Adrenal Chromaffin Cell Calmodulin: Its Subcellular Distribution and Binding to Chromaffin Granule Membrane Proteins

Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane-containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane-bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I-calmodulin indicated the presence of high-affinity binding sites (KD = 1.3 X 10(-8) M; Bmax = 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I-calmodulin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin-binding polypeptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus-secretion coupling.     

A topography-based model of forest cover at the alpine tree line in the tropical Andes

Aim  To present a method that assesses the influence of environmental variables, including climate, substrate, topography, and anthropogenic disturbances, on the distribution of Andean forest at the tree line, and to compare this forest distribution between areas.
Location  Sangay National Park, on the eastern slopes of the Andes in central Ecuador.
Methods  A logistic regression model was built using topographical variables and environmental indices, derived from a digital elevation model, to explain forest cover, derived from a Landsat ETM image, in a zone around the average tree line altitude.
Results  The model shows that after altitude, which can explain about 80% of forest cover, wetness has the next strongest effect (areas accumulating water, but also cold air, were devoid of forest, resulting in inverted tree lines), followed by eastness (western slopes had forest to higher altitudes). Application of the model in two nearby areas showed that the real tree line was lower than the predicted tree line in both areas, probably owing mainly to macroclimatic differences in one area, and partly also to human land use in the other. The locations with the largest deviations could be the focus of further research concerning human impacts on tree line vegetation.
Main conclusions  The tree line is located at lower altitudes on east-facing slopes, which may be because high levels of radiation are received by east-facing slopes in the clear mornings, resulting in the photoinhibition of tree seedlings in the páramo. In spite of the limitations of the quality and resolution of the remote sensing data, the presented method provides indications for important ecological factors at the tree line. The method also allows the detection of differences in tree line position between areas, which may reflect climatic differences or the location of anthropogenic disturbances.     

Mevalonate kinase in lysates of cultured human fibroblasts and lymphoblasts: kinetic properties, assay conditions, carrier detection and measurement of residual activity in a patient with mevalonic aciduria

An assay has been developed for the measurement of mevalonate kinase activity in extracts of cultured human fibroblasts and lymphoblasts. Individual elements of the assay were investigated in order to achieve optimum conditions. Apparent Michaelis constants (KMapp) for the substrates mevalonic acid and adenosine-5'-triphosphate were 22 +/- 10 mumol/l and 0.42-0.53 mmol/l, respectively, in lysates of control fibroblast lines. The same values in lysates of a control lymphoblast line were 17 mumol/l and 0.23 mmol/l, respectively. Mevalonate kinase activity in extracts of cultured fibroblasts derived from 6 control individuals was 3.24 +/- (SD) 0.91 nmol/min/mg protein. The activity in extracts of fibroblasts derived from a patient with mevalonic aciduria was 0.15 +/- 0.10 nmol/min/mg protein, approximately 5% of the control mean. The parents and brother of the patient displayed mevalonate kinase activities in fibroblast extracts approximating 38-42% of the control mean. Substantially higher mevalonate kinase activity was documented in extracts of cultured lymphoblasts. When assayed on various occasions, the mean activity of mevalonate kinase in extracts of lymphoblasts derived from the parents, brother and maternal grandmother of the patient ranged from 27 to 32% of the mean activity of 9.8 +/- (SD) 3.4 nmol/min/mg protein measured in a parallel control lymphoblast line, while the mean activity in a maternal and paternal uncle approximated 65-89% of the same control mean. The mean activity in extracts of lymphoblasts derived from the patient approximated 2% of the control mean. The data suggest that the parents, brother and maternal grandmother are carriers of the defective gene responsible for mevalonate kinase deficiency, consistent with an autosomal recessive mode of inheritance.     

Phosphoinositide kinase signaling controls ER-PM cross-talk

Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca²⁺-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase–mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions.     

Plant-derived xenomiRs and cancer: Cross-kingdom gene regulation

Exosomal microRNAs (miRNAs) critically regulate several major intracellular and metabolic activities, including cancer evolution. Currently, increasing evidence indicates that exosome harbor and transport these miRNAs from donor cells to neighboring and distantly related recipient cells, often in a cross-species manner. Several studies have reported that plant-based miRNAs can be absorbed into the serum of humans, where they hinder the expression of human disease-related genes. Moreover, few recent studies have demonstrated the role of these xenomiRs in cancer development and progression. However, the cross-kingdom gene regulation hypothesis remains highly debatable, and many follow up studies fail to reproduce the same. There are reports that show no effect of plant-derived miRNAs on mammalian cancers. The foremost cause of this controversy remains the lack of reproducibility of the results. Here, we reassess the latest developments in the field of cross-kingdom transference of miRNAs, emphasizing on the role of the diet-based xenomiRs on cancer progression.