Cellular and Vacuolar Fusion of Protoplasts Electrofused Using Platinum Microelectrodes

Protoplasts isolated from cultured cells of Coptis japonicaand Euphorbia millii were electrically fused using platinummicroelectrodes. The process involved two stages, cellular andvacuolar fusion, which are characterized respectively by transientwrinkling of the membrane and the formation of a dark-red precipitate. (Received June 12, 1987; Accepted October 13, 1987)     

A neurochemical study of rat brain maldevelopment induced by MAM treatment at different stages of gestation

The regional levels of several cell marker proteins in the brain and the ability of operant discrimination learning on a multiple fixed ratio (FR), fixed interval (FI) schedule were determined in rats with microencephaly induced by prenatal treatment with methylazoxymethanol (MAM), an antimitotic agent, on the 11 th to 13 th days (Group A) or on the 15 th day (Group B) of gestation. The cell marker proteins were determined with a sensitive enzyme immunoassay. Neuron-specific enolase (NSE; gamma gamma-enolase) had a significantly lowered level in the neocortex anterior in Group A. Non-neuronal enolase (NNE; alpha alpha-enolase) was significantly reduced in the superior colliculus, lateral geniculate body and optic nerve, but increased 1.5 fold in the retina in Group A. S-100b protein, a marker of astroglial cells, showed no significant change. As for the learning performance, the Group B animals showed an elevated behavioral activity and made evident discrimination between the FI and FR schedule. But Group A animals had prolonged FR components requiring responses to light on, and their spontaneous activity counts recorded by Automex showed an inhibition of behavior in light environments. These findings suggest a causative role of some developmental abnormality in the central visual system, indicated by the aberrant cell marker levels, in the disturbed learning ability of the Group A animals.     

Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101

In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of N-glycolyneuraminic acid-containing glycosphingolipids from a Marek's disease lymphoma-derived chicken cell line, MSB1, as tumor-associated heterophile Hanganutziu-Deicher antigens

Heterophile, Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid-containing glycosphingolipids (GSLs) were detected as tumor-associated foreign antigens of a Marek's disease lymphoma-derived cell line, MSB1, by enzyme-immunoassay with chicken antibody against N-glycolylneuraminyl-lactosylceramide (anti-NeuGc-LacCer). At least three species of HD antigen-active GSLs were detected by two-dimensional thin-layer chromatography (TLC) combined with enzyme-immunoassay. The reactivities of the GSLs with anti-NeuGc-LacCer, their behaviors on two-dimensional TLC and the results of an endo-beta-galactosidase digestion study indicated that these three GSLs were NeuGc-LacCer (NeuGc alpha 2-2Gal beta 1-4Glc-Cer), NeuGc-nLcOse4Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and NeuGc-nLcOse6Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer).     

Investigation of actin in Tetrahymena cells. A comparison with skeletal muscle actin by a devised two-dimensional gel electrophoresis method

Total protein constituents of Tetrahymena thermophila strain B1868 III were studied by two-dimensional agarose-polyacrylamide gel electrophoresis to detect actin among the constituents. In the attempts to prepare a whole-cell extract of Tetrahymena, it was found that protease activity in the extract was so high that high molecular components were quickly digested with the endogenous protease into small peptides unless the homogenization and heat-treatment in a sodium dodecylsulfate solution were performed within 5 s. It was eventually found that employment of 8 M guanidine hydrochloride (HCl) in the homogenization of cells perfectly prevented the degradation of protein components, even through a long preparation procedure. A devised two-dimensional agarose-polyacrylamide gel electrophoresis of the guanidine HCl extract gave a 'protein map' on which most proteins were located in their respective positions, including proteins with more than 200,000 mol. wt. Addition of rabbit skeletal muscle actin on the protein map revealed that no protein with isoelectric point and molecular weight identical with those of the actin was contained in the whole Tetrahymena extract, suggesting that Tetrahymena actin may have characteristics far different from those of skeletal muscle actin.     

Structure of chicken 16-kDa beta-galactoside-binding lectin. Complete amino acid sequence, cloning of cDNA, and production of recombinant lectin

The complete primary structure of chicken 16-kDa beta-galactoside-binding lectin (C-16) was determined. It was composed of 134 amino acid residues and has an acetylated NH2 terminus. A cDNA was also cloned, but no signal sequence was found in the initiator region. The initiator methionine remained as the NH2 terminus of the mature lectin. Although C-16 is distinct from chicken 14-kDa beta-galactoside-binding lectin (C-14), it proved to be a member of the vertebrate 14-kDa-type lectin family. Comparison of the primary structures between the vertebrate 14-kDa-type lectins suggests that C-14 and C-16 were produced by gene duplication of an ancestral lectin gene at a time close to the divergence of birds and mammals. Northern and Southern blot analysis indicated that these isolectins are encoded by individual genes which are differently regulated during the development of the embryo. A recombinant C-16 lectin was produced in Escherichia coli. The product was indistinguishable from the authentic C-16 lectin except that the NH2 terminus of the former was found to begin with free methionine.