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During the formation of skeletal muscle, secondary fibres form, by cell fusion, on the surface of primary fibres. Three-dimensional reconstructions of primary fibres with the second- ary fibres and cells on their surfaces were pro- duced from spaced serial transmission electron micrographs. Reconstructions were made of fi- bres from embryonic day (E) 17, E19, and E21 of the Extensor Digitorum muscle of Balbk mice. Cell distribution was analysed in two ways. Firstly, nearest neighbour analysis was used to see if cells were randomly arranged or clustered. Secondly, the association of cells and secondary fibres was tested by measuring the distances be- tween cells and each secondary fibre. Cells were found to be randomly distributed on the surface of primaries at El7 but significantly clustered, and associated with smaller secondary fibres at El9 and E21. Cells were not associated with the ends of secondary fibres. Cells associated with second- ary fibres lay in the groove formed by contact be- tween adjacent primary and secondary fibres. This apparent response to the topography of the surface on which the cells are lying has previously been mimicked in vitro by growing cells on grooved surfaces. It is likely that cells associated with secondaries will fuse laterally with these fi- bres and their response to topography is part of the process of bringing them into correct align- ment with the fibre before fusion. 0 1996 Wiley-Liss, Inc.

The rice green semi looper, Naranga aenescens Moore (Lepidoptera: Noctuidae) causes severe damage to rice fields in Eastern Asia and Middle East. We demonstrate that two types of serine proteases are active in the midgut of the third instar larvae of N. aenescens, but trypsin-like proteases are considerably more active than chymotrypsin-like proteases. To develop better control strategies, purification and biochemical characterization of a major trypsin-like digestive protease from the midgut of the third instar larvae of N. aenescens was achieved by gel filtration and anion exchange chromatography. After the final purification step, the enzyme was purified 9.62-fold with a recovery of 16.1% and a specific activity of 4.12 U/mg protein and a molecular mass of approximately 88.5 kDa. Biochemical characterization indicated that the purified protease had highest activity at pH 10 and 30°C and was stable for up to 6 h under those conditions. Divalent cations, especially Ca2+, Mg2+, and Cu2+, increased the enzyme activity and synthetic inhibitors that target trypsin-like activity caused a significant reduction in caseinolytic activity. These data may be used to develop inhibitors that decrease the damage of N. aenescens to rice cultivars in the field.

Ca2+ dependent exocytosis inParamecium involves the release of numerous secretory organelles known as “trichocysts”. According toGarofalo et al. (1983) trichocysts pass through three stages of condensed (tmxI), partially expanded (stage II) before release, and fully expanded (stage III) or released trichocysts. We have therefore investigated the effect of two widely used Ca2+ channel blockers, verapamil and nifedipine and the muscular relaxant, dantrolene-Na, on the process of trichocyst release. Verapamil and nifedipine inhibited secretion in a dose dependent manner, but dantrolene-Na and the solvent, PEG-400, did not abolish it. Electron microscopic study of preincubatedParamecium cells in verapamil resulted in the appearance of tmxI, whereas untreated controls remained in partially expanded stage II. Pretreatment of isolated membrane free trichocysts with verapamil did not inhibit matrix expansion in the presence of increasing Ca2+ concentration. In a separate experiment, cells were pretreated with verapamil and the anti calmodulin compound, trifluoperazine. The cells were then induced to release their secretory contents by picric acid-Ca2+ treatment. Electron microscopic examination of cells captured by quick fixation with osmium tetroxide revealed that verapamil treated cells manifested the inhibition of membrane fusion, whereas in TFP treated cells there was no sign of a traceable exocytotic opening formation after membrane fusion. Based on present results we propose a role for calmodulin in the formation of exocytotic openings.