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Tomokazu Matsuura

Publications and source records attributed to Tomokazu Matsuura.

16 recordsLinked to original sources
PubMed2006

All-trans retinoic acid down-regulates human albumin gene expression through the induction of C/EBPbeta-LIP.

ATRA (all-trans retinoic acid), which is a major bioactive metabolite of vitamin A and a potent regulator of development and differentiation, mediates down-regulation of the human albumin gene. However, the mechanism of ATRA-mediated down-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrate that ATRA causes a marked decrease in the activity of the albumin promoter, the region between nt -367 and -167 from the transcription start site, where C/EBP (CCAAT/enhancer-binding protein)-binding sites are tightly packed, is indispensable for ATRA-mediated down-regulation. ChIP (chromatin immunoprecipitation) assays revealed that in vivo binding of C/EBPalpha to the region markedly decreases upon incubation with ATRA, whereas ATRA treatment marginally increases the recruitment of C/EBPbeta. We found that ATRA has the ability to differentially and directly induce expression of a truncated isoform of C/EBPbeta, which is an LIP (liver-enriched transcriptional inhibitory protein) that lacks a transactivation domain, and to increase the binding activity of C/EBPbeta-LIP to its response element. Overexpression of C/EBPbeta-LIP negatively regulates the endogenous expression of albumin, as well as the activity of the albumin promoter induced by C/EBP transactivators such as C/EBPalpha and full-length C/EBPbeta. In conclusion, we propose a novel model for down-regulation of the albumin gene, in which ATRA triggers an increase in the translation of C/EBPbeta-LIP that antagonizes C/EBP transactivators by interacting with their binding sites in the albumin promoter.

Albumins↗
PubMed2006

Reconstruction of liver organoid using a bioreactor.

AIM: To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver. METHODS: We previously reported that a radial-flow bioreactor (RFB) could provide a three-dimensional high-density culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) line M1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB. Two x 10(7) FLC-5 cells were incubated in the RFB. After 5 d, 2 x 10(7) A7 cells were added in a similar manner followed by another addition of 10(7) M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB. RESULTS: By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles (VVO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased. CONCLUSION: Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.

Albumins↗
PubMed2006

Serum vitamin A concentrations in asthmatic children in Japan.

BACKGROUND: Vitamin A is an essential micronutrient with important roles in immunity and maintenance of normal epithelial cell differentiation. Little information is available regarding the relationship between vitamin A concentrations and asthma despite the repair of epithelial and other structural changes being of utmost importance for the relief of symptoms and control of the disease. The authors evaluated vitamin A and vitamin E concentrations in well-nourished children with asthma. METHODS: The serum vitamin A and vitamin E concentrations were measured by high performance liquid chromatography methods. Statistical analysis was performed using the Mann-Whitney U-test and Peason's correlation coefficient test. RESULTS: According to these methods, the mean serum vitamin A concentrations were significantly lower (19.41+/-7.45 microg/dL, mean+/-SD) in asthmatic children than controls (29.52+/-11.34 microg/dL, P=0.0001). To compare the correlation of C-reactive protein and serum vitamin A concentrations, there was also significant difference between the two groups. CONCLUSION: The data suggest that there is a correlation between vitamin A deficiency and the mechanism of asthmatic response. These data support that the mechanism of hypovitaminosis A in asthmatic children may involve not only the acute phase response but also the various degrees of chronic epitherial damage of airways.

Asthma↗
PubMed2006

Bioreactors for 3-dimensional high-density culture of human cells.

A bioreactor was developed as an instrument to culture human or animal cells that require attachment in a large quantity or at a high density. The purpose for developing such a bioreactor is two-fold: to produce a large quantity of animal or human cells that have been modified by gene recombination technology to accommodate manufacture of physiologically-active substances or human proteins on an industrial scale; and for research to culture animal cells to form a high-density 3-dimensional structure as a morphological or functional tissue or organ entity. In the current report, the circulatory flow bioreactor and radial flow bioreactor (RFB) are introduced, in which the former can be scaled up. As a small bioreactor produced for the latter purpose, a rotary cell culture system and novel multicoaxial hollow-fiber bioreactor are introduced. Finally, a small RFB culture system that was scaled down by the present author and his collaborators for the study of a 3-dimensional high density culture system is described. The RFB can be readily scaled up for manufacturing or scaled down for research purposes. This is a cell culturing system that can induce the functions of human tissues by preparing a high density 3-dimensional organization of cells of human origin.

