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The Trans-Activator of Transcription (Tat) of Human Immunodeficiency Virus (HIV-1) is involved in virus replication and infection and can promote oxidative stress in human astroglial cells. In response, host cells activate transcription of antioxidant genes, including a subunit of System Xc− cystine/glutamate antiporter which, in turn, can trigger glutamate-mediated excitotoxicity. Here, we present data on the efficacy of bovine Lactoferrin (bLf), both in its native (Nat-bLf) and iron-saturated (Holo-bLf) forms, in counteracting oxidative stress in U373 human astroglial cells constitutively expressing the viral protein (U373-Tat). Our results show that, dependent on iron saturation, both Nat-bLf and Holo-bLf can boost host antioxidant response by up-regulating System Xc− and the cell iron exporter Ferroportin via the Nuclear factor erythroid 2-related factor (Nrf2) pathway, thus reducing Reactive Oxygen Species (ROS)-mediated lipid peroxidation and DNA damage in astrocytes. In U373-Tat cells, both forms of bLf restore the physiological internalization of Transferrin (Tf) Receptor 1, the molecular gate for Tf-bound iron uptake. The involvement of astrocytic antioxidant response in Tat-mediated neurotoxicity was evaluated in co-cultures of U373-Tat with human neuronal SH-SY5Y cells. The results show that the Holo-bLf exacerbates Tat-induced excitotoxicity on SH-SY5Y, which is directly dependent on System-Xc− upregulation, thus highlighting the mechanistic role of iron in the biological activities of the glycoprotein.

Several biological levels are considered as signals of environmental perturbations so that integrative biological responses have been proposed in environmental monitoring activities. We evaluated the use of markers in Mytilus galloprovincialis at two different levels (protein nitration and S-nitrosylation, and geometric morphometrics) to investigate the sea coastal environmental status. We studied adductor muscle, digestive gland and valves of mussels collected from 3 sampling sites (2 disturbed + 1 control) in eastern Tyrrhenian Sea, and 2 (1 + 1) in western Ionian Sea. Molecular and morphometric markers showed significant variations in relation to environmental conditions, but the most interesting result was evidenced by the analysis of covariation between both protein post-translational modifications and geometric morphometric descriptors. The main interesting finding regards the relationship between protein nitration values (an irreversible molecular marker) of both adductor muscle and digestive gland, and geometric morphometric descriptors (that are Procrustes’ distances) of valve shapes. Molecular and morphometric markers revealed useful warning systems in detecting environmental changes, even if causes and mechanisms altering both cellular metabolism and shape morphology are still unclear. Our findings suggested that pollutants affected the normal cellular metabolic pathways altering the correct functionality at higher biological levels and producing developmental detrimental noise visible on the shell.

In the field of research for designing and preparing innovative nanostructured systems, these systems are able to reveal the presence of heavy metals in water samples, and can efficiently and selectively interact with them, allowing for future applications in the field of water remediation. We investigated the electronic and molecular structure, as well as the morphology, of silver nanoparticles stabilized by mixed biocompatible ligands (the amino acid L-cysteine and the organic molecule citrate) in the presence of cadmium and arsenic ions. The molecular, electronic, and local structure at the ligands/silver nanoparticles interface was probed by the complementary synchrotron radiation-induced techniques (SR-XPS, NEXAFS and XAS). The optical absorption (in the UV-Vis range) of the nanosystem was investigated in the presence of Cd(II) and As(III) and the observed behavior suggested a selective interaction with cadmium. In addition, the toxicological profile of the innovative nanosystem was assessed in vitro using a human epithelial cell line HEK293T. We analyzed the viability of the cells treated with silver nanoparticles, as well as the activation of antioxidant response.

