Degradation of protein kinase Cα and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions: Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC

Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-5Y cells with 2-O-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-5Y PKCα by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKCα. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an ‘unregulated’ kinase, since its persistence is apparently strictly regulated by proteolysis.     

Accumulation of Inositol Phosphates Induced by Chlorpromazine in C6 Glioma Cells

Abstract: Chlorpromazine, a cationic amphiphilic drug known to affect phospholipid metabolism, greatly increases the generation of inositol phosphates in C6 glioma cells. When a pulse-chase protocol with myo-[2-³H]inositol as the radioactive precursor was used, the peak increase in radioactivity of inositol phosphates was observed at 20 min. The drug decreased inositol tetrakisphosphate labeling as a percentage of inositol trisphosphate in a dose-dependent manner. It also increased the labeling of the inositol-containing phospholipids, the precursors of the inositol phosphates. The increase in radioactivity of both phospholipids and inositol phosphates was dose-dependent, but appeared also to be a function of the time of exposure of the cultures to the drug, suggesting that the concentration of chlorpromazine in the cell, and not that in the medium, is the critical factor. The optimum concentration for maximum phospholipid labeling was lower than that eliciting maximum generation of inositol phosphates. The data suggest that the mechanism probably does not involve cell-surface receptors, but rather may consist of a direct effect of chlorpromazine on phosphoinositidase C and possibly other enzymatic reactions concerned with the metabolism of inositol phosphates.     

The RBC morphological dependence of the RBC disaggregability

The aim of this study was to determine the red blood cell (RBC) disaggregability dependence upon the RBC shape. The study concentrated on stored blood during bank storage and on suspensions of artificially induced echinocytes. Measurements was performed in autologous plasma of hematocrit 0.45 and at constant plasmatic content. Rheological studies using stationary viscometry, nonstationary viscometry and rheoscopy were made in order to assess different stages of the disaggregability process. Whatever the method of measurement used, the morphological interpretation of the results reveal that beyond 75% of echinocytes within the sample, the disaggregation process is altered. The shear stresses required to dissociate the echinocyte aggregates are significantly higher than those required to disaggregate normal RBC rouleaux.     

Influence of tree cover on the diversity of herbaceous communities in subalpine wooded pastures

Abstract. The relationships between several diversity indices and tree cover in subalpine wooded pastures with Larix de-cidua were analysed at two spatial scales: phytocoenoses (2500-m² plots) and herbaceous synusia (1-m² quadrats). Diversity indices have a non-linear relationship to Larix cover; the best fit was obtained with Gaussian regressions. Species richness was influenced by Larix cover more than evenness. At the phytocoenosis level, the optimum for species and synusial richness in the herb layer was near 25 % tree cover. At the synusia level, species richness of the herb layer showed an optimum when mean distance to the four nearest Larix trees was ca. 30 m, or when the potential number of sunshine hours between April and September was close to 1250 h. Canonical Correspondence Analysis confirmed the influence of Larix cover on the composition of phytocoenoses and herbaceous synusiae, but included also the role of altitude, slope and aspect.