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Articles by N Takahashi
Total Records ( 7 ) for N Takahashi
  M Miura , N Takahashi , M Nara , N Fujishima , H Kagaya , Y Kameoka , H Saitoh , H Tagawa and K. Sawada
  Background

A steady-state trough plasma itraconazole concentration greater than 500 ng/mL is a therapeutic target for itraconazole. A simple, rapid and sensitive high-performance liquid chromatography-based method was developed for quantitation of itraconazole and hydroxyitraconazole in human plasma.

Methods

Itraconazole and hydroxyitraconazole were separated using a mobile phase of 0.5% KH2PO4 (pH 6.0)-acetonitrile (30:70, v/v) on a CAPCELLPAK C18 MGII column at a flow rate of 0.5 mL/min and ultraviolet absorbance at 260 nm.

Results

The analysis required 200 µL of plasma and involved a rapid, simple solid-phase extraction with an Oasis HLB cartridge, which resulted in recoveries of 87–92% for itraconazole and 91–94% for hydroxyitraconazole. The lower limit of quantification for itraconazole and hydroxyitraconazole was 5 ng/mL each. Intra- and interday coefficients of variation for itraconazole and hydroxyitraconazole were less than 11.3% and 12.2%, respectively, and accuracies were within 11.7% and 4.5% over the linear range, respectively. Although the steady-state plasma concentrations of itraconazole and hydroxyitraconazole ranged from 506 to 2482 ng/mL and from 766 to 2444 ng/mL, respectively, after a two-day loading dose of 400 mg/day intravenous itraconazole followed by the administration of 200 mg/day itraconazole oral solution, calibration curves of itraconazole and hydroxyitraconazole showed positive linearity in a concentration range of 5–2500 and 50–2500 ng/mL, respectively.

Conclusions

Our results indicate that this method is applicable for the monitoring of plasma levels of itraconazole and hydroxyitraconazole in a clinical setting. Furthermore, the regimen presented here might also be effective in preventing infection, but further studies with large sample sizes are necessary to investigate this avenue.

  Y Niimura , T Moue , N Takahashi and K. i. Nagai
 

Heat stress on Madin–Darby canine kidney cells increased ceramide content to 187% at 40°C for 24 h, and the de novo synthesis from serine increased to 146%. Glucosylceramide (GlcCer) and galactosylceramide (GalCer) synthesis from ceramide, the first glycosylation step of sphingolipid metabolism in kidney cells, increased to 290% (GalCer) and 143% (GlcCer) after metabolic labeling with 14C-glucose at 42°C for 20 h. The more complex glycolipid lactosylceramide also increased to 151%, whereas sulfatide and ganglioside GM3 decreased to 21% and 43%, respectively. Sulfatide (SM4s) showed optimal sulfation at 37°C, whereas cholesterol sulfate was optimally sulfated at 40°C. The gene expression of ceramide glucosyltransferase (GluT), ceramide galactosyltransferase, and GalCer sulfotransferase (GST) after 24 h culture at 42°C significantly increased to 714%, 221%, and 174%, respectively. Another kidney cell line, COS7, showed less activation of these transferases by heat stress. Although GST gene expression was higher under heat stress, SM4s synthesis decreased, which may have been due to increased GST sensitivity to a temperature higher than 37°C. When we introduced the HSP70 gene into the expression vector and transfected the plasmid (pCDM-dHSP70) into kidney cells, GlcCer synthesis increased significantly. From these results, we speculated that HSP70 may play a role in GluT gene expression to increase GlcCer and decrease intracellular ceramide level.

  A Soeda , Y Morita Hoshi , H Makiyama , C Morizane , H Ueno , M Ikeda , T Okusaka , S Yamagata , N Takahashi , I Hyodo , Y Takaue and Y. Heike
  Objective

Chemotherapy and immunotherapy often seem to contradict each other. However, recent reports suggested that the anticancer effects in some chemotherapeutic agents were concerned with immune response. This study was designed to evaluate the immunological reaction by gemcitabine for future clinical trial of combination therapy with gemcitabine and cancer vaccines.

