Calmodulin and cell proliferation

The calmodulin content of synchronized Chinese hamster ovary (CHO-K₁) cells was determined at each phase of the cell cycle. The calmodulin content was minimum in the G₁ phase, increased after the cells entered S phase and reached the maximum level at the late G₂ or early M phase. When 30 μM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G₂ or early M phase. The addition of W-7 also prevented the morphological changes caused by cholera toxin. These results suggest that calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis.     

Bradykinin-induced translocation of protein kinases C in neuroblastoma NCB-20 cell: dependence on 1,2-diacylglycerol content and free calcium

Bradykinin (BK)-induced production of 1,2-diacylglycerol (1,2-DG) and translocation of protein kinases C (PKCs) were examined in neuroblastoma-derived hybrid NCB-20 cells. Mass analysis of 1,2-DG exhibited a biphasic increase by 1 microM BK stimulation: the first transient phase and the second broad sustained phase. Among three subspecies of PKC expressed in these cells, types II and III were observed to translocate from cytosol to membrane in response to BK as well as PBt2 by Western blotting analysis. Type II translocated more rapidly and distinctly than type III. However, after treatment with quin 2/AM, the second phase of 1,2-DG formation completely disappeared and PKCs translocation by BK or PBt2 was completely abolished. BK-induced IP3 (1,4,5) formation was temporally consistent with the first transient phase of 1,2-DG formation. These findings suggest that PKCs translocation by BK stimulation is caused by 1,2-DG produced not only via phosphoinositide metabolism, but via other phospholipid breakdown which is Ca2+-dependent.     

Monoclonal antibody to galactosylceramide: discrimination of structural difference in the ceramide moiety

A mouse monoclonal antibody (mAb) was developed against monohexaosylceramide. This mAb differentially reacted on thin-layer chromatograms with 3 types of galactosylceramide (GalCer) obtained from bovine brain. Structural analysis of the 3 glycolipids revealed that they consisted of the same galactose and sphingosine but of apparently different fatty acids. Among the GalCers, the mAb reacted with teh two GalCers which contained alpha-hydroxy fatty acids, but not with GalCer composed of nonhydroxy fatty acids. These findings suggest that not only that the mAb discriminated the fatty acid composition in the ceramide moiety of GalCer, but also that the ceramide structure defines the immunological epitope as it is known to do for the carbohydrate moiety of glycosphingolipid.