Biomedical subjects
Kazuyo Nishihara
Publications and source records attributed to Kazuyo Nishihara.
Altered expression and function of P-glycoprotein in dextran sodium sulfate-induced colitis in mice.
P-glycoprotein (P-gp), the product of the multiple drug resistance (mdr) gene, can actively pump toxic drugs out of cells, but its pathophysiologic role is not yet fully understood. In this study, we examined the expression of P-gp in dextran sodium sulfate (DSS)-induced colitis in mice. Eight-week-old Balb/c female mice were given drinking water containing 7% DSS ad libitum for 7 days. Mice receiving DSS were sacrificed on days 3, 5, and 7 for histopathologic study. Tissue samples were examined by hematoxylin and eosin (HE) staining, and immunostained against mdr, CD4+, CD8+, and B220+. RNA was isolated from the large intestine and the expression of mdr1a was determined by RT-PCR. The function of P-gp was evaluated by rhodamine123 efflux using the everted sac method. The induction of colitis in mice was confirmed by body weight changes, HE staining and immunohistological grading of the large intestine with reference to CD4+, CD8+, and B220+ after 7 days of treatment. Severe inflammation was observed in the large, but not the small, intestine on day 7. The expression of mdr1a in the large intestine was reduced on days 3, 5, and 7. In addition, the P-gp function and the expression of PXR were also reduced in the large intestine of DSS-treated mice on day 3. This reduction was consistent with the immunohistologic observations. The expression of the mdr1a gene was reduced before severe symptoms appeared. These results suggest that P-gp expression may be related to the pathology of colitis.
Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli.
Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18 degrees C), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.
Simultaneous assessment of the in vivo amount of CYP1A2 and CYP3A2 by the PKCYP-test using theophylline in rats.
Recently, we developed a method for assessing in vivo drug metabolism capacity by pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP-test), in which an apparent liver-to-blood free concentration gradient in vivo (qg) is introduced. The qg value can be alternatively defined as the ratio of the in vivo-in vitro clearance by a single CYP isoform. In this study, we examined the application of the PKCYP-test to drugs metabolized by multiple CYP isoforms in a rat model with fluctuating CYP1A2 levels using theophylline as a model drug. In control rats, the estimated qg values for each CYP1A2 and CYP3A2 based on the in vivo hepatic intrinsic clearance, in vitro Michaelis constant (K(m)) and maximal rate of metabolism (V(max)) values for liver slices agreed well. Moreover, the qg value for CYP1A2 determined by the K(m) and V(max) values for recombinant CYP1A2 was compatible with that based on liver slices. These qg values also agreed with that of rats pretreated with 3-methylcholanthrene. The time-course of theophylline concentrations in serum simulated by a physiologically-based pharmacokinetic model incorporating the hepatic clearance determined by the PKCYP-test agreed with the observed values. These results demonstrate that the qg value in the PKCYP-test is applicable to drugs metabolized by multiple CYP isoforms.