Animals↗
PubMed2005

Macrophage migration inhibitory factor increases neuronal delayed rectifier K+ current.

Macrophage migration inhibitory factor (MIF) has widespread actions in the immune, endocrine, and nervous systems. Previously, we reported that increases in the intracellular levels of MIF depress the firing of hypothalamus/brain stem neurons in culture, including the chronotropic actions of angiotensin II. The objective of this study was to investigate the effects of MIF on delayed rectifier K+ current (I(Kv)), one of the component currents whose activity contributes to neuronal firing. Intracellular perfusion of MIF (80 nM) into Sprague-Dawley rat neuronal cultures caused a significant increase in I(Kv), as measured by patch-clamp recordings. This effect was apparent by 3 min, and was maximal after 20-30 min. I(Kv) current density (pA/pF) increased from 31.58 +/- 2.36 in controls to 41.88 +/- 3.76 in MIF-treated neurons (mean +/- SE; n = 9; P < 0.01). MIF that had been inactivated by boiling did not alter I(Kv), and MIF-neutralizing antibodies abolished the action of recombinant MIF (rMIF). The stimulatory effect of MIF on I(Kv) current density was mimicked by intracellular application of either P1S-MIF (80 nM) or the peptide MIF-(50-65) (0.8-8 microM), both of which harbor the thiol-protein oxidoreductase (TPOR) activity of the MIF molecule. Conversely, neither C60S-MIF (80 nM) nor the MIF homologue D-dopachrome tautomerase (80 nM), both of which lack TPOR activity, altered I(Kv). Finally, the increase in I(Kv) produced by rMIF was abolished by the superoxide scavenger Tiron (1 mM). These studies indicate that the neuronal action of MIF includes a stimulatory action on I(Kv) that may be mediated by a TPOR/superoxide-scavenging mechanism.

Action Potentials↗
PubMed2005

Electrophysiological properties of rostral ventrolateral medulla neurons in angiotensin II 1a receptor knockout mice.

We compared the electrophysiological properties of neurons in the rostral ventrolateral medulla of neonatal angiotensin II type 1a receptor knockout mice and wild-type mice with responses to angiotensin II, its type-1 receptor blocker candesartan, and its type-2 receptor blocker PD123319. Using the whole-cell patch-clamp technique, we examined the characteristics of rostral ventrolateral medulla neurons in brain stem-spinal cord preparations in which the sympathetic neuronal network is preserved. Baseline membrane potential and firing rate were almost similar between angiotensin II type 1a receptor knockout mice and wild-type mice. Superfusion with angiotensin II depolarized rostral ventrolateral medulla bulbospinal neurons in wild-type mice, whereas it hyperpolarized those in angiotensin II type 1a receptor knockout mice. Because pretreatment with candesartan significantly prevented the angiotensin II-induced depolarization in wild-type mice, the angiotensin II type 1 receptor is crucial for this depolarization. Superfusion with PD123319 depolarized rostral ventrolateral medulla bulbospinal neurons in angiotensin II type 1a receptor knockout mice. PD123319 prevented the angiotensin II-induced hyperpolarization in angiotensin II type 1a receptor knockout mice, and, rather, it induced depolarization. These results suggest that the angiotensin II type 2 receptor in rostral ventrolateral medulla plays an antagonistic role against the angiotensin II type 1a receptor in controlling the neuronal activity of rostral ventrolateral medulla.

Angiotensin II↗
PubMed2005

Adenoviral-mediated neuron specific transduction of angiotensin II type 2 receptors.