Background Meloidae (blister beetles) are known to synthetize cantharidin (CA), a toxic and defensive terpene mainly stored in male accessory glands (MAG) and emitted outward through reflex-bleeding. Recent progresses in understanding CA biosynthesis and production organ(s) in Meloidae have been made, but the way in which self-protection is achieved from the hazardous accumulation and release of CA in blister beetles has been experimentally neglected. To provide hints on this pending question, a comparative de novo assembly transcriptomic approach was performed by targeting two tissues where CA is largely accumulated and regularly circulates in Meloidae: the male reproductive tract (MRT) and the haemolymph. Differential gene expression profiles in these tissues were examined in two blister beetle species, Lydus trimaculatus (Fabricius, 1775) (tribe Lyttini) and Mylabris variabilis (Pallas, 1781) (tribe Mylabrini). Upregulated transcripts were compared between the two species to identify conserved genes possibly involved in CA detoxification and transport. Results Based on our results, we hypothesize that, to avoid auto-intoxication, ABC, MFS or other solute transporters might sequester purported glycosylated CA precursors into MAG, and lipocalins could bind CA and mitigate its reactivity when released into the haemolymph during the autohaemorrhaging response. We also found an over-representation in haemolymph of protein-domains related to coagulation and integument repairing mechanisms that likely reflects the need to limit fluid loss during reflex-bleeding. Conclusions The de novo assembled transcriptomes of L. trimaculatus and M. variabilis here provided represent valuable genetic resources to further explore the mechanisms employed to cope with toxicity of CA in blister beetle tissues. These, if revealed, might help conceiving safe and effective drug-delivery approaches to enhance the use of CA in medicine.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

Blister beetles owe their name to their ability to release cantharidin, a blistering terpene, the highest concentration of which is retained in male accessory glands. The anatomy and ultrastructure of the three pairs of male reproductive accessory glands and the glandular region of the two vasa deferentia of Meloe proscarabaeus were investigated using light, electron and ion beam microscopy. All of the mesodermal glands here analysed share a common structural organization with an outer muscular layer and an inner glandular epithelium facing a broad lumen in which the secretory products are released. Developed rough endoplasmic reticulum, Golgi systems, abundant mitochondria, numerous secretory vesicles and a microvillated apical membrane are commonly found in the cells of different glandular epithelia, suggesting that all accessory gland pairs as well as the vasa deferentia are involved in an active synthesis. Nevertheless, each pair of glands appears specialized in the production of a specific set of substances, as suggested by the peculiarities in cellular ultrastructure and by the different aspect of the secretions stored in their glandular lumen. The above cited features of male accessory glands of M. proscarabaeus are compared with those of other beetles and some hints on their potential role in producing and/or concentrating cantharidin are provided.

Aims. Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. Results. Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. Conclusions. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.

HIV-associated neurocognitive disorders (HANDs) affect a large part of HIV-infected patients, despite highly active antiretroviral therapy. HANDs occur in the absence of a direct infection of neurons. Nevertheless, viral proteins (e.g., Tat) are capable to cause neuronal dysfunction via oxidative stress, but the cellular pathways leading to HANDs are not yet fully defined. Here, we investigated the effects of Tat on Nrf2-mediated antioxidant response and system xc- expression in U373 human astroglial cells. Moreover, the effect of Tat-producing astrocytes on neuronal cell viability was assessed using SH-SY5Y cells as a culture model. We demonstrated that Tat produced by astrocytes was able to induce Nrf2 activation and system xc- expression in astrocytes, thus reducing cell viability of co-cultured neuronal cells. Furthermore, sulfasalazine, a specific system xc- inhibitor, was able to reduce extracellular glutamate and to prevent the reduction of neuronal viability, thus demonstrating that the neurotoxic effect was dependent on an increased glutamate release through the transporter. Our findings provide evidence of the involvement of astroglial Nrf2/system xc- pathway in the neurotoxicity induced by HIV-1 Tat protein, thereby suggesting how astrocytes may exacerbate neurodegeneration through the conversion of an antioxidant response to excitotoxicity.

