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Kaori Murakami

Publications and source records attributed to Kaori Murakami.

8 recordsLinked to original sources
PubMed2006

A new strategy of high-speed screening and quantitative structure-activity relationship analysis to evaluate human ATP-binding cassette transporter ABCG2-drug interactions.

The human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR1/ABCP) plays a critical role in cellular protection against xenobiotics as well as pharmacokinetics of drugs in our body. In the present study, we aimed to analyze the quantitative structure-activity relationship (QSAR) latently residing in ABCG2-drug interactions. We first established standard methods for expression of human ABCG2 in insect cells, quality control of plasma membrane samples by using electron microscopy techniques, and high-speed screening of ABCG2 inhibition with test compounds. Plasma membrane vesicles prepared from ABCG2-expressing Sf9 cells were used as a model system to measure the ATP-dependent transport of [3H]methotrexate (MTX). Forty-nine different therapeutic drugs and natural compounds were tested for their ability to inhibit ABCG2-mediated MTX transport. Based on their inhibition profiles, we performed QSAR analysis using chemical fragmentation codes deduced from the structures of test compounds. Multiple linear regression analysis delineated a relationship between the structural components and the extent of ABCG2 inhibition, allowing us to identify one set of structure-specific chemical fragmentation codes that are closely correlated with the inhibition of ABCG2 transport activity. Based on the QSAR analysis data, we predicted the potency of gefitinib to inhibit ABCG2. The validity of our QSAR-based prediction for gefitinib was examined by actual experiments. Our kinetic analysis experiments suggest that the ABCG2-ATP complex binds gefitinib. The present study provides a new strategy for analyzing ABCG2-drug interactions. This strategy is considered to be practical and useful for the molecular designing of new ABCG2 modulators.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Proteomic identification of differentially-expressed genes in human gastric carcinomas.

Although genetic alterations in proto-oncogenes, tumor-suppressor genes, cell cycle regulators, and cell growth factors have been implicated in the process of human gastric carcinogenesis, the principle carcinogenic mechanisms are not fully understood. In this study, we used a proteomic approach to search for genes that may be involved in gastric carcinogenesis and that might serve as diagnostic markers. We identified nine proteins with increased expression and 13 proteins with decreased expression in gastric carcinomas. The two most notable groups included proteins involved in mitotic checkpoint (MAD1L1 and EB1) and mitochondrial functions (CLPP, COX5A, and ECH1). This suggested that there are links between dysfunctions in these processes and gastric carcinogenesis. We also observed the differential expression of HSP27 and CYR61 proteins in gastric carcinoma, whose expression is known to be altered in other types of tumors. Furthermore, the study identified proteins whose function in gastric carcinomas was previously unsuspected and that may serve as new molecular markers for gastric carcinomas. Importantly, immunohistochemical analyses confirmed that the levels of expression of MAD1L1, HSP27, and CYR61 were altered in gastric carcinoma tissues. Therefore, our study suggested not only that the proteins identified in this study can be useful diagnostic markers but also that a proteomics-based approach is useful for developing a more complete picture of the pathogenesis and function of gastric carcinomas.

Blotting, Western↗
PubMed2004

Different types of glutathionylation of hemoglobin can exist in intact erythrocytes.

Glutathionylation of hemoglobin (Hb) was studied by incubation of intact human erythrocytes with 1 mM tert-butylhydroperoxide (tBHP). Electrophoresis of the membranes showed a time dependent increase of membrane-bound Hb alpha chain until 10 min, and immunoblotting study showed that membrane-bound Hb alpha chain reacted with anti-glutathione antibody only after 10 min. Concomitant with the Hb alpha chain, membrane associated actin, spectrin, and glyceraldehyde 3-phosphate dehydrogenase reacted with the antibody. Cytosolic Hb of the control erythrocytes reacted with anti-glutathione antibody. Together with our previous paper, the present study indicates that at least three different types of glutathionylation of Hb can exist in erythrocytes. The first type is a mixed disulfide bond between reduced glutathione (GSH) and normal Hb. The second type is a disulfide bond between the cysteine 93 of metHb beta chain and oxidized glutathione (GSSG), and the third type is a disulfide bond between the other cysteine residues of metHb alpha chain and/or metHb beta chain and GSSG.

Actins↗
PubMed2004

Absence of correlation between glycated hemoglobin and lipid composition of erythrocyte membrane in type 2 diabetic patients.

