Cytochalasin D and cationized ferritin as probes for the morphological investigation of blebbing in two human cell lines

Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration (1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters. CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli, at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related 6-nm microfilaments. The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical Center, is also greatly appreciated.     

Evidence for an association between calmodulin and membrane patches containing gonadotropin-releasing hormone — receptor complexes in cultured gonadotropes

Summary Participation of calmodulin, clathrin, and actin in receptor mediated endocytosis of gonadotropin-releasing hormone (GnRH) was studied in an in vitro system of dispersed pituitary cells with a triple staining procedure. Cells were incubated in D-Lys⁶-Pro⁹-Des¹⁰-GnRH-biotin and stained with avidin-peroxidase-diaminobenzidine. Calmodulin, clathrin, and actin as well as luteinizing hormone were identified by indirect immunofluorescence with FITC- and rhodamine-labeled second antibody. The results indicate a close spatial association of calmodulin, but not of clathrin and actin, with GnRH-containing plasma membrane patches.Supported by PHS grants NIH NS1761401, HS 09914, and HD 19899     

The inhibition of acetate oxidation by ethanol in Acetobacter aceti

Ethanol grown Acetobacter aceti differed from acetate grown. In ethanol grown cells, acetate uptake, caused by the oxidation of acetate, was completely inhibited by ethanol, in acetate grown cells only to 20%. This was correlated with a 65-fold higher specific activity of the membrane bound NAD(P)-independent alcohol dehydrogenase in ethanol grown than in acetate grown cells. In comparison with ethanol grown cells, acetate grown cells showed a 3-fold higher acetate respiration rate and 3-fold higher specific activities of some tricarboxylic acid cycle enzymes tested. Both adaptations were due to induction by the homologous and not to repression by the heterologous growth substrate. A. aceti showed a membrane bound NAD(P)-independent malate dehydrogenase and no activity of a soluble NAD(P)-dependent one, as was known before from A. xylinum. A hypothesis was proposed explaining the observed inhibition of malate dehydrogenase and of functioning of the tricarboxylic acid cycle in the presence of ethanol or butanol or glucose by a competition of two electron currents for a common link in the convergent electron transport chains. The electrons coming from the quinoproteins, alcohol dehydrogenase and glucose dehydrogenase on the one side and those coming from the flavoproteins, malate dehydrogenase and succinate dehydrogenase via ubiquinonecytochrome c reductase on the other side are meeting at cytochrome c. Here the quinoproteins may be favoured by higher affinity and so inhibit the flavoproteins. Inhibition could be alleviated in the cell free system by increasing the oxygen supply.Dedicated to Professor Carl Martius on the occasion of his 80th birthday, March 1st 1986     

Late enzymes of vindoline biosynthesis

From differentiated plants of Catharanthus roseus (L.) G. Don we have isolated a specific enzyme of the vindoline biosynthetic pathway catalysing the S-adenosylmethionine-dependent methylation of 11-O-demethyl-17-O-deacetyl-vindoline. The enzyme we named S-adenosyl-L-methionine : 11-O-demethyl-17-O-deacetylvindoline 11-O-methyltransferase. This transferase exhibits a high substrate specificity. Obviously the O-methylation at C-11 precedes the O-acetylation at the C-17 position during the biosynthesis of vindoline.A second enzyme was detected which hydrolyses the acetyl function of vindoline. The distribution of this acetylesterase in C. roseus plants demonstrates that the enzyme is not specifically associated with the vindoline distribution in the plant material. Most probably this enzyme plays no essential role in the biosynthesis of vindoline.     

Late enzymes of vindoline biosynthesis. Acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyl-transferase

From differentiated plants of Catharanthus roseus (L.) G. Don, a specific enzyme was isolated and named acetyl-CoA : 17-O-deacetylvindoline 17-O-acetyltransferase, acting on the biosynthetic formation of the Aspidosperma type alkaloid vindoline.The enzyme shows a high selectivity towards different substrates. The acetyl-CoA-dependent transferase also catalyses the reverse reaction by hydrolysis of the 17-O-acetyl group of vindoline in the presence of free CoA. This enzyme is localized only in vindoline-containing plant parts, but was so far not detectable in cell suspension cultures of C. roseus. The enzyme allows the synthesis of labelled vindoline with high specific activity, applicable for instance as tracer for radioimmunoassays of vindoline.     

Geschlechtsspezifische Unterschiede beim Nahrungserwerb des Fichtenkreuzschnabels (Loxia curvirostra)

Summary By recording the rate of glancing (i. e. looking up), the number of seeds taken by a crossbill per unit time from a larch cone (Larix X eurolepis) was evaluated. Female crossbills took more seeds per unit time from the cones than males do. Because of similar bill (and body) sizes the sexual difference could be explained in the following ways: a) during the breeding season the females need more energy for egg production than males do for spermatogenesis; b) females are more vigilant than males.     

Filter mesh size and food particle uptake by Daphnia

Food size selection of four Daphnia, species (D. magna, D. hyalina, D. galeata, D. pulicaria) was investigated using spherical plastic beads as artificial food and with small bacteria. The size of the particles ranged from 0.1 to 35 m with special emphasis to the particle diameters between 0.1 and 1 m. In one set of experiments a mixture of differently sized particles was offered as food suspension and the selectivity of filtering was determined by comparing the size spectrum of the particles found in the gut contents with the spectrum in the food suspension. In a second series of experiments suspensions of uniformly sized particles were offered to single animals and their feeding activity was observed directly. In both types of experiments the mesh sizes of the filtering apparatus of the respective animals studied were measured after the experiments by, scanning electron microscopy. The mean sizes of the filter meshes were about 0.4–0.7 m. In all experiments the size of the particles found in the gut or those which caused high feeding activities were larger than the smallest mesh sizes of the filters. As a consequence simple mechanical sieving provides a sufficient explanation for the mechanism of particle retention of the filtering process in Daphnia. D. magna was found to feed with high efficiency on suspended freshwater bacteria, the residual species investigated showed low filtering efficiencies when bacteria were offered as food.The present study was supported by Deutscher Akademischer Austauschdients