Excitotoxic stress has been associated with several different neurological disorders, and it is one of the main causes of neuronal degeneration and death. To identify new potential proteins that could represent key factors in excitotoxic stress and to study the relationship between polyamine catabolism and excitotoxic damage, a novel transgenic mouse line overexpressing spermine oxidase enzyme in the neocortex (Dach-SMOX) has been engineered. These transgenic mice are more susceptible to excitotoxic injury and display a higher oxidative stress, highlighted by 8-Oxo-2'-deoxyguanosine increase and activation of defense mechanisms, as demonstrated by the increase of nuclear factor erythroid 2-related factor 2 (Nrf-2) in the nucleus. In Dach-SMOX astrocytes and neurons, an alteration of the phosphorylated and non-phosphorylated subunits of glutamate receptors increases the kainic acid response in these mice. Moreover, a decrease in excitatory amino acid transporters and an increase in the system xc- transporter, a Nrf-2 target, was observed. Sulfasalazine, a system xc- transporter inhibitor, was shown to revert the increased susceptibility of Dach-SMOX mice treated with kainic acid. We demonstrated that astrocytes play a crucial role in this process: neuronal spermine oxidase overexpression resulted in an alteration of glutamate excitability, in glutamate uptake and efflux in astrocytes involved in the synapse. Considering the involvement of oxidative stress in many neurodegenerative diseases, Dach-SMOX transgenic mouse can be considered as a suitable in vivo genetic model to study the involvement of spermine oxidase in excitotoxicity, which can be considered as a possible therapeutic target.

The impact of emerging chemical pollutants, on both status and functionality of aquatic ecosystems is worldwide recognized as a relevant issue of concern that should be assessed and managed by researchers, policymakers, and all relevant stakeholders. In Europe, the Reach Regulation has registered more than 100.000 chemical substances daily released in the environment. Furthermore, the effects related to the mixture of substances present in aquatic ecosystems may not be predictable on the basis of chemical analyses alone. This evidence, coupled with the dramatic effects of climate changes on water resources through water scarcity and flooding, makes urgent the application of innovative, fast and reliable monitoring methods. In this context, Effect-Based Methods (EBMs) have been applied in the urban stretch of the Tiber River (Central Italy) with the aim of understanding if detrimental pressures affect aquatic environmental health. In particular, different eco-genotoxicological assays have been used in order to detect genotoxic activity of chemicals present in the river, concurrently characterized by chemical analysis. Teratogenicity and embryo-toxicity have been studied in order to cover additional endpoints. The EBMs have highlighted the presence of diffuse chemical pollution and ecotoxicological effects in the three sampling stations, genotoxicological effects have been also detected through the use of different tests and organisms. The chemical analyses confirmed that in the aquatic ecosystems there is a diffuse presence, even at low concentrations, of emerging contaminants such as pharmaceuticals, not routinely monitored pesticides, personal care products, PFAS. The results of this study can help to identify an appropriate battery of EBMs for future studies and the application of more appropriate measures in order to monitor, mitigate or eliminate chemical contamination and remediate its adverse/detrimental effects on the ecosystem health.

In the field of tissue engineering, recombinant protein-based biomaterials made up of block polypeptides with tunable properties arising from the functionalities of the individual domains are appealing candidates for the construction of medical devices. In this work, we focused our attention on the preparation and structural characterization of nanofibers from a chimeric-polypeptide-containing resilin and elastin domain, designed on purpose to enhance its cell-binding ability by introducing a specific fibronectin-derived Arg-Gly-Asp (RGD) sequence. The polypeptide ability to self-assemble was investigated. The molecular and supramolecular structure was characterized by Scanning Electronic Microscopy (SEM) and Atomic Force Microscopy (AFM), circular dichroism, state-of-the-art synchrotron radiation-induced techniques X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The attained complementary results allow us to assess as H-bonds influence the morphology of the aggregates obtained after the self-assembling of the chimeric polypeptide. Finally, a preliminary investigation of the potential cytotoxicity of the polypeptide was performed by culturing human fetal foreskin fibroblast (HFFF2) for its use as biomedical device.

HIV-associated neurocognitive disorders (HANDs) affect a large part of HIV-infected patients, despite highly active antiretroviral therapy. HANDs occur in the absence of a direct infection of neurons. Nevertheless, viral proteins (e.g., Tat) are capable to cause neuronal dysfunction via oxidative stress, but the cellular pathways leading to HANDs are not yet fully defined. Here, we investigated the effects of Tat on Nrf2-mediated antioxidant response and system x

Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, "mesenchymal stem cells") in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells.

