Kiyoto Yachi

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Comparison of bile acid binding to sinusoidal and bile canalicular membranes isolated from rat liver

Simon et al. (J. Clin. Invest., 70 (1982) 401) studied cholate binding to crude liver plasma membrane vesicles and suggested that the binding may represent mainly the binding to the receptor (carrier) on the canalicular membrane. This hypothesis was supported by finding a good correlation between the number of cholate binding sites on liver plasma membrane and the maximal rate of biliary secretion (Tm) for taurocholate. We studied bile acid binding to sinusoidal and canalicular membrane vesicles isolated from rat liver by a rapid filtration technique. Scatchard analysis of the saturation kinetics showed both [3H]cholate and [3H]chenodeoxycholate bind to two classes of binding site on each membrane. However, little difference was observed between the binding to sinusoidal and canalicular membrane vesicles for each bile acid (cholate, Kd1 = 10.4 and 19.8 microM, n1 = 31.0 23.6 pmol/mg protein, Kd2 = 1.32 and 1.73 mM, n2 = 13.1 and 23.4 nmol/mg protein; and chenodeoxycholate, Kd1 = 0.207 and 0.328 microM, n1 = 36.7 and 27.4 pmol/mg protein, Kd2 = 1.16 and 2.26 mM, and n2 = 20.6 and 24.2 nmol/mg protein; numbers show the mean values sinusoidal and canalicular membrane vesicles, respectively). Chenodeoxycholate binding to sinusoidal membrane vesicles was markedly inhibited by cholate but not by Rose bengal, an organic anion dye. These studies indicate that both membranes (sinusoidal and canalicular membrane vesicles) have two kinds of binding site for bile acids, although no clear difference in the binding properties was observed between the two membranes. Consequently, the cholate binding Simon detected may represent the binding not only to canalicular membrane vesicles but also to sinusoidal membrane vesicles.

Screening and biological evaluation of liposomal formulations containing Adriamycin

Abstract Liposomal Adriamycin® (L-ADM) suppressed phagocytic activity for more than 1 week. This would contribute to the variation in the biodistribution and toxicological characteristics of L-ADM after repeat administration. This finding suggests that the interval in repeat administration of L-ADM should be more than 1 week. Blood toxicity of L-ADM was affected by the administration schedule. It is expected that the blood toxicity of Adriamycin® (ADM) would be reduced by liposomal encapsulation in clinical use where administration of antitumor agents is usually repeated at intervals of 2–3 weeks. L-ADM had greater antitumor activity against a liver metastatic model, M5076, than ADM. However, no significant enhancement of the effects of liposomal encapsulation was observed in another model, L5178Y-ML. Based on these findings, the problems in exploratory and preclinical studies of liposomal Adriamycin® (L-ADM) are discussed.

STUDIES ON TECHNIQUES FOR INTRACELLULAR INTRODUCTION OF MACROMOLECULES

Publisher Summary This chapter discusses the use of the silkworm Bombyx mori nuclear polyhedrosis virus (BmNPV) system for the hyperproduction of ts-src, v-myc, v-erbB, and v-sis products as a fusion protein with the viral protein polyhedrin (NP). Fluoresceinisothiocyanate (FITC)-bovine serum albumin (BSA) and FITC-dextran are used as markers, and these markers are introduced into L cells in high frequency with improved liposome techniques. Chimeric oncogenes with the polyhedrin gene of BmNPV can be highly expressed as fused proteins. In case of v-src and v-erbB genes, their products show tyrosine kinase activity even in a fused form. Purification of the products seems to be a little bit difficult as they are insoluble in water. Cleavage of the domain of the oncogene from a fused protein and its purification without loss of biological function remains to be solved. The chapter describes the effort to improve the liposome methods and presents the development of new methods with which constant and high efficiency introduction can be obtained.