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Insulin-like growth factor (IGF-I) is known to synergistically stimulate the proliferation of hematopoietic cells in combination with other hematopoietic growth factors. However, the precise mechanism underlying the cooperative effects of IGF-I is unknown. In a human interleukin-3 or erythropoietin (EPO)-dependent cell line, F-36P, IGF-I alone failed to stimulate DNA synthesis but did augment the EPO-dependent DNA synthesis of F-36P cells. The treatment of F-36P cells with a combination of EPO and IGF-I (EPO/IGF-I) was found to enhance EPO-induced tyrosine phosphorylation of STAT5, whereas IGF-I alone did not. Furthermore, c-CIS mRNA expression, one of the target molecules of STAT5, was more effectively induced by EPO/IGF-I than by EPO alone. To examine the mechanisms of the EPO- and EPO/IGF-I-induced proliferation of F-36P cells, we expressed dominant negative (dn) mutants of STAT5 and Ras in an inducible system. The EPO-induced DNA synthesis and the cooperative effect of EPO/IGF-I were significantly inhibited by the inducible expression of dn-STAT5 or dn-Ras. In addition, the inducible expression of dn-Ras abolished the IGF-I-enhanced tyrosine phosphorylation of STAT5. These results suggest that IGF-I may augment EPO-induced proliferation by enhancing tyrosine phosphorylation of STAT5 and raise the possibility that Ras may be involved in the augmentation of STAT5 tyrosyl phosphorylation.

Staurosporin, a broad-spectrum kinase inhibitor, induced cell spreading in a human colon cancer cell line, Colo 201. On collagen and laminin, cell spreading was induced in more than 90% of the cells and was dependent on very late activation antigen-3, as shown by an antibody inhibition assay. Cell spreading required divalent cations and showed the order of preference Mn2+ > Mg2+ > Ca2+. On fibronectin, only about 30% of the cells were observed to spread, and spreading occurred via a non-integrin, RGD-independent pathway. Staurosporin-induced spreading was inhibited by treatment with tyrosine kinase inhibitors herbimycin A and methyl 2,5-dihydroxycinnamate. Despite the presence of staurosporin, seven proteins (220, 175, 150, 98, 62, 58, and 45 kDa) showed increased levels of tyrosine phosphorylation in association with cell adhesion. Two of these (58 and 220 kDa) were identified by immunoprecipitation as Src product and tensin, respectively. Flow cytometric analysis showed that the Colo 201 cells expressed the alpha 2, alpha 3, alpha 6, and beta 1 chains of integrin, but expression of these chains was not influenced by staurosporin. Immunofluorescence microscopy revealed that the alpha 3 chain, diffusely expressed on the cell surface in the absence of staurosporin, was concentrated at focal adhesion plaques after staurosporin treatment. Neither alpha 2 nor alpha 6 was focalized by the treatment.

The heterogeneous asparagine-linked sugar chains of bovine and human immunoglobulins G were separated into 12 components by reversed-phase high performance liquid chromatography, and their structures were determined by 1H NMR spectroscopy. Both immunoglobulin (Ig) G sources contained eight non-bisected biantennary complexes and four bisected biantennary complexes. In the non-bisected sugar chains of bovine IgG, galactosylation of the Man alpha 1-3 branch predominated over that of the Man alpha 1-6, whereas in the bisected complexes galactosylation of the Man alpha 1-6 branch predominated. This difference can be explained by the substrate specificities of the galactosyl-transferases and of the N-acetylglucosaminyltransferase III involved in their synthesis. The sugar chains of human IgG1 differs in the distribution of its galactose residues from bovine IgG and human IgG2. The Man alpha 1-6 branch of all IgG1s was more highly galactosylated than the Man alpha 1-3 branch even in the non-bisected complexes. Such findings are in conflict with the substrate specificities of galactosyltransferases. Whereas these enzymes derivatized more of the Man alpha 1-6 branch of native human IgG1, in denatured protein more of the Man alpha 1-3 branch was galactosylated. Thus, protein conformation may influence the structure of its sugar chains.