Determinants of the anesthetic sensitivity of two-pore domain acid-sensitive potassium channels: molecular cloning of an anesthetic-activated potassium channel from Lymnaea stagnalis

Certain two-pore domain K(+) channels are plausible targets for volatile general anesthetics, yet little is known at the molecular level about how these simple agents cause channel activation. The first anesthetic-activated K(+) current I(K(An)) that was characterized was discovered in the mollusk Lymnaea stagnalis and is remarkable for both its sensitivity to general anesthetics and its stereoselective responses to anesthetic enantiomers (Franks, N. P., and Lieb, W. R. (1988) Nature 333, 662-664 and Franks, N. P., and Lieb, W. R. (1991) Science 254, 427-430). Here we report the molecular cloning of a two-pore domain K(+) channel LyTASK from L. stagnalis and show that, when expressed in HEK-293 cells, it displays the same biophysical characteristics as the anesthetic-activated K(+) current I(K(An)). Sequence analysis and functional properties show it to be a member of the TASK family of channels with approximately 47% identity at the amino acid level when compared with human TASK-1 and TASK-3. By using chimeric channel constructs and site-directed mutagenesis we have identified the specific amino acid 159 to be a critical determinant of anesthetic sensitivity, which, when mutated to alanine, essentially eliminates anesthetic activation in the human channels and greatly reduces activation in LyTASK. The L159A mutation in LyTASK disrupts the stereoselective response to isoflurane while having no effect on the pH sensitivity of the channel, suggesting this critical amino acid may form part of an anesthetic binding site.     

(R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1

The multidrug-resistant protein MRP1 (involved in the cancer cell multidrug resistance phenotype) has been found to be modulated by racemic verapamil (through stimulation of glutathione transport), inducing apoptosis of human MRP1 cDNA-transfected baby hamster kidney 21 (BHK-21) cells and not of control BHK-21 cells. In this study, we show that the two enantiomers of verapamil have different effects on MRP1 activity. Only the S-isomer (not the R-isomer) potently induced the death of MRP1-transfected BHK-21 cells. The decrease in cellular glutathione content induced by the S-isomer, which was not observed with the R-isomer, was stronger than that induced by the racemic mixture, indicating that the R-isomer antagonized the S-isomer effect. Both enantiomers altered leukotriene C(4) and calcein transport by MRP1. Thus, the R-isomer behaved as an inhibitor, which was confirmed by its ability to revert the multidrug resistance phenotype toward vincristine. Molecular studies on purified MRP1 using fluorescence spectroscopy showed that both enantiomers bound to MRP1 with high affinity, with the binding being prevented by glutathione. Furthermore, conformational changes induced by the two enantiomers (monitored by sodium iodide accessibility of MRP1 tryptophan residues) were quite different, correlating with their distinct effects. (S)-Verapamil induces the death of potentially resistant tumor cells, whereas (R)-verapamil sensitizes MRP1-overexpressing cells to chemotherapeutics. These results might be of great potential interest in the design of new compounds able to modulate MRP1 in chemotherapy.     

Iron deficiency differently affects metabolic responses in soybean roots

Iron deficiency responses were investigated in roots of soybean, a Strategy I plant species. Soybean responds to iron deficiency by decreasing growth, both at the root and shoot level. Chlorotic symptoms in younger leaves were evident after a few days of iron deficiency, with chlorophyll content being dramatically decreased. Moreover, several important differences were found as compared with other species belonging to the same Strategy I. The main differences are (i) a lower capacity to acidify the hydroponic culture medium, that was also reflected by a lower H(+)-ATPase activity as determined in a plasma membrane-enriched fraction isolated from the roots; (ii) a drastically reduced activity of the phosphoenolpyruvate carboxylase enzyme; (iii) a decrease in both cytosolic and vacuolar pHs; (iv) an increase in the vacuolar phosphate concentration, and (v) an increased exudation of organic carbon, particularly citrate, phenolics, and amino acids. Apparently, in soybean roots, some of the responses to iron deficiency, such as the acidification of the rhizosphere and other related processes, do not occur or occur only at a lower degree. These results suggest that the biochemical mechanisms induced by this nutritional disorder are differently regulated in this plant. A possible role of inorganic phosphate in the balance of intracellular pHs is also discussed.     