Correlation of glycated hemoglobin (HbA(1c)) level with degrees of certain peroxidative changes in erythrocyte membrane lipids in diabetic patients have been reported. In the present study, peroxidation of erythrocyte lipids was assessed by changes in tocopherols (Toc), phospholipids (PL), and malondialdehyde (MDA). Membrane cholesterol, Toc, and PL were determined from the same lipid extract. Toc and cholesterol were measured simultaneously by high-performance liquid chromatography (HPLC), and each PL class was determined by a single HPLC elution with ultraviolet light (UV) detection. The detection of PL with UV depends primarily on double bonds in fatty acids and shows a decrease in fatty acids by peroxidation. Changes in Toc and each PL were calculated on the basis of cholesterol and SM, respectively, since cholesterol and sphingomyelin (SM) in the cell membrane are not prone to peroxidation. MDA was measured by an HPLC method with fluorescence detection. These methods for assessment for peroxidation of membrane lipids in intact erythrocytes were validated by experiments with 2, 2-azobis(2-amidinopropane)dihydrochloride (AAPH) and tert-butylhydroperoxide (tBHP); nevertheless, significant differences in the levels of Toc, each PL class, and MDA between a high-HbA(1c) group and a low-HbA(1c) group were not detected.

Adult↗
PubMed2003

Oxidation of hemoglobin to methemoglobin in intact erythrocyte by a hydroperoxide induces formation of glutathionyl hemoglobin and binding of alpha-hemoglobin to membrane.

Biochemical consequences of oxidation of hemoglobin (Hb) in intact human erythrocytes were studied. The incubation of washed erythrocyte with 1mM tert-butylhydroperoxide induced an increase in glutathionyl-Hb (G-Hb). The formation of G-Hb occurred linearly until 10 min in parallel with the formation of methemoglobin (metHb) after exhaustion of reduced glutathione. The results show that metHb, but not normal Hb, reacts with oxidized glutathione to form G-Hb. G-Hb was confirmed by immunoblotting with anti-glutathione antibody and the formation of G-Hb was accompanied by parallel decrease in beta-globin detected with a reversed phase HPLC. Electrophoretic studies showed time-dependent increase in membrane-associated alpha-Hb until 10 min, indicating that a part of unpaired alpha-Hb bound to the membrane. Pre-beta-globin increased despite the decrease in beta-globin and a part of the increase was independent of the decrease in beta-globin. Pre-beta-globin reacted with anti-glutathione antibody, but it differs from G-Hb in many features.

Cells, Cultured↗
PubMed2003

High-cholesterol diets induce changes in lipid composition of rat erythrocyte membrane including decrease in cholesterol, increase in alpha-tocopherol and changes in fatty acids of phospholipids.

Effects of high dietary cholesterol on erythrocyte membrane lipids were studied. Feeding rats with a diet containing 0.5% cholesterol and 0.15% sodium cholate for two weeks induced changes in erythrocyte membrane lipids including a decrease in cholesterol, an increase in alpha-tocopherol (alpha-Toc) and changes in the fatty acid composition of phospholipids. Oleic acid and linoleic acid increased, while arachidonic acid decreased in phosphatidylcholine. Saturated fatty acids decreased and unsaturated fatty acids increased in phosphatidylethanolamine. Almost the same changes in membrane lipids were also noted after six weeks of feeding rats with the diet. A diet containing 0.5% cholesterol but without sodium cholate caused a decrease in erythrocyte cholesterol and an increase in erythrocyte alpha-Toc after two weeks of feeding, as compared to the basal diet, indicating that high dietary cholesterol, but not sodium cholate, was responsible for these changes in the erythrocyte membrane.

Animals↗
PubMed2003

Enhancement of cisplatin-induced cytotoxicity by ROCK inhibitor through suppression of focal adhesion kinase-independent mechanism in lung carcinoma cells.

Intracellular signaling through Rho-associated coiled-coil forming kinase (ROCK) is a target of antimetastatic therapy and is proposed to be involved in carcinogenesis. Focal adhesion kinase (FAK) functions downstream of ROCK and participates in anti-apoptotic signaling. We hypothesized that a specific ROCK inhibitor, Y-27632, may exert a pro-apoptotic effect in combination with anticancer agents through the suppression of FAK. A549 lung carcinoma cells were treated with Y-27632 and cisplatin. The simultaneous combination did not exert any additional effect, whereas sequential treatment, in which cisplatin followed Y-27632, enhanced cytotoxicity in concentration- and time-dependent manner. Y-27632 did not suppress tyrosine phosphorylation of FAK in A549-FAK, the active form of FAK expressing A549 cells, as observed in parental cells. Nevertheless, the pretreatment of A549-FAK cells with Y-27632 still enhanced cisplatin-induced cytotoxicity. It was concluded that the ROCK inhibitor enhances cisplatin-induced cytotoxicity through FAK suppression-independent mechanism(s). These observations raise the possibility that the inhibition of the ROCK-mediated signal enhances the effect of anti-cancer agents in addition to its antimetastatic property.

Amides↗