Telomeres are protein-bound tandemly repeated simple DNA sequences placed at the chromosome ends, their role being essential for maintaining genome integrity. Severe telomere damage can trigger potential carcinogenic events such as chromosome fusions, whilst telomere shortening results (at least in mammals) in a protective mechanism known as 'replicative senescence'. In most Metazoa, telomere shortening is avoided by the ribonucleoprotein telomerase. In this brief overview, we focused on the evolutionary conservation of telomeres and telomerase in basal Metazoa (Ctenophora, Porifera, Placozoa, Cnidaria). In all these taxa, telomerase seems to be active and telomeres show the canonical TTAGGG sequence. Presence of telomerase activity and absence of telomere shortening is in accordance with the lack of senescence seen in basal Metazoa, although a clear correspondence between the “demographic senescence” and “replicative senescence” remains to be elucidated. Nonetheless, basal Metazoa can be useful as model organisms in studies on the evolution of telomere biology, Evo-Devo investigations on the control of telomerase activity, the emergence of senescence, as well as telomere-independent effects of telomerase., Invertebrate Survival Journal, Vol 14 No 1 (2017)

A structural model of human ferroportin has been built using two Escherichia coli proteins belonging to the major facilitator superfamily of transporters. A potential iron binding site was identified in the inward-open conformation of the model, and its relevance was tested through measurement of iron export of HEK293T cells expressing wild-type or mutated ferroportin. Aspartates 39 and 181 were found to be essential for the transport ability of the protein. Noteworthy, the D181V mutation is naturally found in type 4 hemochromatosis with reticuloendothelial system iron retention phenotype. The outward-open conformation of ferroportin was also predicted, and showed that significant conformational changes must occur in the inward- to outward-open transition of ferroportin. In particular, putative iron ligands move several ångströms away from each other, leading to the logical conclusion that the iron binding site is not occupied by the metal in the outward-open conformation of ferroportin. © 2014 FEBS.

Previously, we reported that HIV-Tat elicits spermine oxidase (SMO) activity upregulation through NMDA receptor (NMDAR) stimulation in human SH-SY5Y neuroblastoma cells, thus increasing ROS generation, which in turn leads to GSH depletion, oxidative stress, and reduced cell viability. In several cell types, ROS can trigger an antioxidant cell response through the transcriptional induction of oxidative stress-responsive genes regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2). Here, we demonstrate that Tat induces both antioxidant gene expression and Nrf2 activation in SH-SY5Y cells, mediated by SMO activity. Furthermore, NMDAR is involved in Tat-induced Nrf2 activation. These findings suggest that the NMDAR/SMO/Nrf2 pathway is an important target for protection against HIV-associated neurocognitive disorders.

Inducible nitric oxide synthase (iNOS) is expressed in several cell types, particularly in inflammatory cells, in response to diverse proinflammatory stimuli, including viral proteins as HIV Tat and gp120. This response is preceded by an early decline in basal nitric oxide (NO) levels, dependent on a signaling leading to inhibition of the constitutive isoform of NO synthase (cNOS). This process requires critical levels of arachidonic acid (AA), generated by Calt;supgt;2+lt;/supgt;-dependent activation of cytosolic phospholipase A2, and is mediated by the downstream tyrosine kinase-dependent phosphorylation of cNOS. Lowering basal NO levels are necessary for the activation of nuclear factor-κB, and thus for the expression of a variety of genes regulated by this transcription factor, which include iNOS. Notably, NO and AA, two small lipid soluble molecules, can trigger the above responses also in distal cells. Thus, AA produced at the very early stages of the inflammatory response is a likely critical signal switching the regulation of the "NO tone" from physiological (i.e., mediated by cNOS) to pathological (i.e., mediated by iNOS). This later phase of the inflammatory response is often accompanied by the onset of deleterious effects in the tissue, in which a critical role is played by iNOS-derived NO (directly or indirectly, i.e., via formation of peroxynitrite) as well as by products of the AA cascade. In this review, the authors discuss the implications of the crosstalk between the NOS isoforms in HIV-associated neuro-pathogenesis highlighting the role of NO and AA as mediators of cytotoxicity.

