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T Kawanishi

Publications and source records attributed to T Kawanishi.

At least 19 recordsLinked to original sources
PubMed2005

Screening of novel nuclear receptor agonists by a convenient reporter gene assay system using green fluorescent protein derivatives.

Nuclear receptors represent a very good family of protein targets for the prevention and treatment of diverse diseases. In this study, we screened natural compounds and their derivatives, and discovered ligands for the retinoic acid receptors (RARs) and the farnesoid X receptor (FXR). In the reporter assay systems of nuclear receptors presented here, two fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), were used for detection of a ligand-based induction and as an internal control, respectively. By optimizing the conditions (e.g., of hormone response elements and promoter genes for reporter plasmids), we established a battery of assay systems for ligands of RARs, retinoid X receptor (RXR) and FXR. The screening using the reporter assay system can be carried out without the addition of co-factors or substrates. As a result of screening of more than 140 compounds, several compounds were detected which activate RARs and/or FXR. Caffeic acid phenylethyl ester (CAPE), known as a component of propolis from honeybee hives, and other derivatives of caffeic acid up-regulated the expression of reporter gene for RARs. Grifolin and ginkgolic acids, which are non-steroidal skeleton compounds purified from mushroom or ginkgo leaves, up-regulated the expression of the reporter gene for FXR.

Animals↗
PubMed2005

CD16+CD57- natural killer cells in multifocal motor neuropathy.

We analyzed the CD16+CD57- lymphocyte subset, which is considered to have strong natural killer (NK) cell activity, in peripheral blood from patients with chronic immune-mediated neuropathies and patients with other neurological diseases. We found that the ratio of CD16+CD57- NK cells to total lymphocytes was increased in 4 of 6 patients with multifocal motor neuropathy (MMN) with persistent conduction block. Since the CD16 molecule is an Fc receptor for immunoglobulin G (IgG), high-dose intravenous immunoglobulin (IVIg) may interfere with CD16+CD57- NK cells via Fc receptor blockade. In addition, cyclophosphamide (Cy) is often used to suppress NK cells. Therefore, our findings may partly account for the effectiveness of IVIg or Cy, which is the current treatment of choice for MMN.

Adult↗
PubMed2004

A study of boronic acid based fluorescent glucose sensors.

Boronic acid based anthracene dyes were designed, synthesized, and immobilized to solid phase, creating a continuous glucose sensor. Glucose sensitivities of dyes can decrease drastically after immobilization, therefore how to immobilize a dye to solid phase without changing the dye property is a key issue in developing the sensor. The glucose sensitivity of the simplest 1st generation sensor, which is based on an immobilized mono-phenylboronate/single-arm type, came short of the sensitivity requirement for practical use, because of the very moderate fluorescence intensity change over the physiological glucose range. However, the 2nd generation, an immobilized bis-phenylboronate/double-arm type sensor, which contained two boronate groups in the dye moiety in expectation of a large intensity change, brought about considerable improvement on its glucose sensitivity. We tried to introduce functional groups onto an anthracene ring in order to improve the dies' fluorescence properties. Acetyl or carboxyl substitution on anthracene contributed to shift the fluorescence wavelength into the more visible range (red-shift) and a divergence of wavelength between an excitation peak and an emission peak. This improvement is advantageous to the design of an optical detection system. Furthermore, single arm immobilization to this carboxyl group, thus linking directly to the fluorophore led to a 3rd generation sensor, an immobilized bis-phenylboronate/single-arm type, that was twice as sensitive as that of the 2nd generation sensor, presumably due to increased mobility of the dye moiety. The results of our study advance closer toward a clinically useful continuous fluorescent glucose sensor.

Anthracenes↗
PubMed2003

Improvement in enzyme activity and stability by addition of low molecular weight polyethylene glycol to sodium bis(2-ethyl-L-hexyl)sulfosuccinate/isooctane reverse micellar system.

