Ichtyotoxic activity of extracts from Mexican marine macroalgae

Seventy-three species of macroalgae from the Mexican Pacific, Atlantic and Caribbean coast were screened for ichtyotoxic activity. Ethanolic, acetonic and aqueous extracts were prepared and tested against the fish Carassius auratus. The extracts were classified on the basis of their effects as: toxic if the fish died in two hours or less; moderately toxic, if the organism behaved abnormally but death did notoccur, and non-toxic if the fish did not display any change. 79% species were ichtyotoxic to some degree. Extracts of 39 species were toxic, with at least one extract with lethal effects, 19 were moderately toxic and 15 species were non-toxic. Only the extracts ofDictyota bartayresiana, Dictyota cervicornis,Lobophora variegata, Bryothamnion triquetrum and Laurencia obtusa were toxic in all three solvents. The acetone and ethanol extracts were more active, and therefore are more suitable for extraction of toxic substances. This revised version was published online in August 2006 with corrections to the Cover Date.     

Helicobacter pylori induces an increase in inositol phosphates in cultured epithelial cells

Abstract Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pyfori -infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or α-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pytori -infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

Isolation of apical plasma membrane in rabbit gallbladder epithelium by Percoll density gradient centrifugation

The apical membranes of rabbit gallbladder epithelial cells were isolated by treating the homogenate with Ca2+ or Mg2+ and centrifuging the suspension in Percoll gradient. In this way brush-border membranes were obtained with enrichment factors ranging between 10 and 20 and yields of 15-30%. A second method is described with which membranes were isolated, without any preliminary treatment, first by differential centrifugation, then with Percoll gradient; the final membrane enrichment was over 15, however the yield was very low (3%). Many possible enzymatic markers of the apical plasma membrane were investigated: L-gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase, sucrase. The first appears to be that of choice. Apical membrane fraction could be also evidenced by autofluorescence or by labeling with Lotus tetragonolobus lectin. Preliminary experiments showed that apical plasma membranes isolated in this way form vesicles.     

Enkephalin regulation of L-valine transport in rabbit ileum

We studied the influence on ionic basal transport (Na+ and Cl-) and L-valine transport of two enkephalins which are not metabolized and act in delta and mu receptors respectively. Transports have been indirectly determined measuring the transepithelial electric potential and the short circuit current. DADLE does not significantly influence ion and amino-acid transport, while DAGO alters both of them in the presence of the myenteric plexus (muscle layers present) or inhibits only L-valine transport in the absence of the plexus (muscle layers removed).     

Processing of SP1 precursor in a cell-free system from poly(A+) mRNA of human placenta

Cell-free translation of polyadenylated mRNA from human term placenta in a wheat germ extract, after immunoprecipitation with antibodies directed against purified pregnant serum SP₁, yielded a single polypeptide of 31 kDa. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of two polypeptides, one of them of 46 kDa and the other of 28 kDa. Both polypeptides were protected from limited proteolysis and when the assay was performed with lytic detergent concentrations in addition to proteases, this protection was abolished indicating that the polypeptides were segregated into the microsomal vesicles. The cleavage of a signal peptide of 3 kDa from the 31 kDa primary translation product gives rise to 28 kDa and accounts for the slight increase in electrophoretic mobility. The treatment of the immunoprecipitated products with Endoglycosidase H and -mannosidase, suggested that only the 46 kDa polypeptide is a glycoprotein.From the results obtained we conclude that SP₁ is synthesized and processed to a glycoprotein of 46 kDa which would be a protomeric form of the oligomers reported in pregnant serum by other authors.Abbreviations PMSF phenylmethyl sulfonylfluoride - TCA trichloroacetic acid - DTT dithiotreitol     

Effect of Drying Medium on Residual Moisture Content and Viability of Freeze-Dried Lactic Acid Bacteria

The effect of various substances on the relationship between residual moisture content and the viability of freeze-dried lactic acid bacteria has been studied. Compounds such as polymers, which display considerable ability in displacing water, showed no protective action during freeze-drying. Adonitol, on the other hand, produced the smallest change in water content at various times during drying and allowed the highest rate of survival.