The angiotensin II (Ang II) type 2 receptor (AT2R) is localized at specific nuclei within adult rat brain. However, a lack of specific approaches for manipulating the activity of neuronal AT2R has meant that the physiological actions of these sites in the brain remain to be established. Therefore, in this study, our aim was to develop a method by which AT2R can be specifically overexpressed in neurons and in rat brain, with the ultimate goal of a producing a system where discrete increases in AT2R levels in brain nuclei could reveal (and be linked to) physiological actions. Here, we have constructed an AT2R recombinant adenoviral vector, Ad5-SYN-AT2R-IRES-EGFP, which contains the AT2R gene and an IRES-linked EGFP reporter gene, both driven by the neuron-specific synapsin I (SYN) gene promoter. This vector efficiently transduces the AT2R into neuronal cells in culture and results in the expression of high levels of AT2R. These expressed receptors are functional in terms of inhibition of Erk mitogen activated protein kinases (Erk MAPK) and stimulation of neuronal K+ current. Furthermore, microinjection of this vector into adult rat brain elicits a long lasting ( approximately 1 month) expression of AT2R within neurons. In summary, we have developed a viral vector that can be used for the efficient transduction of AT2R into neurons both in vitro and in vivo, the use of which may help to define the physiological functions of brain AT2R in adult rats.

Adenoviridae↗
PubMed2003

Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor.

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.

Amino Acid Sequence↗
PubMed2003

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver.

CYP3A is responsible for approximately 50% of the therapeutic drug-metabolizing activity in the liver. The present study was undertaken to establish the CYP3A4 inducible model for analysis of human drug metabolism using a bioartificial liver composed of the functional hepatocellular carcinoma cell (HCC) line FLC-5. A radial-flow bioreactor (RFB), which is a carrier-filled type bioreactor, was used for 3-dimensional perfusion culture of FLC-5 cells. The CYP3A4 messenger RNA (mRNA) expression level 48 hours after rifampicin treatment in the RBF was approximately 100 times higher than that in a monolayer culture. Western blot analysis also demonstrated an increase in expression of the CYP3A protein. When testosterone, a substrate for CYP3A4, was added to the rifampicin-treated cell culture, 6 beta-hydroxy testosterone as a metabolite was formed. Electrophoretic mobility shift assay (EMSA) with a CYP3A4 ER6 probe demonstrated that relatively high molecular weight complex containing pregnane X receptor (PXR)/retinoid X receptor alpha(RXR alpha), compared with that in the monolayer culture, is possibly generated in the RFB culture of FLC-5 treated with rifampicin. Similarly, the assay with a probe of HNF-4 alpha-binding motif indicated the formation of a large protein complex in the RFB culture. Because it is known that PXR transactivates CYP3A4 gene via its response element and expression of PXR is regulated by HNF-4 alpha, the large complexes binding to response elements of PXR or HNF-4 alpha in the RFB culture may contribute to up-regulation of CYP3A4 mRNA. In conclusion, the bioartificial liver composed of human functional HCC cell line was useful in studying drug interactions during induction of human CYP3A4.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2002

Potentiated sympathetic nervous and renin-angiotensin systems reduce nonlinear correlation between sympathetic activity and blood pressure in conscious spontaneously hypertensive rats.

BACKGROUND: Patients with a reduced nonlinear component of heart rate regulation have a poorer outcome. METHODS AND RESULTS: We investigated whether a nonlinear correlation between renal sympathetic nerve activity (RSNA) and blood pressure or renal blood flow is reduced in conscious, spontaneously hypertensive rats (SHR) by comparing them with normotensive Wistar-Kyoto rats (WKY). We also determined the linearity and nonlinearity of the correlation in SHR who were given an angiotensin II receptor blocker, candesartan, orally for 2 weeks. The RSNA value was higher in SHR than in WKY, and coherence peaks of transfer function were found at 0.05 and 0.80 Hz (ie, below respiratory- and cardiac-related fluctuations). The coherence (linearity) of the transfer function was significantly higher and gain was smaller in SHR than in WKY. Because mutual information values (linear and nonlinear correlation) were similar in both strains, we found the nonlinear correlation to be lower in SHR than in WKY. Time delay values calculated by the mutual information method demonstrated that RSNA preceded blood pressure and renal blood flow by 0.5 to 1.0 s. In SHR given candesartan, the RSNA value was lower, and the linearity was lower and nonlinearity higher than SHR given vehicle. CONCLUSIONS: Linear correlation between RSNA and blood pressure or renal blood flow was higher in SHR than in WKY, whereas the nonlinear correlation was lower. Oral treatment with candesartan increased the nonlinearity and reduced the linearity in SHR. Increased RSNA and the renin-angiotensin system may be responsible for the lower nonlinearity and higher linearity in hypertension.

Administration, Oral↗
PubMed2002

Ultrastructure of human pheochromocytoma cells cultured for long periods.