The activity and stability of Chromobacterium viscosum lipase (glycerolester hydrolase, EC 3.1.1.3)-catalyzed olive oil hydrolysis in sodium bis (2-ethyl-l-hexyl)sulfosuccinate (AOT)/isooctane reverse micelles is increased appreciably when low molecular weight polyethylene glycol (PEG 400) is added to the reverse micelles. To understand the effect of PEG 400 on the phase behavior of the reverse micellar system, the phase diagram of AOT/ PEG 400/water/isooctane system was studied. The influences of relevant parameters on the catalytic activity in AOT/PEG 400 reverse micelles were investigated and compared with the results in the simple AOT reverse micelles. In the presence of PEG 400, the linear decreasing trend of the lipase activity with AOT concentration, which is observed in the simple AOT reverse micelles, disappeared. Enzyme entrapped in AOT/PEG reverse micelles was very stable, retaining >75% of its initial activity after 60 d, whereas the half-life in simple AOT reverse micelles was 38 d. The kinetics parameter maximum velocity (Vmax) exhibiting the temperature dependence and the activation energy obtained by Arrhenius plot was suppressed significantly by the addition of PEG 400.

Chromobacterium↗
PubMed2002

Nitrate removal rate in a continuous column denitrification reactor using hydrogen generated by electrolysis with carbon anodes and stainless cathodes.

An autotrophic continuous denitrification process, using hydrogen generated by electrolysis with activated carbon anodes, was experimentally demonstrated to be an effective nitrate removal process. Several fixed bed columns with polypropylene packing and honeycomb shaped activated carbon anodes and stainless rod cathodes were set in a thermostat chamber at 30 degrees C, and potassium nitrate enriched tap water as influent was supplied at various flow rates and electric currents. Although the anode is in the same column where microbial biomass grows, sufficient nitrate removal was observed. For example, almost complete removal of nitrate and nitrite was observed at a hydraulic retention time (HRT) as short as 1.8 h. A model assuming successive denitrification reactions and plug-flow process, nitrate reduction rate = k1 [NO3-] [H2], and nitrite reduction rate = k2 [NO2-] [H2](1.5) was proposed. Calculated results with k1 = 1.3 mmol(-1) h(-1) and k2 = 3.3 mmol(-1.5) x h(-1) agreed well with all the experimental results.

Biofilms↗
PubMed2002

Use of nitrogen stable isotope ratio of periphyton for monitoring nitrogen sources in a river system.

In order to confirm the usefulness of the N stable isotope ratio of periphyton (mainly composed of attached algae) as an indicator for monitoring the N sources in river watersheds, we measured the isotope ratio of periphyton along the Chikuma River. In the river, both the concentrations of dissolved total nitrogen (DTN) and the delta15N values of periphyton increased downstream. Specific nitrogen loading rates (SNLR) calculated from administrative data also showed an increase downstream from 7 to 11 kg N ha(-1) yr(-1), with the increasing contribution by sewage and livestock waste from 6 to 40% to total N loading. There are significant positive relationships between the DTN concentration and the SNLR (r2=0.54, P<0.05), and the delta15N values of periphyton and the SNLR (r2=0.78, P<0.05). The increase in DTN concentration reflected the increase in input of N loading. The increase in delta15N of periphyton might reflect the increase in relative contribution by sewage and livestock waste down the river, especially the increase in sewage. The present study indicates the usefulness of the N stable isotope ratio of periphyton as an indicator for monitoring N sources in a river system.

Animals↗
PubMed2001

Involvement of Ca2+ waves in excitation-contraction coupling of rat atrial cardiomyocytes.

Two-dimensional and line-scan analyses of the early phase Ca2+ transients in rat cardiomyocytes were performed with a rapid-scanning laser confocal microscope and fluo-3 to elucidate the mechanism of activation of Ca2+ release from the sarcoplasmic reticulum in atrial myocytes which lack a well developed T-tubular network. On electrical stimulation of ventricular myocytes, Ca2+ concentration began to rise earliest at the Z-line level and became uniform throughout the cytoplasm within about 10 msec. In contrast, on stimulation of atrial myocytes, the earliest rise in Ca2+ occurred at the cell periphery and then spread to the cell interior; cytoplasmic Ca2+ became uniform after more than 30msec. The velocity of the propagation of rise in Ca2+ was 112 +/- 5.1 microm/sec (n = 10), which was similar to that of spontaneous Ca2+ waves observed in atrial and ventricular myocytes. No difference in frequency, amplitude and kinetics of spontaneous Ca2+ sparks was observed between the subsarcolemmal and central regions of atrial myocytes. Ryanodine concentration-dependently decreased the contractile force of isolated rat atrial and ventricular tissue preparations; the sensitivity was higher in atrial myocytes. The present study visualized the involvement of a propagated Ca2+-induced-Ca+ release mechanism in atrial but not ventricular myocytes. This difference may underlie some of the atrioventricular difference in response to physiological and pharmacological stimuli.

