Biochemical and immunocytochemical characterization of two types of myosins in cultured tobacco bright yellow-2 cells

We have isolated a myosin (referred to as 170-kD myosin) from lily pollen tubes, which consists of 170-kD heavy chain and calmodulin (CaM) light chain and is responsible for cytoplasmic streaming. A 170-kD polypeptide that has similar antigenicity to the 170-kD myosin heavy chain of lily pollen tubes was also present in cultured tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells, and possessed the ability to interact with F-actin in an ATP-dependent manner. In addition to this myosin, we identified biochemically another kind of myosin in BY-2 cells. This myosin consisted of a CaM light chain and a 175-kD heavy chain with antigenicity different from the 170-kD myosin heavy chain. In the present study, we referred to this myosin as 175-kD myosin. This myosin was able to translocate rhodamine-phalloidin (RP)-labeled F-actin at an average velocity of about 9 &mgr;m/s in the motility assay in vitro. In contrast, the sliding velocity of RP-labeled F-actin translocated by fractions containing the 170-kD myosin was 3 to 4 &mgr;m/s. The velocity of cytoplasmic streaming in living BY-2 cells ranged from 2 to 9 &mgr;m/s. The motile activity of 175-kD myosin in vitro was inhibited by Ca(2+) at concentrations higher than 10(-6) M. Immunoblot analyses using an antiserum against the heavy chain of 170- or 175-kD myosin revealed that in tobacco plants, the 175-kD myosin was expressed in leaf, stem, and root, but not in germinating pollen, while 170-kD myosin was present in all of these plant parts and in germinating pollen. These results suggest that the two types of myosins, 170 and 175 kD, presumably participate in cytoplasmic streaming in BY-2 cells and other somatic cells of tobacco plants.

Lack of mutagenicity of a red food colour impurity (1-carboxyl-5,7-dibromo-6-hydroxy-2,3,4-trichloroxanthone) in phloxine B.

To evaluate the mutagenicity of 1-carboxyl-5,7-dibromo-6-hydroxy-2,3,4-trichloroxanthone (HXCA), which is an impurity present in Food Red No. 104 (FR104, Phloxine B, the Japanese counterpart of D&C Red No. 28), HXCA was isolatedfrom FR104 using pH-zone-refining counter-current chromatography and preparative HPLC. A large amount of HXCA was synthesized to perform the Ames test, and its identity was confirmed by comparison of its HPLC retention time, UV-Vis, MS and NMR spectra with those of HXCA isolated from FR104. The results of the Ames test using synthetic HXCA showed that it did not possess mutagenic activity. The results of the mutagenicity test for HXCA will provide useful information for the establishment of an upper limit for HXCA in FR104 (or D&C Red No. 28) for use in regulatory considerations.