We conducted ultrastructural analysis of human pheochromocytoma (PC) cells maintained in primary culture for about 10 months. The cells were first isolated by the enzymatic treatment of a surgically resected tissue specimen obtained from a 37-year-old man with PC, a condition which is characterized by elevated blood levels of adrenaline and noradrenaline. It was found that noradrenaline production in the medium continued until the 90th day of culture (1330 pg/ml). The production level decreased to 20 pg/ml on the 180th day, and to 18 pg/ml on the 300th day. Examination under a transmission electron microscope (TEM) at 4 weeks of culture revealed electron-dense granules (about 200 nm in size and, presumably, rich in catecholamines), which were also observed in the tumor cells from the original PC tissue. Neurite-like processes grew at around 1 week of culture, and were still maintained at 6 months of culture. But, after 6 months of culture, the neurite-like processes contained a rosary-like elevated structure, which was suggestive of cell degeneration, as determined by a plasma polymerization replica method and observed with a scanning electron microscope. When cells were examined under the TEM, fewer electron-dense granules were observed in the cell bodies, with more numerous lipofuscin-like granules and filaments. Thus, electron-dense granules, which, presumably, contain catecholamines, were seen in a long-term culture of human PC cells. These granules decreased in number in parallel with the decrease in catecholamine levels in the culture.

Adrenal Gland Neoplasms↗
PubMed2002

Rostral ventrolateral medulla neurons of neonatal Wistar-Kyoto and spontaneously hypertensive rats.

We compared the electrophysiological properties of neurons in the rostral ventrolateral medulla (RVLM) of neonatal Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), and responses to angiotensin II and its type 1 receptor antagonist candesartan. Using the whole-cell patch-clamp technique, we examined the characteristics of RVLM neurons in brainstem-spinal cord preparations with a preserved sympathetic neuronal network. The baseline membrane potential of irregularly firing neurons was less negative (-50.1+/-0.6 versus -52.0+/-0.6 mV) and the firing rate was faster (3.0+/-0.2 versus 2.0+/-0.2 Hz) in SHR (n=56) than in WKY (n=38). Superfusion with angiotensin II (6 micromol/L) significantly depolarized the RVLM bulbospinal neurons in SHR (5.4+/-1.1 mV, n=15) but not in WKY. In contrast, candesartan (0.12 micromol/L) induced a significant membrane hyperpolarization (-3.7+/-0.4 mV; n=14) and a decrease in the firing rate in RVLM bulbospinal neurons of SHR but not of WKY. These results suggest that endogenously generated angiotensin II binds to type 1 receptors on RVLM bulbospinal neurons, thus tonically contributing to a higher membrane potential and a faster firing rate in SHR. The electrophysiological properties of RVLM neurons and their responses to angiotensin II and candesartan differ between neonatal WKY and SHR. These differences in RVLM neurons suggest a mechanism that possibly leads to elevation in blood pressure.

Action Potentials↗
PubMed2002

[Comparison of ARB and ACEI for renoprotection in chronic glomerulonephritis].

This 1.5-year prospective study compared the effects of angiotensin II receptor blocker, candesartan(n = 21), and ACE inhibitor(ACEI, n = 23) on proteinuria and renal function in patients with moderate renal impairment due to chronic glomerulonephritis. Blood pressure reductions were comparable between both groups. Proteinuria was significantly reduced from 2.1 +/- 0.4 (mean +/- SE) to 0.6 +/- 0.2 g/day(p < 0.001) with candesartan, and the reduction was significantly larger than with ACEI(p < 0.01). Serum creatinine did not increase with candesartan or ACEI, suggesting the renoprotective effect. Serum potassium increased significantly with both drugs from 6 months. Plasma aldosterone concentrations(PAC) showed a stronger suppression with candesartan(150 pg/ml before treatment and 51 at 1.5 years) than with ACEI(104 pg/ml at 1.5 years). Plasma renin activity with candesartan(1.0 ng/ml/hr before treatment) decreased from 3.9 ng/ml/hr at 6 months to 1.3 at 1.5 years, whereas PRA remained elevated with ACEI. We speculate that the stronger reduction of PAC contributed to suppression of growth of mesangial matrix and the interstitial fibrosis in the candesartan group.

Angiotensin Receptor Antagonists↗