Citrate-silver nanoparticles and their impact on some environmental beneficial fungi.

Colloidal suspensions of silver nanoparticles (AgNPs) with surface modified by capping with citrate ions were synthesized by chemical reduction method. Transmission and Scanning Electron Microscopy as well as darkfield Optical Microscopy provided information on the nanoparticle morphology, with fine symmetrical grains and log-normal fitted size distribution. Small Angle X-ray Scattering method allowed theoretical confirmation of colloidal silver nanoparticle fine granularity, based on measurements in the native fluid sample. UV-Vis spectrophotometry allowed studying the Localized Surface Plasmon Resonance band versus the stability of the citrate-AgNP sample after storage and after UV-C exposure. The colloidal AgNP impact on Phanerochaete chrysosporium environmental microorganisms was studied by specific biochemical investigations. Silver released from the colloidal suspension of AgNPs was supposed to induce changes in some antioxidant enzymes and in some enzymes of Krebs' cycle. Catalase activity was moderately changed (an increase with over 50%) as well as superoxide dismutase activity, while the diminution of the activities of four dehydrogenases synthesized in the fungus mycelium was emphasized also: a decrease with about 60% for malate dehydrogenase, with over 50% for isocitrate dehydrogenase and succinate dehydrogenase and with about 40% for alpha-ketoglutarate dehydrogenase. These findings suggested the nano-toxicological issues of citrate-AgNPs impact on the environmental beneficial microorganisms.

The effect of visible light on gold nanoparticles and some bioeffects on environmental fungi.

The oxidative stress induced by light exposed gold nanoparticles in some microorganism cells was investigated. Gold nanoparticles are currently used in biomedical and pharmaceutical research. For this study citrate-gold nanoparticles were synthesized in alkaline conditions at constant temperature of 85°C under magnetic stirring. Equal volumes of such prepared colloidal solution, were exposed to visible light at different wavelengths for 90min at room temperature. The spectra in the visible and ultraviolet range have revealed an increase in the intensity of the absorption band for gold nanoparticles exposed to light, due to the effect of surface plasmon resonance. Versatility of gold nanoparticles photocatalytic action was shown by means of manipulating wavelengths of incident light, which evidenced differences in the bioeffects induced in cellulolytic fungi - known for their environmental role but also for other applications such as in cosmetics industry. The comparative analysis of fungal response to gold nanoparticle stressors has revealed different enzyme activity and lipid peroxidation when fungi were supplied with gold nanoparticles exposed to different wavelength lights. The activity of catalase and superoxide dismutase were remarkably increased for green light exposure of gold nanoparticles suggesting fungi adaption to increased oxidative stress induced by irradiated particles; increased level of lipid peroxidation was showed by high concentration of malondialdehyde for white light exposed gold particles since antioxidant enzymes were less active.

Structure of NPP1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification.

Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) converts extracellular nucleotides into inorganic pyrophosphate, whereas its close relative NPP2/autotaxin hydrolyzes lysophospholipids. NPP1 regulates calcification in mineralization-competent tissues, and a lack of NPP1 function underlies calcification disorders. Here, we show that NPP1 forms homodimers via intramembrane disulfide bonding, but is also processed intracellularly to a secreted monomer. The structure of secreted NPP1 reveals a characteristic bimetallic active site and a nucleotide-binding groove, but it lacks the lipid-binding pocket and open tunnel present in NPP2. A loop adjacent to the nucleotide-binding site, which is disordered in NPP2, is well ordered in NPP1 and might promote nucleotide binding. Remarkably, the N-terminal somatomedin B-like domains of NPP1, unlike those in NPP2, are flexible and do not contact the catalytic domain. Our results provide a structural basis for the nucleotide pyrophosphatase activity of NPP1 and help to understand how disease-causing mutations may affect NPP1 structure and function.

Structural basis of substrate discrimination and integrin binding by autotaxin.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

Rapid clearance of the circulating metastatic factor autotaxin by the scavenger receptors of liver sinusoidal endothelial cells.

Autotaxin, also known as NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2), is a secreted lysophospholipase-D that generates lysophosphatidic acid and thereby promotes the metastatic and invasive properties of tumor cell as well as angiogenesis. We show here that, in mice, NPP2 is cleared from the circulation within minutes and is retained by the liver sinusoidal endothelial cells (LSECs). The binding of NPP2 to isolated LSECs resulted in its degradation and could be competed for with ligands of the scavenger receptor family. Our finding that circulating NPP2 has a rapid turnover has important implications for its development as an anti-cancer target.

Domain interplay mediated by an essential disulfide linkage is critical for the activity and secretion of the metastasis-promoting enzyme autotaxin.

