Calmodulin and acidic compounds alter basic protein phosphorylation by the protein kinase from human platelets

A cyclic nucleotide-independent protein kinase of human platelets, which phosphorylated histones, myelin basic protein and protamine and did not catalyze the phosphorylation of acidic proteins such as casein, phosvitin and myosin light chain, has been purified approx. 1,500-fold from the crude extract by steps of DEAE-cellulose, Sephadex G-200, hydroxylapatite and phosphoryl cellulose column chromatography. The substrate phosphorylation by this kinase was markedly enhanced by calmodulin even in the absence of Ca²⁺, when mixed histone was used as a substrate. The interaction of the kinase with mixed histone resulted in an irreversible inactivation of the enzyme. Calmodulin prevented this inactivation, and this compound produced an apparent increase in histone phosphorylation by the kinase. It should be noted that acidic polypeptides such as troponin-C, phospholipids and nucleic acids have a similar ability. The addition of Ca²⁺ reduced the effect of calmodulin more than the effects of other acidic compounds.     

Effects of Chronic Ethanol Administration on Rat Brain Phospholipid Metabolism

Alterations in brain phospholipid metabolism were observed after chronic ethanol administration for 16 days to developing rats. Animals were injected intraperitoneally with 32Pi 16 h prior to killing. Overall uptake of 32Pi by brain did not differ between the control and ethanol-treated groups, which were killed 2 h and 24 h after the last ethanol feeding. Except for an increase in the labeling of myelin after ethanol treatment, the amount of radioactivity recovered in the synaptosomal-mitochondrial and plasma membrane fractions of control and ethanol-treated groups was not different. Relative to the radioactivity of phosphatidylcholines, which indicated no change, there were increases (20-44%) in labeling of ethanolamine plasmalogens, phosphatidic acids, and phosphatidylinositols in cortical synaptosomes from the 2-h ethanol-treated group. In the plasma membrane fractions, however, increases (9-14%) in labeling of phosphatidylserines and phosphatidylinositols were observed in both 2- and 24-h ethanol-treated groups. In both membrane fractions, there was an obvious increase (44-86%) in labeling of polyphosphoinositides at 24 h after withdrawal from ethanol. Results thus indicate an adaptive increase in the biosynthesis of ethanolamine plasmalogen and brain acidic phospholipids due to chronic ethanol administration. Furthermore, the increase in labeling of polyphosphoinositides in the 24-h withdrawal group may reflect the hypoactivity associated with ethanol withdrawal.     

Cytoplasmic Signals Mediate Apical Early Endosomal Targeting of Endotubin in MDCK Cells

Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin–Darby canine kidney cells. We show that both the apically targeting wild-type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.     

The preparative isolation of endosome fractions: a review

The endosome is an intracellular, acidic, membrane-bound, subcellular compartment to which endocytosed ligands, receptors and plasma membrane proteins are conveyed before sorting and delivery to destinations elsewhere in the cell. The preparative isolation of elements of this compartment has been achieved successfully using various appropriate combinations of density gradient ultracentrifugation, electrophoretic, gel filtration and immunoaffinity techniques. These methods for isolating endosome fractions are reviewed together with the difficulties of establishing markers for such fractions. The isolation of an endosome fraction from the pathway of polymeric IgA transcytosis in rat liver is discussed to exemplify successful isolation procedures and appropriate subcellular markers.     

Effects of Low pH on the Induction of Root Hair Formation in Young Lettuce (Lactuca sativa L. cv. Grand Rapids) Seedlings

Received 13 August 1999/ Accepted in revised form 22 December 1999     

Actin on trafficking

During an infection, neutrophils are the first immune cells to arrive armed to clear the invading pathogen. In order to do so, neutrophils need to transmigrate from the peripheral blood through the endothelial layer toward the site of inflammation. This process is in most cases dependent on integrins, adhesion molecules present on all immune cells. These molecules are functionally regulated by “inside-out” signaling, where stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs and many factors have been linked to regulation of integrins on neutrophils. Control of integrin conformation and clustering is of pivotal importance for proper cell adhesion, migration, and bacterial clearance. Recently, gelsolin was found to be involved in β₁-integrin affinity regulation and cell adhesion. Here, I summarize the role of neutrophil integrin regulation in the essential steps to reach the site of inflammation and clearance of bacterial pathogens.     

