Volume-regulated Cl- channels in human pleural mesothelioma cells

A novel xyloglucan-specific endo-β-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-β-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.     

Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s

Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by pluteus. On the contrary, the same treatment with cadmium induces continuous HSP70/72 synthesis and produces irregular gastrula embryos which then degenerate. Moreover, a long treatment induces over control embryos a slight increase in the amount of constitutive HSP75 during development while lead treatment depresses constitutive HSP75 at early stages and doubles its quantity at late stages.     

Role of multiple drug resistance protein 1 in neutral but not acidic glycosphingolipid biosynthesis

Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.     

Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5)

NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes.     

cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells

We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.     

The effects of Tynnanthus fasciculatus (Bignoniaceae) infusion on testicular parenchyma of adult Wistar rats

Traditional medicine provides strong guidance for scientific experiments involving plant products used by the Brazilian people. The species "cipó-cravo" (Tynnanthus fasciculatus) is a plant commonly used either to combat indigestion and stomach aches, or as a general stimulant and aphrodisiac. In this study, the effects of "cipó-cravo" infusion were investigated within the testicular parenchyma of adult Wistar rats. Rats were divided into 3 groups: a control (distilled water) and two treated groups, which received the plant infusion (100 and 200mg/animal/day). The 200mg dose promoted a significant increase of the testicular parenchyma weight and of the volume and total length of the seminiferous tubules, as well as in total daily sperm production and sperm production per gram of testis.     

No need for particle tracing: From accumulating fluid properties to novel blood coagulation model in the lattice Boltzmann method

The accumulation of a fluid property from the standpoint of a particle moving with non-steady fluid flow (i.e., platelet/blood-cell damage index in pulsating blood flow) is a challenged computational problem due to the current need for particle-tracing methods. The method we developed (dubbed VPI) enables the approximation of the Lagrangian integral in real-time for any point in space and time for the entire domain and which is easily integrated into the the lattice Boltzmann method. As an illustrative numerical example we applied our method to a blood coagulation model which was shown to accurately capture the coagulation characteristics observed in experiments, and therefore opening a door for more detailed study of systems which are currently hard to study using particle tracing methods.     

60-kDa heat shock protein of Chlamydia pneumoniae promotes a T helper type 1 immune response through IL-12/IL-23 production in monocyte-derived dendritic cells

Infection, in particular by Chlamydia pneumoniae (Cp), has been associated with atherosclerosis and coronary heart disease. Immune reactions to heat shock proteins (HSPs) have been advocated to link infection to atherosclerosis and its acute sequelae based on molecular mimicry with host HSPs. We have here evaluated the role played by recombinant Cp-HSP60 and Cp-HSP10 for their ability to induce maturation of human monocyte-derived dendritic cells (MDDC) and T cell polarization. Cp-HSP60, but not Cp-HSP10, induced a strong MDCC maturation, as assessed by the expression of co-stimulatory molecules and other markers. Secretion of regulatory cytokines and enhancement of antigen presenting ability of mature (m)MDDC toward a clear T helper (Th) 1 pattern were also induced by Cp-HSP60. An analysis of the IL-12 cytokine family demonstrated that Cp-HSP60-matured MDDC were able to express p35 and p40 mRNA subunits to form IL-12, and p19 and p40 subunits to form IL-23. Thus, preferential Th1 polarization of immune response induced by Cp-HSP60-matured MDDC appears to be due to the concomitant expression of IL-12 and IL-23. Our data suggest that Cp-HSP60-matured DC may contribute to T-cell mediated immunopathology of atherosclerosis via a chronic stimulation of Th1 immune responses.     

Surface layer proteins from Clostridium difficile induce inflammatory and regulatory cytokines in human monocytes and dendritic cells

Clostridium difficile, an etiological agent of most cases of antibiotic-associated diarrhea, exerts its pathological action mainly by the activity of toxin A and toxin B. Less known is the role that S-layer proteins (SLPs), predominant surface components of the bacterium, may play in pathogenesis. Here, we evaluate the ability of SLPs to modulate the function of human monocytes and dendritic cells (DC) and to induce inflammatory and regulatory cytokines, influencing the natural and adaptive immune response. To this aim, SLPs were extracted from the clinical isolate C253 and characterized for their effects on immune cells. SLPs induced the release of elevated amounts of interleukin (IL)-1β and IL-6 pro-inflammatory cytokines by resting monocytes, induced maturation of human monocyte-derived DC (MDDC), and enhanced proliferation of allogeneic T cells. C253-SLP-treated MDDC also secreted large amounts of IL-10 and IL-12p70 and induced a mixed Th1/Th2 orientation of immune response in naïve CD4 T cells. In conclusion, C. difficile SLPs may contribute to the pathogenicity of the bacterium by perturbing the fine balance of inflammatory and regulatory cytokines. These data are of interest also in the light of the possible use of SLPs in a multicomponent vaccine against C. difficile infections for high-risk patients.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at