consumption of cells.

Animals↗

PubMed2005

Separation of 2-aminobenzoic acid-derivatized glycosaminoglycans and asparagine-linked glycans by capillary electrophoresis.

A capillary electrophoresis method was developed for the analysis of oligosaccharides combined with derivatization with 2-aminobenzoic acid. Glycosaminoglycan delta-disaccharides were effectively resolved on a fused-silica capillary tube using 150 mM borate, pH 8.5, as a running electrolyte solution. This analytical method was applied to the identification of glycosaminoglycan in combination with enzymatic digestion. The separation of N-glycans or glucose-oligomers was performed with a phosphate buffer containing polyethylene glycol or borate as an electrolyte solution. This method is expected to be useful in the determination of oligosaccharide structures in a glycoprotein.

Animals↗

PubMed2004

Microchip-based enzyme-linked immunosorbent assay (microELISA) system with thermal lens detection.

A microchip-based enzyme-linked immunosorbent assay (microELISA) system was developed and interferon-gamma was successfully determined. The system was composed of a microchip with a Y-shaped microchannel and a dam structure, polystyrene microbeads, and a thermal lens microscope (TLM). All reactions required for the immunoassay were done in the microchannel by successive introduction of a sample and regents. The enzyme reaction product, in a liquid phase, was detected downstream in the channel using the TLM as substrate solution was injected. The antigen-antibody reaction time was shortened by the microchip integration. The limit of the determination was improved by adopting the enzyme label. Moreover, detection procedures were greatly simplified and required time for the detection was significantly cut. The system has good potential to be developed as a small and automated high throughput analyzer.

Enzyme-Linked Immunosorbent Assay↗

PubMed2004

New diagnostic approaches for early detection of gastric cancer.

Detection of gastric cancer in early stages is vitally important for ascertaining better prognosis and quality of life for the patients. Therefore, every endoscopist should be trained to master enough diagnostic skills to identify early gastric cancer that often shows minimal alteration from the surrounding mucosa, easily evading detection. For the first step, it is essential that endoscopists understand the normal gastric mucosa as well as the mucosal changes caused by chronic H. pylori infection, a high-risk condition for the development of gastric cancer. Once a suspicious lesion is identified, use of a dye-spraying method may be useful to clarify structural alteration caused by neoplastic changes and facilitate the biopsy sampling. Development of zoom (magnifying) endoscopy enabling 80x magnification with a one-touch switch from conventional endoscopic observation helps to identify the detailed surface structure as well as the vascular architecture of the mucosa without tissue biopsy. Combined with chromoendoscopy, this powerful endoscopic method can be used to identify small cancer foci or delineate the margin of early gastric cancer that can be treatable by mucosal dissection. Other new modalities using a variety of optical devices have been developed but the real value of their utility still remains to be proven in the actual clinical settings.

Diagnosis, Differential↗

PubMed2004

Integration of an immunoassay system into a microchip for high-throughput assay.

An immunosorbent assay system was integrated into a glass microchip. The scale merits of liquid microspace for molecular behavior contributed remarkably to reduced assay time, and troublesome operations required for conventional immunosorbent assays could be replaced by simple operations. Moreover, a micro-immunoassay system suitable for simultaneous assay of multiple samples was constructed on a microchip. The chip had branching multichannels and four reaction and detection regions; thus the constructed system could process four samples at a time with only one pump unit. A higher throughput assay was realized with the branching structure chip.

Animals↗

PubMed2004

Drug response assay system in a microchip using human hepatoma cells.

A microchip-based cell response assay system to an anticancer agent was developed. The hepatoma cell line HepG2 was used to assess the effects of an anticancer agent, doxorubicin. The required cell number was reduced by two orders, and the observation of the time course of cell response became possible. The system clearly showed that treatment with higher doses of the drug or longer exposure times gave more effects to cells. The possibilities of novel drug response studies or toxicity assay system were demonstrated.

Antibiotics, Antineoplastic↗