Methods

We evaluated several immunological parameters in patients with advanced pancreatic cancer who received a conventional dose of gemcitabine for 2 months. Twenty-eight patients with metastasis or locally advanced tumor, including 18 gemcitabine-naïve and 10 with a history of preceding gemcitabine treatment, were enrolled in this study. The patients received gemcitabine 1000 mg/m2 for 3 weeks, followed by 1 week of rest. We monitored the kinetics of lymphocytes, natural killer cells, monocytes, dendritic cells (DC), human leukocyte antigen (HLA)-multimer conjugated with CMV or WT1 peptide, and intracellular cytokine production of interferon- and interleukin-4 by flow cytometry. The T cell receptor (TCR) repertoire was also analyzed.

Results

The absolute number and percentage of CD14+ monocytes and CD11c+ (myeloid) DC increased with gemcitabine treatment (P = 0.033 and P = 0.021). The percentage of CD123+ (plasmacytoid) DC also increased (P = 0.034), whereas no significant change was observed in other immune parameters, including multimer, intracellular cytokine production and TCR repertoire.

Conclusions

Our finding that gemcitabine treatment induced the proliferation of CD14+ monocytes and CD11c+ DC could support combination therapy with gemcitabine and specific immunotherapy such as peptide vaccination against pancreatic cancers.

  H Kimura , X Li , K Torii , T Okada , K Kamiyama , D Mikami , N Takahashi and H. Yoshida
 

Background. Long-term treatment with glucocorticoids (GCs) reportedly exaggerates renal fibrosis in chronic progressive inflammatory kidney disease. GCs induce the gene expression of plasminogen activator inhibitor-1 (PAI-1), a fibrosis enhancer in non-renal cells. Tumour necrosis factor-alpha (TNF-) reduces the gene expression of 11β-hydroxysteroid dehydrogenase (HSD) 2, an inactivator of GCs, and may enhance GC activity. However, the individual and collective effects of adrenal steroids, TNF- and HSD2 status on PAI-1 production are unknown in human proximal renal tubular epithelial cells (HPTECs).

Methods. Confluent HPTECs were treated with adrenal steroids (10 nM to 10 µM) or TNF- (10 ng/ml) for up to 48 h. The mRNA amounts of the target genes were determined by TaqMan quantitative PCR, and the PAI-1 protein amounts were measured by an immunoassay.

Results. Dexamethasone (DXA) maximally increased the amounts of PAI-1 mRNA and protein at 100 nM. Aldosterone (Ald) increased PAI-1 expression dose dependently, but the effect was over 100-fold weaker than that of DXA. The PAI-1-increasing effects of DXA and Ald were abolished completely by U-486, a specific inhibitor of the glucocorticoid receptor (GR) but not by spironolactone, a specific inhibitor of the mineralocorticoid receptor. The effect of DXA was also blocked partially by AG1478 and herbimycin A, tyrosine kinase inhibitors. DXA further increased TNF--stimulated PAI-1 expression via the GR. Although TNF- treatment caused an 80% reduction in the gene expression of HSD2, an inactivator of GCs, HSD2 inhibition did not enhance DXA-induced PAI-1 production.

Conclusions. DXA induces basal and TNF--stimulated PAI-1 expression via the GR pathway, regardless of HSD2 status in HPTECs. Excess GCs may serve as a pro-fibrotic factor in chronic inflammatory kidney diseases.

