Abi mutants in Dictyostelium reveal specific roles for the SCAR/WAVE complex in cytokinesis.

Actin polymerization drives multiple cell processes involving movement and shape change. SCAR/WAVE proteins connect signaling to actin polymerization through the activation of the Arp2/3 complex. SCAR/WAVE is normally found in a complex with four other proteins: PIR121, Nap1, Abi2,and HSPC300 (Figure S1A available online) [1-3]. However,there is no consensus as to whether the complex functions as an unchanging unit or if it alters its composition in response to stimulation, as originally proposed by Edenet al. [1]. It also is unclear whether complex members exclusively regulate SCAR/WAVEs or if they have additional targets [4-6]. Here, we analyze the roles of the unique Dictyostelium Abi. We find that abiA null mutants show less severe defects in motility than do scar null cells, indicating--unexpectedly--that SCAR retains partial activity in the absence of Abi. Furthermore, abiA null mutants have a serious defect in cytokinesis, which is not seen in other SCAR complex mutants and is seen only when SCAR itself is present. Detailed examination reveals that normal cytokinesis requires SCAR activity, apparently regulated through multiple pathways.

Endothelial cell-specific ROS production increases susceptibility to aortic dissection

Background—Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. Methods and Results—A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II–mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45+ inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II–induced vascular smooth muscle cell ROS production. Conclusions—These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II–mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole and ivermectin in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.

Modulatory effect of fatty acids on fungicidal activity, respiratory burst and TNF-alpha and IL-6 production in J774 murine macrophages

The reported effects of different families of fatty acids (FA; SFA, MUFA, n-3 and n-6 PUFA) on human health and the importance of macrophage respiratory burst and cytokine release to immune defence led us to examine the influence of palmitic acid (PA), oleic acid (OA), linoleic acid, arachidonic acid, EPA and DHA on macrophage function. We determined fungicidal activity, reactive oxygen species (ROS) and cytokine production after the treatment of J774 cells with non-toxic concentrations of the FA. PA had a late and discrete stimulating effect on ROS production, which may be associated with the reduced fungicidal activity of the cells after treatment with this FA. OA presented a sustained stimulatory effect on ROS production and increased fungicidal activity of the cells, suggesting that enrichment of diets with OA may be beneficial for pathogen elimination. The effects of PUFA on ROS production were time-and dose-dependently regulated, with no evident differences between n-3 and n-6 PUFA. It was worth noting that most changes induced after stimulation of the cells with lipopolysaccharide were suppressed by the FA. The present results suggest that supplementation of the diet with specific FA, not classes of FA, might enable an improvement in host defence mechanisms or a reduction in adverse immunological reactions.

Lack of photoreceptor signaling alters the expression of specific synaptic proteins in the retina

Synaptic modulation by activity-dependent changes constitutes a cellular mechanism for neuronal plasticity. However, it is not clear how the complete lack of neuronal signaling specifically affects elements involved in the communication between neurons. In the retina, it is now well established that both chemical and electrical synapses are essential to mediate the transmission of visual signaling triggered by the photoreceptors. In this study, we compared the expression of synaptic proteins in the retinas of wild-type (WT) vs. rd/rd mice, an animal model that displays inherited and specific ablation of photoreceptors caused by a mutation in the gene encoding the beta-subunit of rod cGMP-phosphodiesterase (Pde6b(rd1)). We specifically examined the expression of connexins (Cx), the proteins that form the gap junction channels of electrical synapses, in addition to synaptophysin and synapsin 1, which are involved in the release of neurotransmitters at chemical synapses. Our results revealed that Cx36 gene expression levels are lower in the retinas of rd/rd when compared with WT. Confocal analysis indicated that Cx36 immunolabeling almost disappeared in the outer plexiform layer without significant changes in protein distribution within the inner plexiform layer of rd/rd retinas. Likewise, synaptophysin expression remarkably decreased in the outer plexiform layer of rd/rd retinas, and this down-regulation was also associated with diminished transcript levels. Furthermore, we observed down-regulation of Cx57 gene expression in rd/rd retinas when compared with WT and also changes in protein distribution. Interestingly, Cx45 and synapsin I expression in rd/rd retinas showed no noticeable changes when compared with WT. Taken together, our results revealed that the loss of photoreceptors leads to decreased expression of some synaptic proteins. More importantly, this study provides evidence that neuronal activity regulates, but is not essential to maintain, the expression of synaptic elements. (c) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) similar to 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.

Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates

Introduction: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes. Methods: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB. Results: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells. Conclusion: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-mu M verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca(2+). Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and maybe considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases. (C) 2010 Elsevier Ltd and ISBI. All rights reserved.

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta 1 integrin

We studied the induction of protease activity by the laminin alpha 1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha 1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M I cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta 1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta 1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta 1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta 1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells. (c) 2008 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Synthesis, Structure, and Phosphatase-Like Activity of a New Trinuclear Gd Complex with the Unsymmetrical Ligand H(3)L As a Model for Nucleases

The new trinuclear gadolinium complex [Gd(3)L(2)(NO(3))(2)(H(2)O)(4)]NO(3)center dot 8H(2)O (1) with the unsymmetrical ligand 2-[N-bis-(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N-bis(2-hydroxy-2-oxoethyl)aminomethyl] phenol (H(3)L) was synthesized and characterized. The new ligand H(3)L was obtained in good yield. Complex I crystallizes in an orthorhombic cell, space group Pcab. Kinetic studies show that complex 1 is highly active in the hydrolysis of the substrate 2,4-bis(dinitrophenyl)phosphate (K(m) = 4.09 mM, V(max) = 2.68 x 10(-2) mM s(-1), and k(cat) = V(max)/[1] = 0.67 s(-1)). Through a potentiometric study and determination of the kinetic behavior of 1 in acetonitrile/water solution, the species present in solution could be identified, and a trinuclear monohydroxo species appears to be the most prominent catalyst under mild conditions. Complex 1 displays high efficiency in DNA hydrolytic cleavage, and complete kinetic studies were carried out (K(m) = 4.57 x 10(-4) M, K(cat)` = 3.42 h(-1), and k(cat)`/K(m) = 7.48 x 10(3) M(-1) h(-1)). Studies with a radical scavenger (dimethyl sulfoxide, DMSO) showed that it did not inhibit the activity, indicating the hydrolytic action of 1 in the cleavage of DNA, and studies on the incubation of distamycin with plasmid DNA suggest that 1 is regio-specific, interacting with the minor groove of DNA.

Adenosine Modulatesa alpha(2)-Adrenergic Receptors within Specific Subnuclei of the Nucleus Tractus Solitarius in Normotensive and Spontaneously Hypertensive Rats

Adenosine Is known to modulate neuronal activity within the nucleus tractus solitarius (NTS). The modulatory effect of adenosine A, receptors (A(1R)) on alpha(2)-adrenoceptors (Adr(2R)) was evaluated using quantitative radioautography within NTS subnuclei and using neuronal culture of normotensive (WKY) and spontaneously hypertensive rats (SHR). Radioautography was used in a saturation experiment to measure Adr2R binding parameters (B(max), K(d)) In the presence of 3 different concentrations of N(6)-cyclopentyladenosine (CPA), an A(1R) agonist. Neuronal culture confirmed our radioautographic results. [(3)H]RX821002, an Adr(2R) antagonist, was used as a ligand for both approaches. The dorsomedial/dorsolateral subnucleus of WKY showed an increase in B(max) values (21%) Induced by 10 nmol/L of CPA. However, the subpostremal subnucleus showed a decrease in Kd values (24%) induced by 10 nmol/L of CPA. SHR showed the same pattern of changes as WKY within the same subnuclei; however, the modulatory effect of CPA was induced by I nmol/L (increased B(max), 17%; decreased K(d), 26%). Cell culture confirmed these results, because 10(-5) and 10(-7) mol/L of CPA promoted an Increase in [3H]RX821002 binding of WKY (53%) and SHR cells (48%), respectively. DPCPX, an AIR antagonist, was used to block the modulatory effect promoted by CPA with respect to Adr2R binding. In conclusion, our study shows for the first time an interaction between A(1R) that increases the binding of Adr2R within specific subnuclei of the NTS. This may be important In understanding the complex autonomic response induced by adenosine within the NTS. In addition, changes in interactions between receptors might be relevant to understanding the development of hypertension. (Hypertens Res 2008; 31: 2177-2186)