Autotaxin or NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2) is a secreted lysophospholipase-D that promotes metastasis and tumor growth by its ability to generate lysophosphatidic acid. Considerable evidence suggests that inhibitors of NPP2 can be used as a novel therapy for the treatment of cancer. Although most attention is currently directed toward the development of inhibitors of the catalytic site, we have explored whether NPP2 can also be targeted through its non-catalytic nuclease-like domain. We demonstrate here that the catalytic and nuclease-like domains are covalently linked by an essential disulfide bridge between Cys(413) and Cys(805). Within the nuclease-like domain, residues 829-850 are involved in the secretion of NPP2, and Lys(852) is required for the expression of catalytic activity. These data show that the nuclease-like domain is crucial for catalysis by NPP2 and is a possible target to generate inhibitors.

Over-expression of Hsp27 does not influence disease in the mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a chronic, adult-onset neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons, resulting in severe atrophy of muscles and death. Although the exact pathogenic mechanism of mutant superoxide dismutase 1 (SOD1) causing familial ALS is still elusive, toxic protein aggregation leading to insufficiency of chaperones is one of the main hypotheses. In this study, we investigated the effect of over-expressing one of these chaperones, heat shock protein 27 (Hsp27), in ALS. Mice over-expressing the human, mutant SOD1(G93A) were crossed with mice that ubiquitously over-expressed human Hsp27. Even though the single transgenic hHsp27 mice showed protection against spinal cord ischemia, the double transgenic SOD1(G93A)/hHsp27 mice did not live longer, and did not show a significant delay in the onset of disease compared to their SOD1(G93A) littermates. There was no protective effect of hHsp27 over-expression on the motor neurons and on the mutant SOD1 aggregates in the double transgenic SOD1(G93A)/hHsp27 mice. In conclusion, despite the protective action against acute motor neuron injury, Hsp27 alone is not sufficient to protect against the chronic motor neuron injury due to the presence of mutant SOD1.

An essential oligomannosidic glycan chain in the catalytic domain of autotaxin, a secreted lysophospholipase-D.

Autotaxin/NPP2, a secreted lysophospholipase-D, promotes cell proliferation, survival, and motility by generating the signaling molecule lysophosphatidic acid. Here we show that ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is N-glycosylated on Asn-53, Asn-410, and Asn-524. Mutagenesis and deglycosylation experiments revealed that only the glycosylation of Asn-524 is essential for the expression of the catalytic and motility-stimulating activities of NPP2. The N-glycan on Asn-524 was identified as Man8/9GlcNAc2, which is rarely present on mature eukaryotic glycoproteins. Additional studies show that this Asn-524-linked glycan is not accessible to alpha-1,2-mannosidase, suggesting that its non-reducing termini are buried inside the folded protein. Consistent with a structural role for the Asn-524-linked glycan, only the mutation of Asn-524 augmented the sensitivity of NPP2 to proteolysis and increased its mobility during Blue Native PAGE. Asn-524 is phylogenetically conserved and maps to the catalytic domain of NPP2, but a structural model of this domain suggests that Asn-524 is remote from the catalytic site. Our study defines an essential role for the Asn-524-linked glycan chain of NPP2.

Ivermectin inhibits AMPA receptor-mediated excitotoxicity in cultured motor neurons and extends the life span of a transgenic mouse model of amyotrophic lateral sclerosis.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitotoxicity contributes to the selective motor neuron death in amyotrophic lateral sclerosis (ALS). In this study, we investigated the effect of P2 receptor-influencing substances on kainate-induced motor neuron death in an in vitro model for AMPA receptor-mediated excitotoxicity. Complete protection was found after preincubation of the motor neurons with ivermectin or Cibacron Blue 3G-A. Preincubation with both P2X4 modulators did not influence the number or Ca2+ permeability of the AMPA receptors and addition during kainate stimulation alone had no effect. Preincubation with a low concentration of ATP, the natural agonist of the P2X4 receptor, also protected the motor neurons against a subsequent excitotoxic stimulation, while high concentrations of ATP were toxic. Moreover, ivermectin increased the toxicity of low ATP concentrations, indicating that ivermectin can potentiate the effect of ATP on its receptor. Ivermectin and ATP also protected against hypoxia/hypoglycemia. To further investigate the relevance of these findings for ALS, we treated SOD1(G93A)-mice, a transgenic animal model for familial ALS, with ivermectin. This resulted in an extension of the life span of these mice with almost 10%. We conclude that ivermectin induces a mechanism in motor neurons, in vivo and in vitro, that protects against subsequent excitotoxic insults. Our in vitro data indicate that this protective mechanism is due to the potentiation by ivermectin of an effect of ATP mediated by the P2X4 receptor.