  A. H Toychiev , R. Z Sabirov , N Takahashi , Y Ando Akatsuka , H Liu , T Shintani , M Noda and Y. Okada
 

The maxi-anion channel with a large single-channel conductance of >300 pS, and unknown molecular identity, is functionally expressed in a large variety of cell types. The channel is activated by a number of experimental maneuvers such as exposing cells to hypotonic or ischemic stress. The most effective and consistent method of activating it is patch membrane excision. However, the activation mechanism of the maxi-anion channel remains poorly understood at present. In the present study, involvement of phosphorylation/dephosphorylation in excision-induced activation was examined. In mouse mammary fibroblastic C127 cells, activity of the channel was suppressed by intracellular application of Mg-ATP, but not Mg-5'-adenylylimidodiphosphate (AMP-PNP), in a concentration-dependent manner. When a cocktail of broad-spectrum tyrosine phosphatase inhibitors was applied, channel activation was completely abolished, whereas inhibitors of serine/threonine protein phosphatases had no effect. On the other hand, protein tyrosine kinase inhibitors brought the channel out of an inactivated state. In mouse adult skin fibroblasts (MAFs) in primary culture, similar maxi-anion channels were found to be activated on membrane excision, in a manner sensitive to tyrosine phosphatase inhibitors. In MAFs isolated from animals deficient in receptor protein tyrosine phosphatase (RPTP), activation of the maxi-anion channel was significantly slower and less prominent compared with that observed in wild-type MAFs; however, channel activation was restored by transfection of the RPTP gene. Thus it is concluded that activation of the maxi-anion channel involves protein dephosphorylation mediated by protein tyrosine phosphatases that include RPTP in mouse fibroblasts, but not in C127 cells.

  H Inoue , N Takahashi , Y Okada and M. Konishi
 

The volume-sensitive outwardly rectifying (VSOR) chloride channel is ubiquitously expressed and involved in cell volume regulation after osmotic swelling, called regulatory volume decrease (RVD), in various cell types. In adipocytes, the expression of the VSOR channel has not been explored to date. Here, by employing the whole-cell patch-clamp technique, we examined whether or not the VSOR channel is expressed in white adipocytes freshly isolated from epididymal fat pads of normal (C57BL/6 or KK) and diabetic (KKAy) mice. Whole cell voltage-clamp recordings revealed that Cl currents were gradually activated upon cell swelling induced by application of a hypotonic solution, both in normal and diabetic adipocytes. Although both the mean cell size (or cell capacitance) and the current magnitude in KKAy adipocytes were larger than those in C57BL/6 cells, the current density was significantly lower in KKAy adipocytes (23.32 ± 1.94 pA in C57BL/6 adipocytes vs. 13.04 ± 2.41 pA in KKAy adipocytes at +100 mV). Similarly, the current density in diabetic KKAy adipocytes was lower than that in adipocytes from KK mice (a parental strain of KKAy mice), which do not present diabetes until an older age. The current was inhibited by Cl channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and glibenclamide, or hypertonic solution, and showed outward rectification and inactivation kinetics at large positive potentials. These electrophysiological and pharmacological properties are consistent with those of the VSOR channel in other cell types. Moreover, adipocytes showed RVD, which was inhibited by NPPB. In KKAy adipocytes, RVD was significantly slower (; 8.42 min in C57BL/6 adipocytes vs. 11.97 min in KKAy adipocytes) and incomplete during the recording period (25 min). It is concluded that the VSOR channel is functionally expressed and involved in volume regulation in white adipocytes. RVD is largely impaired in adipocytes from diabetic mice, presumably as a consequence of the lower density of the functional VSOR channel in the plasma membrane.

  M Kawahara , S Morita , N Takahashi and T. Kono
 

Parental genome functions in ontogeny are determined by interactions among transcripts from the maternal and paternal genomes, which contain many genes whose expression is strictly dependent on their parental origin as a result of genomic imprinting. Comprehensive recognition of the interactions between parental genomes is important for understanding genomic imprinting in mammalian development. The placenta is a key organ for exploring the biological significance of genomic imprinting. To decipher the unknown roles of paternally methylated imprinted genes on chromosomes 7 and 12 in mouse placentation, we performed a transcriptomic analysis on placentae in three types of bimaternal conceptuses that contained genomes derived from both non-growing and fully grown oocytes. Furthermore, we used the Ingenuity pathway analysis software to predict key networks and identify functions specific to paternally methylated imprinted genes regulated by the Igf2-H19 imprinting control region and Dlk1-Dio3 imprinting control region. The data suggested that dynamic conversion of the gene expression profile by restoring the expression of paternally methylated imprinted genes resulted in phenotypic improvements in bimaternal placentae. These results provide a framework to further explore the role of epigenetic modifications in paternal genome during mouse placentation.

 
 
 
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