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

A short proregion of trialysin, a pore-forming protein of Triatoma infestans salivary glands, controls activity by folding the N-terminal lytic motif

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect that transmits the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas` disease. Its saliva contains trialysin, a protein that forms pores in membranes. Peptides based on the N-terminus of trialysin lyse cells and fold into alpha-helical amphipathic segments resembling antimicrobial peptides. Using a specific antiserum against trialysin, we show here that trialysin is synthesized as a precursor that is less active than the protein released after saliva secretion. A synthetic peptide flanked by a fluorophore and a quencher including the acidic proregion and the lytic N-terminus of the protein is also less active against cells and liposomes, increasing activity upon proteolysis. Activation changes the peptide conformation as observed by fluorescence increase and CD spectroscopy. This mechanism of activation could provide a way to impair the toxic effects of trialysin inside the salivary glands, thus restricting damaging lytic activity to the bite site.

Fluorimetric study of the pro-oxidant activity of EUK8 in the presence of hydrogen peroxide

The catalase mimetic complex Mn(III)-salen chloride (EUK8) was found to be pro-oxidant under low hydrogen peroxide concentrations. The increase in the fluorescence rate of the probe 1,2,3-dihydrorhodamine (DHR) in solution, as well as the carbonyl content of human serum albumin were found to be maximum at H(2)O(2):EUK8 molar ratios ranging from 0 to 2, supporting previous findings regarding the mechanism of EUK8 catalase activity and the formation of highly oxidative Mn(V)-O(2-) species. This pro-oxidant effect is precluded by the presence of glutathione. Cytotoxicity to HeLa cells, as probed by increased rate of oxidation of intracellular DHR, was not observed. Our findings suggest that the combination of H(2)O(2) and EUK8 at specific molar ratios, in the absence of reductants/antioxidants, induces the oxidation of organic molecules. It is shown that the fluorimetric determination of pro-oxidant activity of metal complexes is more sensitive than the colorimetric quantification of protein carbonyl content. The implications of our findings with respect to the somewhat confusing results arising from in vivo studies of EUK8 and other Mn(III) anti-oxidant metal complexes are discussed.

Influence of neodymium ions on photocatalytic activity of TiO(2) synthesized by sol-gel and precipitation methods

Titanium dioxide with and without the addition of neodymium ions was prepared using sol-gel and precipitation methods. The resulting catalysts were characterized by thermal analysis, X-ray diffraction and BET specific surface area. Neodymium addition exerted a remarkable influence on the phase transition temperature and the surface properties of the TiO(2) matrix. TiO(2) samples synthesized by precipitation exhibit an exothermic event related from the amorphous to anatase phase transition at 510 degrees C, whereas in Nd-doped TiO(2) this transition occurred at 527 degrees C. A similar effect was observed in samples obtained using sol-gel method. The photocatalytic reactivity of the catalysts was evaluated by photodegradation of Remazol Black B (RB) under ultraviolet irradiation. Nd-doped TiO(2) showed enhanced photodegradation ability compared to undoped TiO(2) samples, independent of the method of synthesis. In samples obtained by sol-gel, RB decoloration was enhanced by 16% for TiO(2) doped with 0.5% neodymium ions. (C) 2010 Elsevier B.V. All rights reserved.