Dynamics of the linuron hydrolase libA gene pool size in response to linuron application and environmental perturbations in agricultural soil and on-farm biopurification systems

libA, a gene encoding a novel type of linuron hydrolase, was recently identified in the linuron-mineralizing Variovorax sp. strain SRS16. In order to assess the contribution of libA to linuron degradation in environmental settings, libA abundance was monitored in response to the application of linuron and to environmental perturbations in agricultural soil microcosms and microcosms simulating the matrix of on-farm biopurification systems. libA numbers were measured by real-time PCR and linked to reported data of Variovorax community composition and linuron mineralization capacity. In the soil microcosms and one biopurification system setup, libA numbers responded to the application of linuron and environmental changes in congruency with the modulation of linuron mineralization capacity and the occurrence of a particular Variovorax phylotype (phylotype A). However, in another biopurification system setup, no such correlations were found. Our data suggest that in the simulated environmental settings, the occurrence of libA can be linked to the linuron mineralization capacity and that libA is primarily hosted by Variovorax phylotype A strains. However, the results also suggest that, apart from libA, other, as-yet-unknown isofunctional genes play an important role in linuron mineralization in the environment.     

Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K⁺, Cl^–, and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl^– current (I_Cl,swell) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl^– or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied I_Cl,swell and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted I_Cl,swell, but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl^–, taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane. caveolae; osmolyte; epithelial cell; chloride channel     

Differential effects of matrix and growth factors on endothelial and fibroblast motility: application of a modified cell migration assay

Cell migration is crucial in virtually every biological process and strongly depends on the nature of the surrounding matrix. An assay that enables real-time studies on the effects of defined matrix components and growth factors on cell migration is not available. We have set up a novel, quantitative migration assay, which enables unharmed cells to migrate along a defined matrix. Here, we used this so-called barrier-assay to define the contribution of fibronectin (FN) and Collagen-I (Col-I) to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and lysophosphatidic acid (LPA)-induced cell migration of endothelial cells (EC) and fibroblasts. In EC, both FN and Col-I stimulated migration, but FN-induced motility was random, while net movement was inhibited. Addition of bFGF and VEGF overcame the effect of FN, with VEGF causing directional movement. In contrast, in 3T3 fibroblasts, FN stimulated motility and this effect was enhanced by bFGF. This motility was more efficient and morphologically completely different compared to LPA stimulation. Strikingly, directional migration of EC was not paralleled by higher amounts of stable microtubules (MT) or an increased reorientation of the microtubule-organizing centre (MTOC). For EC, the FN effect appeared concentration dependent; high FN was able to induce migration, while for fibroblasts both low and high concentrations of FN induced motility. Besides showing distinct responses of the different cells to the same factors, these results address contradictive reports on FN and show that the interplay between matrix components and growth factors determines both pattern and regulation of cell migration. J. Cell. Biochem. 99: 1536-1552, 2006. (c) 2006 Wiley-Liss, Inc.     

Seed Dispersal by Golden-headed Lion Tamarins Leontopithecus chrysomelas in Southern Bahian Atlantic Forest, Brazil

Seed dispersal by small primates may be particularly relevant in areas where populations of larger frugivores have been reduced or extinguished by hunting and/or habitat disturbance. In this context, the aim of this study was to evaluate the role of the golden-headed lion tamarin Leontopithecus chrysomelas as a seed disperser in Atlantic forest remnants in Brazil. To this end, we opportunistically collected feces deposited during observations on the feeding behavior of two groups of golden-headed lion tamarins ranging in the degraded areas of the Una Biological Reserve, Bahia, Brazil, from February 2006 to January 2007. We collected 587 fecal samples, of which 524 contained seeds from 24 plant species, distributed over 13 families. Disregarding seeds of<3 mm, the majority of seeds recovered were bromeliad seeds. In general, ingestion of seeds by golden-headed lion tamarins did not improve the germination proportion or decrease the germination delay of seeds, with the exception of Aechmea spp. seeds. The tamarins encountered different habitats during their daily activity period, while feeding and defecating. Consequently, some seeds were transported to different habitats including disturbed areas. Thus, the role of seed dispersal in combination with the daily movement pattern of L. chrysomelas contributes to the persistence of fruit plants and epiphyte species and to the natural regeneration process within Atlantic forest remnants.     

Polymorphisms in the CD28/CTLA4/ICOS genes: role in malignant melanoma susceptibility and prognosis?

The appearance of vitiligo and spontaneous regression of the primary lesion in melanoma patients illustrate a relationship between tumor immunity and autoimmunity. T lymphocytes play a major role both in tumor immunity and autoimmunity. CD28, Cytotoxic T lymphocyte antigen 4 (CTLA4) and inducible costimulator (ICOS) molecules are important secondary signal molecules in the T lymphocyte activation. Single nucleotide polymorphisms (SNPs) in the CD28/CTLA4/ICOS gene region were reported to be associated with several autoimmune diseases including, type-1 diabetes, SLE, autoimmune thyroid diseases and celiac disease. In this study, we investigated the association of SNPs in the CD28, CTLA4 and ICOS genes with the risk of melanoma. We also assessed the prognostic effect of the different polymorphisms in melanoma patients. Twenty-four tagging SNPs across the three genes and four additional SNPs were genotyped in a cohort of 763 German melanoma patients and 734 healthy German controls. Influence on prognosis was determined in 587 melanoma cases belonging to stage I or II of the disease. In general, no differences in genotype or allele frequencies were detected between melanoma patients and controls. However, the variant alleles for two polymorphisms in the CD28 gene were differentially distributed in cases and controls. Similarly no association of any polymorphism with prognosis, except for the rs3181098 polymorphism in the CD28 gene, was observed. In addition, individuals with AA genotype for rs11571323 polymorphism in the ICOS gene showed reduced overall survival. However, keeping in view the correction for multiple hypothesis testing our results suggest that the polymorphisms in the CD28, CTLA4 and ICOS genes at least do not modulate risk of melanoma and nor do those influence the disease prognosis in the investigated population.     