Role of phospholipid in the low affinity reactions between cytochrome c and cytochrome oxidase

The steady-state oxidation of ferrocytochrome c by cytochrome oxidase monitored spectrophotometrically showed that: (1) the kinetics were strictly biphasic with purified enzyme, while mitochondrial membrane-bound enzyme exhibited multiphasic kinetics with extended low affinity phases; (2) the TNmax for the highest affinity phase was as slow as 5-10 electron X s-1 for both preparations, while for the low affinity phases it was about 45 electron X s-1 for the purified enzyme and 150 electron X s-1 for the mitochondrial membrane-bound enzyme; (3) reconstitution of purified enzyme into acidic phospholipid vesicles partially repleted the extended low affinity phases, while reconstitution into uncharged vesicles had no effect.     

Phenotype modulation in primary cultures of rat aortic smooth muscle cells

The transition of adult rat aortic smooth muscle cells from a contractile to a synthetic phenotype during the first week of primary culture on a substrate of fibronectin in serum-free medium was studied by light and electron microscopy. The weak base chloroquine and the carboxylic ionophore monensin were both found to inhibit the spreading of the cells and the accompanying changes in cellular fine structure. The exchange of myofilament bundles for a prominent rough endoplasmic reticulum and Golgi complex was delayed and vacuoles filled with incompetely degraded material accumulated in the cytoplasm. The microtubule-disruptive drugs colchicine and nocodazole likewise opposed the spreading and fine structural reorganization of the cells. Most typically, the Golgi stacks were small and widely dispersed. In addition, vacuoles of the type mentioned above increased in number. On the other hand, there was surprisingly little effect of cytochalasin B, a drug that is supposed to interfere with the assembly of actin filaments. The observations suggest that the phenotypic modulation of arterial smooth muscle cells is dependent on: (a) lysosomal degradation of discarded cellular constituents, (b) active vesicular transport along the exocytic pathway to provide the expanding cell surface with new membrane, and (c) a normal microtubular cytoskeleton to ensure the establishment of a new and functionally efficient intracellular organization.     

Response of leaf surfaces and gas exchange to wind stress and acid mist in birch (Betula pubescens)

 This research demonstrates that a leaf’s response to acid mist is dependent on the integrity of the leaf cuticle and that significant differences in the structural and physiological disturbances in leaves can be attributable to different types of wind action. Betula pubescens Ehrh. plants were located at adjacent, but contrasting, sites to create different wind treatments: (i) direct wind action, (ii) indirect wind action and (iii) shelter from wind action (control). In combination with the wind treatments, acidic (pHs 5 and 3) or neutral (pH 7) mists were applied weekly. Wind action significantly increased visible leaf injury, microscopic cuticular lesions and cuticular conductance (g _c ), but reduced photosynthetic rate (P _N ) and stomatal conductance (g _s ) compared to shelter. Wind action combined with acid mist was more injurious than wind action alone, but leaves sheltered from wind action were highly resistant to the damaging effects of acid mist. Direct wind action combined with pH 3 mist resulted in the highest values of g _c and the greatest number of cuticular lesions. By contrast, indirect wind action combined with pH 3 mist induced most visible injury, but relatively low values of g _c and few microscopic cuticular lesions. Acid mist reduced P _N only when leaves had been damaged by wind action. Higher values of g _c were associated both with increases in the area of visible leaf injury and with the number of cuticular lesions. Compensatory increase in P _N of healthy tissue was evident in leaves exposed to combinations of wind action and acid mist. Received: 10 November 1997 / Accepted: 6 March 1998