Comparison of the expression of cell surface poly-N-acetyllactosamine-type oligosaccharides in PC12 cells with those in its variant PC12D.

To explore the biological role of carbohydrate chains in the process of nerve cell differentiation, we carried out a characterization of the carbohydrate structure of glycoproteins by comparing conventional PC12 cells with variant cells (PC12D). In vitro metabolic labeling of cells with either [(3)H] glucosamine or [(3)H] threonine, together with tomato lectin staining, revealed that nerve growth factor (NGF) stimulation caused a decrease in the poly-N-acetyllactosamine synthesis of high-molecular-weight glycopeptides from PC12 cells. By comparison, the amount of glycopeptides with poly-N-acetyllactosamine from PC12D cells was already significantly low and it was not changed by NGF stimulation. By assaying the glycosyltransferases that participate in poly-N-acetyllactosamine synthesis, the decrease in the amount of the poly-N-acetyllactosamine in PC12D cells as well as NGF-stimulated PC12 cells could be accounted for by a reduction in the activity of poly-N-acetyllactosamine extension enzyme (GnT-i), because the amount of poly-N-acetyllactosamine in both cells precisely correlated with changes in GnT-i activity, whereas the activities of N-acetylglucosaminyltransferase V (GnT-V) and beta 1-4 galactosyltransferase remained unchanged. These results demonstrate that the decrease in poly-N-acetyllactosamine synthesis in PC12 cells occurred prior to neurite formation, whereas PC12D cells were insensitive to this effect. Next, we showed that GnT-i but not GnT-V catalyzed a rate-limiting reaction in the expression of poly-N-acetyllactosamine chains, especially in pheochromocytoma.     

Hyperplastic cellular components of a hemangiopericytoma. An ultrastructural study

Based on ultrastructural features of cellular components of a hemangiopericytoma, hyperplastic cells are classifiable into fibroblast-like (group I), endotheloid (group II) and pericyte-like (group III) cells. The transformation of the group I cells to the group II, or to the group III cells, is pronounced in our electron micrographs and this may imply that the group I cell is the principal cell of origin in this neoplasm. The smooth muscle-like (group IV) cells comprising the media of the arteries and veins in this neoplasm may represent modified, possibly de-differentiated smooth muscle cells reacted to the neoplastic proliferation of the surrounding adventitial (group I) cells.     

Regulation of Drosophila myosin ATPase activity by phosphorylation of myosin light chains--II. Flightless mfd- fly

1. The Ca2(+)-activated and Mg2+ actin-activated myosin ATPase activities of flightless mfd- mutant Drosophila flight muscle myosin were one-half and one-third of those of the wild-type fly muscle myosin, respectively. 2. In the two-dimensional gel electrophoresis, the spots corresponding to phosphorylated myosin light chains, Lfl and Ltl, were hardly detected in mfd- mutant myosin. 3. These results support not only the conclusion that phosphorylation of myosin light chains regulates Drosophila myosin ATPase activity but also the assumption that the phosphorylation of myosin light chains is directly involved in flight function of the Drosophila fly.     

Physarum myosin light chain interacts with actin in a Ca2+-dependent manner

Actin-modulating activity was analysed with the 16,131-dalton calcium-binding light chain (CaLc, Kobayashi et al. (1988) J. Biol. Chem. 263, 305-313) of Physarum myosin, which is under an inhibitory Ca-control (Kohama and Kendrick-Jones (1986) J. Biochem. 99, 1433-1446). When skeletal muscle actin was polymerized in the presence of CaLc and Ca2+, increases in both viscosity and birefringence were reduced under high shear conditions. However, CaLc did not inhibit actin polymerization under no or low shearing forces, which was demonstrated by a variety of methods including fluorescence intensity measurements using pyrenyl actin. We propose that actin polymerized in the presence of CaLc and Ca2+ is easily fragmented under high shearing forces to produce the changes in viscosity and birefringence.     

Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C

The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.     

Isolation and characterization of cDNA for chicken muscle adenylate kinase

A cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly(A)+ RNA, and the DNA sequence was determined. The cDNA insert had 854 nucleotides, which consisted of the 5'-untranslated sequence of 57 nucleotides, the sequence of 582 nucleotides coding for 194 amino acids, and the 3'-untranslated sequence of 163 nucleotides and the poly(A) tail of 52 nucleotides. The amino acid sequence predicted from the nucleotide sequence was highly homologous with the reported sequences of human, calf, porcine, and rabbit muscle adenylate kinases. RNA blot analysis of poly(A)+ RNA from various chicken tissues revealed a single species of mRNA of approximately 850 nucleotides and its tissue-specific distribution. The induction of muscle adenylate kinase mRNA synthesis during the chick embryogenesis was also demonstrated by the blot analysis. Southern blot analysis indicated a single gene for muscle adenylate kinase in the chicken genome.     

Distinctions between the multiple cationic forms of rat liver glutathione S-transferase

Three cationic glutathione S-transferase forms isolated from rat liver were characterized as dimers that originated from different combinations of two subunit types, Ya and Yc. The cationic forms were purified using lysyl glutathione affinity matrices and were chromatographically resolved from anionic glutathione S-transferases that contain Yb subunits. The three classes of cationic transferase exhibited similar specific activities with 1-chloro-2,4-dinitrobenzene as a substrate, all forms cross-reacted with antibodies to glutathione S-transferase B, and all had comparable secondary structures and tryptophan fluorescence properties. In spite of those similarities, the Yc-containing forms were clearly distinguishable from Ya forms on the basis of characteristic differences in circular dichroic patterns associated with their aromatic side chains. All cationic transferases bound bilirubin with stoichiometric ratios of 1 mol/dimeric protein molecule, but discrete differences in mode of binding were ascribed to forms containing Ya subunits as compared to Yc dimers. Binding to Yc forms was of lower affinity and may be associated with the catalytic region of the protein since glutathione effectively displaced bilirubin from the Yc component.