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.     

FAM-MDR: A Flexible Family-Based Multifactor Dimensionality Reduction Technique to Detect Epistasis Using Related Individuals

We propose a novel multifactor dimensionality reduction method for epistasis detection in small or extended pedigrees, FAM-MDR. It combines features of the Genome-wide Rapid Association using Mixed Model And Regression approach (GRAMMAR) with Model-Based MDR (MB-MDR). We focus on continuous traits, although the method is general and can be used for outcomes of any type, including binary and censored traits. When comparing FAM-MDR with Pedigree-based Generalized MDR (PGMDR), which is a generalization of Multifactor Dimensionality Reduction (MDR) to continuous traits and related individuals, FAM-MDR was found to outperform PGMDR in terms of power, in most of the considered simulated scenarios. Additional simulations revealed that PGMDR does not appropriately deal with multiple testing and consequently gives rise to overly optimistic results. FAM-MDR adequately deals with multiple testing in epistasis screens and is in contrast rather conservative, by construction. Furthermore, simulations show that correcting for lower order (main) effects is of utmost importance when claiming epistasis. As Type 2 Diabetes Mellitus (T2DM) is a complex phenotype likely influenced by gene-gene interactions, we applied FAM-MDR to examine data on glucose area-under-the-curve (GAUC), an endophenotype of T2DM for which multiple independent genetic associations have been observed, in the Amish Family Diabetes Study (AFDS). This application reveals that FAM-MDR makes more efficient use of the available data than PGMDR and can deal with multi-generational pedigrees more easily. In conclusion, we have validated FAM-MDR and compared it to PGMDR, the current state-of-the-art MDR method for family data, using both simulations and a practical dataset. FAM-MDR is found to outperform PGMDR in that it handles the multiple testing issue more correctly, has increased power, and efficiently uses all available information.     

The regulatory role of CD45 on rat NK cells in target cell lysis.

To investigate the role of CD45 in rat NK cell function, we developed new mAbs directed against rat CD45. mAb ANK12 binds to a high molecular isoform of CD45 and mAb ANK74 binds to the common part on all known CD45 isoforms, as has been described for the anti-rat CD45 mAb OX1. The ability of these mAbs to affect NK cell-mediated lysis was tested using the Fc receptor-positive target cell line P815. mAb ANK12 was found to significantly enhance the lysis of P815, whereas ANK74 and the anti-CD45 mAb OX1 did not. In addition, cross-linking of the CD45 isoform by ANK12 induced tyrosine phosphorylation of specific proteins in NK cells. Subsequently, the involvement of CD45 in the negative signaling after "self" MHC class I recognition by rat NK cells was investigated. The anti-CD45 mAbs were found to affect NK cell-mediated lysis of syngeneic tumor cell lines, depending upon the expression level of MHC class I on target cells. mAbs ANK74 and OX1 only inhibited lysis of the syngeneic tumor cell lines that expressed low levels of MHC class I. Furthermore, both mAbs caused an inhibition of NK cell-mediated lysis of these tumor cell lines when MHC class I molecules on the tumor cell lines were masked by an Ab. These results suggest that CD45 regulates the inhibitory signal pathway after self MHC class I recognition, supposedly by dephosphorylation of proteins.     

Toxic effects caused by heavy metals in the yeast Saccharomyces cerevisiae: a comparative study

The decreasing order of toxicity of select heavy metals on the yeast Saccharomyces cerevisiae, in 10 mM MES (2-(N-morpholino)ethanesulfonic acid) pH buffer at pH 6.0, was found to be copper, lead, and nickel. Heavy metal (200 microM) induced a decrease in the number of viable cells by about 50% in the first 5 min for copper and in 4 h for lead, while nickel was not toxic up to a 200 microM concentration over a period of 48 h. Glucose (25 mM) strongly enhanced the toxic effect of 50 microM copper but had little or no effect on the toxicity of 200 microM lead or nickel. Copper, lead, and nickel induced the leakage of UV260-absorbing compounds from cells with different kinetics. The addition of 0.5 mM calcium, before addition of 200 microM copper, showed a protective action against cell death and decreased the release of UV-absorbing compounds, while no effect was observed against lead or nickel toxic effects. Copper complexation capacities of the filtrates of cells exposed for 2 h in 200 microM copper and 24 h in 200 microM lead were 51 and 14 microM, respectively. The implication of the complexation shown by these soluble compounds in the bioavailability of heavy metals is discussed.