Animals↗
PubMed2001

Ganglioside GD1a inhibits HGF-induced motility and scattering of cancer cells through suppression of tyrosine phosphorylation of c-Met.

We previously reported that ganglioside GD1a, which is highly expressed in poorly metastatic FBJ-S1 cells, inhibits the serum-induced motility of FBJ-LL cells and that the metastatic potential of FBJ-LL cells is completely suppressed by enforced GD1a expression (Hyuga et al., Int J Cancer 1999;83:685-91). We recently discovered that hepatocyte growth factor (HGF) induces FBJ-LL cell motility. In the present study, the HGF-induced motility of FBJ-S1 cells was found to be one-thirtieth that of FBJ-LL cells. This motility of GD1a-expressing transfectants, which were produced by transfection of FBJ-LL cells with GM2/GD2 synthase cDNA, decreased with increases in their GD1a expression and HGF induced almost no motility in GD1a-pretreated FBJ-LL cells, indicating that GD1a inhibits the HGF-induced motility of FBJ-LL cells. The expression of the HGF receptor c-Met on FBJ-S1 cells, FBJ-LL cells, transfectants and a mock-transfectant was almost the same. The level of tyrosine phosphorylation of c-Met after HGF stimulation in FBJ-S1 cells, GD1a-pretreated FBJ-LL cells and a GD1a-expressing transfectant was significantly lower than in FBJ-LL cells and a mock-transfectant. These findings suggested that GD1a inhibits the HGF-induced motility of FBJ-LL cells through suppression of tyrosine phosphorylation of c-Met. HepG2 cells, a human hepatoma cell line, were used to investigate whether GD1a interferes with other cancer cells expressing c-Met. HepG2 cells did not express GD1a. HGF induced cell scattering of HepG2 cells and the scattering was inhibited by pretreating the cells with GD1a. The c-Met in the cells was autophosphorylated by stimulation with HGF, but after treating the cells with GD1a, the HGF-induced autophosphorylation of c-Met was suppressed. These results suggest that GD1a acts as a negative regulator of c-Met in cancer cells.

Actins↗
PubMed2001

A mechanism of Ca2+ release from Ca2+ stores coupling to the Na+/Ca2+ exchanger in cultured smooth muscle cells.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.

Animals↗
PubMed2001

Effect of tributyltin chloride on the release of calcium ion from intracellular calcium stores in rat hepatocytes.

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca(2+) from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca(2+) was measured, using a fluorescent Ca(2+) dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min, the extracellular Ca(2+) concentration was high, but the inositol 1,4,5-trisphosphate (InsP(3))-induced increase in Ca(2+) concentration was suppressed, suggesting that the extracellular release of Ca(2+) in response to TBT treatment was from intracellular stores. Images of the Ca(2+) concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca(2+) was released. When the hepatocytes were treated with 4.0 microM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 microM TBT and 2.0 microM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca(2+) from intracellular stores. When the hepatocytes were preincubated with 0.1 microM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP(3)-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca(2+) from the intracellular stores at high concentrations, and suppressed the InsP(3)-induced Ca(2+) release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca(2+) response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54-61).

Animals↗
PubMed2001

[Studies on quality and safety control of drugs for human use from transgenic animals/clone animals].