Tight junction formation in early <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos: identification and ultrastructural characterization of junctional crests and junctional vesicles

How tight junctions (TJ) form during early amphibian embryogenesis is still an open question. We used time-lapse video microscopy, scanning electron microscopy (SEM), TEM and freeze-fracture to gain new insight into TJ biogenesis in early clevages of Xenopus laevis. Video analysis suggests three phases in junction formation between blastomeres. A first "waiting" phase, where new unpigmented lateral membranes are generated. A second "mixing" phase, where the unpigmented lateral membrane is separated from the pigmented apical membrane by an area showing a limited degree of intermingling of cortical pigment. And a third "sealing" phase, characterized by the formation of cingulin-containing boundaries between membrane domains, and their rapid directional adhesion in a zipper-like fashion. By SEM, we characterized these boundaries ("junctional crests", JC) as arrays of villiform protrusions at the border between old and new membranes. In the 2-cell embryo, JC are deeply located, and thus not visible at the surface, but they become increasingly more superficial as cleavages progress. After adjacent blastomeres have adhered to each other, fractured JC display linear arrays of junctional vesicles (JV) of 1-3 mum diameter. TEM analysis shows that JV are symmetrically located near the apposed membranes of adjacent blastomeres, and that the membranes near the JV display focal sites of intimate contact, typical of TJ. Freeze-fracture analysis confirms that intramembrane fibrils, typical of TJ, are present at adhesion sites. We conclude that TJ are formed following the sealing of JC, through the recruitment, sorting and assembly of membrane and cytoplasmic proteins at or near JV.     

Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.     

Observations on future sea level changes in the Venice lagoon

The identification of the environmental conditions inducing different ecophysiological responses in the different strains and populations of the brine shrimp Artemia should improve the understanding of their biogeographic distribution. Nauplii from two Argentinean brine shrimp populations, Artemia persimilis from Salinas Grandes de Hidalgo (province of La Pampa) and Artemia franciscana from Laguna Mar Chiquita (province of Cordoba), were grown up until adulthood at different salinities (30, 60, 90, 120 gL⁻¹) and temperatures (12, 21, 28°C). The aim was to assess the effects of these different conditions on prereproductive life span and reproductive traits. Results evidenced that at 21 and 28°C, at any salinity, A. franciscana from Laguna Mar Chiquita attained higher survival and fecundity, after a shorter prereproductive period, than A. persimilis from Salinas Grandes de Hidalgo. These data support that A. franciscana, considered a superspecies, exhibits higher phenotypic plasticity than A. persimilis, and that A. persimilis is better adapted to lower temperatures than A. franciscana. These differences in temperature and salinity tolerance could explain the present distribution of these two species in the South Cone in South America. Handling Editor: J. Melack     

Natural dibenzoxazepinones from leaves of Carex distachya: Structural elucidation and radical scavenging activity

Two new dibenzoxazepinones have been isolated from the leaves of Carex distachya, an herbaceous plant growing in the Mediterranean area. The structures have been elucidated on the basis of their spectroscopic properties. Bidimensional NMR (DQ-COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC) furnished important data useful for the characterization of the molecules. The compounds have been assayed, for the antioxidant activity, by measuring its capacity to scavenge the DPPH, the superoxide anion, and nitric oxide radicals.     

Natural feruloyl monoglyceride macrocycles as protecting factors against free-radical damage of lipidic membranes

Nine novel natural feruloyl monoglyceride (MGs) macrocycles have been isolated from the leaves of Carex distachya, an herbaceous plant growing in the Mediterranean maquis. All the structures have been elucidated on the basis of their spectroscopic features, especially two-dimensional NMR (DQ-COSY, TOCSY, NOESY, ROESY, HSQC, HMBC, HSQC-TOCSY) and ESI-MS. All the compounds have been assayed as protecting factors against the radical damage of the lipids by using different antioxidant tests.     

Taxonomic features ofSesleria calabrica (Poaceae), a neglected species from southern Italy

A taxonomic study of theSesleria juncifolia complex in the Italian Peninsula is presented, with a focus on southern Italian populations here named asSesleria calabrica (Deyl)Di Pietro comb. nov. et stat. nov. Karylological, morphological and anatomical characters were used for comparative analyses with populations of closely related species, such asS. juncifolia Suffren andSesleria apennina Ujhelyi occurring in the Italian Peninsula. Multivariate and univariate morphometric analyses, and qualitative morphological characters showed a distinct position ofS. calabrica. Populations ofS. calabrica differ in respect to various morphological characters of leaf and spikelet, such as the glume, lemma and palea length, stem length, the leaf sheath, leaf width, leaf thickness, and the total number of vascular bundles. Furthermore, previous karyological studies demonstrated that all the populations of bothS. juncifolia andS. apennina examined are octopolid (2n=8x=56), whereasS. calabrica was found to be dodecaploid (2n=12x=84).S. calabrica is strictly confined to the Pollino massif and the Orsomarso mountains in southern Italy, which represent the southernmost limit of theSesleria juncifolia complex distribution area both in Italy and in Europe. In addition toS. juncifolia andS. calabrica, the recognition ofS. apennina as a taxon distinct fromS. juncifolia is supported, at least for the area of the Apuan Alps.