Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

Cell interactions in the generation of chaetae pattern inDrosophila

Summary We have already shown that theachaetae-scute complex (AS-C) ofDrosophila is regulated by two genes,hairy andextramacrochaetae. Using mutants in these genes, we have analysed how different levels of expression of AS-C affect the pattern of chaetae. The results indicate that the spatial distribution of chaetae results from cell interactions, probably by a mechanism of lateral inhibition. The results are discussed in view of the different theories of pattern formation.     

Multiparametric comparison of mesenchymal stromal cells obtained from trabecular bone by using a novel isolation method with those obtained by iliac crest aspiration from the same subjects

Trabecular bone fragments from femoral heads are sometimes used as bone grafts and have been described as a source of mesenchymal progenitor cells. Nevertheless, mesenchymal stromal cells (MSC) from trabecular bone have not been directly compared with MSC obtained under standard conditions from iliac crest aspiration of the same patients. This is the ideal control to avoid inter-individual variation. We have obtained MSC by a novel method (grinding bone fragments with a bone mill without enzymatic digestion) from the femoral heads of 11 patients undergoing hip replacement surgery and compared them with MSC obtained by standard iliac crest aspiration of bone marrow from the same patients. We have shown that trabecular bone MSC obtained by mechanically fragmented femoral heads fulfil the immunophenotypic and multilineage (adipogenic, osteogenic and chondrogenic) differentiation criteria used to define MSC. We have also differentially compared cellular yields, growth kinetics, cell cycle assessment, and colony-forming unit-fibroblast content of MSC from both sources and conclude that these parameters do not significantly differ. Nevertheless, the finding of slight differences, such as a higher expression of the immature marker CD90, a lower expansion time through the different passages, and a higher percentage of cycling cells in the trabecular bone MSC, warrants further studies with the isolation method proposed here in order to gain further knowledge of the status of MSC in this setting. The present study was partially supported by grant HUS01B07 from the Consejería de Educación and by grant SAN196/SA13/07 from the Consejería de Sanidad, Junta de Castilla y León, Spain.     

Identification of novel genomic regions associated with resistance to <Emphasis Type="Italic">Pyrenophora tritici</Emphasis>-<Emphasis Type="Italic">repentis</Emphasis> races 1 and 5 in spring wheat landraces using association analysis

Tan spot, caused by Pyrenophora tritici-repentis, is a major foliar disease of wheat worldwide. Host plant resistance is the best strategy to manage this disease. Traditionally, bi-parental mapping populations have been used to identify and map quantitative trait loci (QTL) affecting tan spot resistance in wheat. The association mapping (AM) could be an alternative approach to identify QTL based on linkage disequilibrium (LD) within a diverse germplasm set. In this study, we assessed resistance to P. tritici-repentis races 1 and 5 in 567 spring wheat landraces from the USDA-ARS National Small Grains Collection (NSGC). Using 832 diversity array technology (DArT) markers, QTL for resistance to P. tritici-repentis races 1 and 5 were identified. A linear model with principal components suggests that at least seven and three DArT markers were significantly associated with resistance to P. tritici-repentis races 1 and 5, respectively. The DArT markers associated with resistance to race 1 were detected on chromosomes 1D, 2A, 2B, 2D, 4A, 5B, and 7D and explained 1.3–3.1% of the phenotypic variance, while markers associated with resistance to race 5 were distributed on 2D, 6A and 7D, and explained 2.2–5.9% of the phenotypic variance. Some of the genomic regions identified in this study correspond to previously identified loci responsible for resistance to P. tritici-repentis, offering validation for our AM approach. Other regions identified were novel and could possess genes useful for resistance breeding. Some DArT markers associated with resistance to race 1 also were localized in the same regions of wheat chromosomes where QTL for resistance to yellow rust, leaf rust and powdery mildew, have been mapped previously. This study demonstrates that AM can be a useful approach to identify and map novel genomic regions involved in resistance to P. tritici-repentis.     

Purification and characterization of an intracellular lipase from pleopods of whiteleg shrimp (Litopenaeus vannamei)

An intracellular lipase present in the whiteleg shrimp Litopenaeus vannamei was detected in pleopods. The lipase from pleopods was purified and characterized by biochemical and kinetic parameters. Purified intracellular lipase has a molecular mass of 196kDa, the polypeptide is assembled by two monomers, 95.26 and 63.36kDa. The enzyme lacks glycosylation, and it has an isoelectric point of 5.0. The enzyme showed the highest activity at a temperature range of 30-40°C at pH 8.0-10.0. Activity was completely inhibited by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate, suggesting that the intracellular lipase is a serine lipase. The lipase hydrolyzes short and long-chain triacylglycerides, as well as naphthol derivatives at comparable rates in contrast to other sources of lipases. Specific activity of 930U mg(-1) and 416.56U mg(-1) was measured using triolein and tristearin at pH 8.0 at 30°C as substrates, respectively. The lipase showed a K(M,app) of 41.03mM and k(cat)/K(M,app) ratio of 4.88 using MUF-butyrate as the substrate. The intracellular lipase described for shrimp has a potential role in hydrolysis of triacylglycerides stored as fat body, as has been shown in humans.     

Simultaneous Detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, Three Major Fish Pathogens, by Multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

Purification and partial characterization of human neutrophil elastase inhibitors from the marine snail Cenchritis muricatus (Mollusca)

Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (K_i) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human neutrophil elastase inhibitors described in a mollusk.