Recently the pharmaceuticals, which were produced using transgenic animals, have been developed, and will be submitted for registration in nearly future in Japan as well as in USA and EU. In addition, clone animals are also thought to be useful for the productions of the pharmaceuticals. This study has been, therefore, undertaken to establish the technical requirement for registration of the pharmaceuticals. They should be evaluated from the following standpoints: 1) Transgene construct and its characterization; 2) Creation and characterization of the transgenic founder animal; 3) Establishment of a reliable and continuous source of transgenic founder animals; 4) Generation and selection of the production animals; 5) Maintenance of transgenic animals; 6) Recovery and purification of products from transgenic animals; 7) Characterization of products; 8) Process validation/evaluation and in-process test; 9) Specification of products; 10) Stability of products; 11) Preclinical safety evaluation and clinical evaluation. Cloning technology by nuclear transfer of a transformed somatic cell has been already applied to the creation of the transgenic founder animal for the production of pharmaceuticals. The pharmaceuticals produced using the clone animals could be evaluated from almost the same standpoints. However, the flexible evaluation will be also needed depending on the development of the technology.

Animals↗
PubMed2000

The 5-HT(1A) receptor agonist, 8-OH-DPAT, attenuates stress-induced anorexia in conjunction with the suppression of hypothalamic serotonin release in rats.

The effect of the selective 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) on stress-induced anorexia and serotonin (5-HT) release in the rat hypothalamus was studied with brain microdialysis. Subcutaneous injection of 8-OH-DPAT (1 mg/kg) significantly attenuated the immobilization-induced anorexia for 3 h, but had no effect during the following 9 h. Injection of 8-OH-DPAT itself had no effect on basal release of 5-HT, while it significantly blocked the immobilization-induced 5-HT release in the lateral hypothalamus. The results suggest that 8-OH-DPAT attenuated the stress-induced anorexia through the activation of 5-HT(1A) autoreceptors in dorsal raphe nucleus.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2000

Increased HTLV type 1 tax specific CD8+ cells in HTLV type 1-asociated myelopathy/tropical spastic paraparesis: correlation with HTLV type 1 proviral load.

Less than 1% of individuals infected with the human T lymphotropic virus type 1 (HTLV-1) develop an inflammatory neurological disorder, termed HTLV-1-associated myelopathy/tropical spastic (HAM/TSP), while the vast majority of those infected remain asymptomatic HTLV-1 carriers (ACs). The fundamental viroimmunological differences between these groups are not well understood. To address this issue, we have investigated HTLV-1-specific T cell responses and measured the proviral load in these groups. Frequencies of HTLV-1-specific CD8(+) cells were demonstrated to be significantly higher in HAM/TSP patients than in ACs by using intracellular cytokine staining and soluble divalent HLA-A2/Ig fusion protein loaded with HTLV-1 Tax 11-19 peptide. It is consistent with the observed increase in HTLV-1-specific cytotoxic T lymphocytes in HAM/TSP patients. These CD8(+) cells produced interferon (IFN)-gamma in recognition of HTLV-1 antigens bound to HLAs on the infected CD4(+) cells. Using phenotypic markers indicative for T cell differentiation, memory and/or effector HTLV-1 Tax-specific CD8(+) cells were found to be increased in HLA-A2 HAM/TSP patients. HTLV-1 proviral load was elevated in HAM/TSP patients when compared to ACs. In addition, the proviral load in HAM/TSP patients correlated with the frequency of HTLV-1-specific IFN-gamma(+)CD8(+) cells or Tax-HLA-A2/Ig(+)CD8(+) cells, especially with the effector cells. In contrast, the proviral load inversely correlated with memory cells. These results suggest that HTLV-1 antigens may continuously stimulate HTLV-1-specific CD8(+) cells and differentiate them from memory cells into effector cells in vivo. These differentiated HTLV-1-specific CD8(+) cells may play a role in the pathogenesis of HAM/TSP.

CD8-Positive T-Lymphocytes↗
PubMed2000

Brewster's scattering angle in scattered waves from slightly rough metal surfaces

Brewster's scattering angle in electromagnetic wave scattering from slightly random metal surfaces is investigated by means of the stochastic functional approach. While there are dips due to Brewster's scattering angle in scattering profiles from dielectric surfaces, Brewster's scattering angle does not exist in scattering from metal surfaces. However, the dips can exist in scattering from rough metal surfaces with the optically denser medium to convert evanescent wave into radiative wave.

