Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland.

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatrography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.

The effects of U.V.-light, ionizing radiation and the carcinogen N-acetoxy-2-fluorenylacetamide on the development in vitro of one- and two-cell mouse embryos.

The development of one- and two-cell mouse embryos to morula-blastula stages was followed in vitro after treatment with low doses of U.V.-light, ionizing radiation or N-acetoxy-2-fluorenylacetamide. Exposure of one-cell embryos to either radiation source 18 and 24 hours after human chorionic gonadotropin injections prevented maturation, most embryos being arrested at the one-cell stage and a few at the two-cell stage. Two-cell embryos, however, were not sensitive to low doses of either U.V. or X-irradiation and developed normally. Treatment of early one-cell embryos with the carcinogen, N-acetoxy-2-fluorenyl-acetamide (0-7 muM), also arrested development, whereas exposure of late one-cell embryos did not completely prevent maturation to morula-blastula stages. Exposure of two-cell embryos to the same concentration of carcinogen had no effect on their development to blastulas. Results with all three agents showed that mouse embryos at the one-cell stage are more sensitive than those at the two-cell stage, as judged by their ability to develop in vitro.

Cleavage furrow formation of Rana amurensis eggs observed with fluorescence pattern photobleaching.

Taking advantage of the extremely slow lateral diffusion of proteins on Rana amurensis egg surface (Xu et al., 1984), it was possible to make a sharp concentric ring pattern on the egg surface by photobleaching of the fluorescein-labelled egg surface. The shape change of the pattern reveals the movement of the egg surface. The surface, even in front of the furrow tip, is drawn towards the furrow centre. The furrow tip differentiates into border and central lines as shown by the distribution of surface protrusions. Between the border and central lines the nascent membrane inserts while the width of furrow tip increases to nearly 100 microns.

[The effects of polylysine on the aggregation of surface and on the cleavage furrow of Rana amurensis eggs].

Polylysine synchronously aggregated pigment granules and egg surface stained uniformly with fluorescein isothiocyanate to form patches (Plate I, fig 1 & 2). Patches always appeared and aggregated first in the opposite grey crescent region, then in grey crescent region and next at animal hemisphere. A fluorescent ring formed at the upper side of the equatorial region. The ring and the patches at the animal hemisphere accumulated to the animal pole to make a cap (Plate I, Fig. 3). Finally the vegetal hemisphere broke due to the contraction of egg surface during aggregation. These indicated that four different domains: opposite grey crescent region, grey crescent region, animal and vegetal hemispheres, were present at the fertilized egg surface. Similar domains also occurred at the unfertilized egg surface as revealed by the same treatment suggesting that the opposite grey crescent region of fertilized egg, which corresponding to the sperm entrance region had existed at the mature egg surface. There were three modes in the early stage of patch formation: (1) the features of granular protrusions did not change greatly (Plate I, Fig. 5-7), (2) the granular protrusions elongated (Plate I, Fig 8-11) and (3) folds radiated from patch (Plate I, Fig. 12, 13). Around each patch new surface membrane arose, which was similar to the nascent membrane of cleavage furrow.(ABSTRACT TRUNCATED AT 250 WORDS)

Detergent, Brij, increasing the area of new surface membrane during the early cleavage of eggs of Rana amurensis.

The exposure of new surface membrane occurred in the cleavage furrow of Rana amurensis eggs enclosed in fertilization membrane immersed in Brij solution. The exposed area increased gradually and reached a maximum while the furrow extended to 240 degrees around the egg surface. At this time, the new membrane area of the treated eggs was significantly larger than that of the control. Afterwards, the exposed new membrane area decreased gradually. This may result from the extent of new membrane increase being less than the extent of contraction of cleavage furrow.