No abstract

Abstract. The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads beating GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca 2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39407 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca2+-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.T ROPOMYOSINS (TMs) ~ are ubiquitous actin-binding proteins found in muscle and nonmuscle cells (25,38,41,44,57). Nonmuscle cells express multiple TM isoforms with a broad range of molecular weight. TMs isolated from rat fibroblasts can be grouped into high (termed TM1, TM2, and TM3) and low molecular weight TM isoforms (termed TM4 and TM5) (42). These nonmuscle TM isoforms are associated with actin in microfilaments. Although their functions in microfilaments are poorly understood, they are proposed to play a regulatory role in defining actin filament assembly and organization during cell motility, cell division, changes in cell shape, and differentiation. This is based on several observations. TM inhibits the actions of vilAddress all correspondence to K. Takenaga, Division of Chemotherapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuoh-ku, Chiba 260 Japan.1. Abbreviations used in this paper: MAP, microtubule-associated protein; TM, tropomyosin. lin (9), gelsolin (15), and actin-depolymerizing proteins (6) toward actin filaments. In transformed and more malignant cells, the synthesis of at least one of the high molecular weight TMs is reduced (7,10,25,26,37,38,41,54,55), which is thought to be responsible, in part, for the disorg...

The internalization of human IgG in human intestine might be, at least in part, due to FcRn-mediated endocytosis, which could occur by a process other than clathrin- and caveolin-dependent mechanisms.

We report the first successful example of a highly enantioselective fluorolactonization with an electrophilic fluorinating reagent, Selectfluor(®), in the presence of a novel bifunctional organocatalyst. The catalyst design includes a carboxylate anion functioning as a phase-transfer agent and a benzyl alcohol unit to capture the substrate through hydrogen bonding. Fluorinated isobenzofuranones were obtained in good yields with up to 94% ee (97:3 er). On the basis of mechanistic studies, we propose a unique reaction mechanism with potential for further applications.

No abstract