Journal Article↗
PubMed2000

HTLV-I specific IFN-gamma+ CD8+ lymphocytes correlate with the proviral load in peripheral blood of infected individuals.

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease caused by HTLV-I infection. It has been shown that HAM/TSP patients have high proviral loads and an extraordinarily high frequency of circulating CD8 + cytotoxic T lymphocytes specific for HTLV-I in their peripheral blood when compared to asymptomatic HTLV-I carriers (AC). We have previously described an intracellular cytokine detection assay, in which interferon-gamma (IFN-gamma) + CD8 + lymphocytes are specific for HTLV-I in infected individuals. Here, we have established a competitive polymerase chain reaction assay to measure the proviral load of patients and investigate a potential relationship between proviral load and virus-specific CD8 + lymphocytes. Genomic DNA was extracted from peripheral blood lymphocytes (PBL) from eight HAM/TSP patients and seven AC for the measurement of HTLV-I measuring proviral loads. The same PBL were analyzed for intracellular IFN-gamma expression by flow cytometry. In the HAM/TSP patients and AC, the average proviral loads were 34,482 and 9784 copy/microg DNA (P = 0.021), and the average of IFN-gamma + CD8 + lymphocytes in total PBL were 1.47 and 0.08% (P = 0.001), respectively. It was confirmed that HAM/TSP patients have both high proviral loads and increased HTLV-I-specific CD8 + lymphocytes. Furthermore, we found a positive correlation between both factors in the patients with HAM/TSP (P = 0.044) but not in the AC (P = 0.508). These findings suggest that the high number of HTLV-I-specific lymphocytes may result from the increased proviral load in HAM/TSP patients.

Aged↗
PubMed2000

Changes in CD4+CD8-/CD4-CD8+ ratio and humoral immune functions in vitamin B12-deficient rats.

To clarify the role of vitamin B12 in the function of cell-mediated and humoral immune functions, the splenocytes expression of CD4, CD8 and serum C3, IgM, IgG concentrations were examined in vitamin B12-deficient rats, and the effect of the administration of methylcobalamin was also studied. The CD4+CD8-/CD4-CD8+ ratio in splenocytes was significantly higher in vitamin B12-deficient rats than in control rats (p < 0.05). The value in the 48 hours after methylcobalamin administration group, was within the normal range (p < 0.05). From these results, the elevation of the CD4+CD8-/CD4-CD8+ ratio by vitamin B12-deficiency was confirmed in rats. The serum C3, IgM and IgG concentrations were lower in the vitamin B12-deficient group than in the control group. These findings suggest that vitamin B12 plays a role in maintaining the immune function in rats.

Animals↗
PubMed2000

Extremely low activity of methionine synthase in vitamin B-12-deficient rats may be related to effects on coenzyme stabilization rather than to changes in coenzyme induction.

Severely vitamin B-12 (B-12)-deficient rats were produced by feeding a B-12-deficient diet. The status of B-12 deficiency was confirmed by an increase in urinary methylmalonate excretion and decreases in liver B-12 concentrations and cobalamin-dependent methionine synthase activity. Rat liver methionine synthase existed almost exclusively as the holoenzyme. In B-12-deficient rats, the level of methionine synthase protein was lower, although the mRNA level was not significantly different from that of control rats. When methylcobalamin, the coenzyme for methionine synthase, was administered to the B-12-deficient rats, growth, liver B-12 concentrations and urinary excretion of methylmalonate were reversed although not always to control (B-12-sufficient) levels in a short period. During this recovery process, methionine synthase activity and its protein level increased, whereas the mRNA level was unaffected. We reported previously that rat apomethionine synthase is very unstable and is stabilized by forming a complex with methylcobalamin. Thus, the extremely low activity of methionine synthase in B-12-deficient rats may be related to effects on "coenzyme stabilization" (stabilization of the enzyme by cobalamin binding) rather than to changes in "coenzyme induction."

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