The ceruloplasmin mutant R701W, that causes a dramatic phenotype in the young heterozygous patient carrying this mutation, has been shown to have profound effects also in cell culture models. Here we show that Golgi rearrangement and degradation of the iron exporter ferroportin, that follow transfection of cells with this mutant, are accompanied by the massive production of reactive oxygen species (ROS) in the cell. Scavenging ROS production with different antioxidants, including reduced glutathione and zinc, restores Golgi morphology and rescues ferroportin on the cell membrane.

The original version of this article unfortunately contained an error in the bars labels of the Fig. 8.

Relaxin, best known for its reproductive effects can be also viewed as a cardiovascular hormone. Its action in- cludes a marked increase in coronary blood flow, exerted through the up-regulation of inducible nitric oxide (NO) syn- thase (NOS II) and NO production in vascular endothelial and smooth muscle cells. This effect seems to involve NF- B, a classical transcription factor controlling NOS II induction by proinflammatory cytokines. The present study was designed to clarify the mechanisms underlying the relaxin-induced up-regulation of NOS II gene in endothelial cells. Rat coronary endothelial (RCE) cells were grown for 30 min, 2, 6 and 12 h in the absence or presence of 60 ng/ml porcine relaxin. Time-course analysis of the expression of NOS II and the proinflammatory cytokines IL-1 and TNF was per- formed. Relaxin induced the expression of NOS II transcript and protein at all these time points. No correlation was ob- served with the expression profiles of the genes for the assayed cytokines: IL-1 expression showed a first peak at 30 min. followed by a decline and a second peak at 12 h, whereas faint TNF- expression was only detected at 2 h. Relaxin re- tained the ability to induce NOS II transcript and to generate NO even in the presence of neutralizing anti- IL1 and/or anti-TNF- antibodies. The current findings suggest that the induction of NOS II by relaxin in coronary endothelial cells is a direct effect of this hormone and does not depend on a primary cytokine-mediated pathway that eventually results in NF- B activation and NOS II induction.

Background: Amyloid beta (A beta)-induced vascular dysfunction significantly contributes to the pathogenesis of Alzheimer's disease (AD). A beta is known to impair endothelial nitric oxide synthase (eNOS) activity, thus inhibiting endothelial nitric oxide production (NO). Method: In this study, we investigated A beta-effects on heat shock protein 90 (HSP90) interaction with eNOS and Akt in cultured vascular endothelial cells and also explored the role of oxidative stress in this process. Results: Treatments of endothelial cells (EC) with A beta promoted the constitutive association of HSP90 with eNOS but abrogated agonist (vascular endothelial growth factor (VEGF))-mediated HSP90 interaction with Akt. This effect resulted in blockade of agonist-mediated phosphorylation of Akt and eNOS at serine 1179. Furthermore, A beta stimulated the production of reactive oxygen species in endothelial cells and concomitant treatments of the cells with the antioxidant N-acetyl-cysteine (NAC) prevented A beta effects in promoting HSP90/eNOS interaction and rescued agonist-mediated Akt and eNOS phosphorylation. Conclusions: The obtained data support the hypothesis that oxidative damage caused by A beta results in altered interaction of HSP90 with Akt and eNOS, therefore promoting vascular dysfunction. This mechanism, by contributing to A beta-mediated blockade of nitric oxide production, may significantly contribute to the cognitive impairment seen in AD patients.

Background The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors. Methodology/Principal Findings We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells. Conclusions These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.

Nitric oxide (NO) regulates key aspects of cell metabolism through reversible inhibition of cytochrome c oxidase (CcOX), the terminal electron acceptor (complex IV) of the mitochondrial respiratory chain, in competition with oxygen. Recently, a constitutive mitochondrial NOS corresponding to a neuronal NOS-I isoform (mtNOS-I) has been identified in several tissues. The role of this enzyme might be to generate NO close enough to its target without a significant overall increase in cellular NO concentrations. An effective, selective, and specific NO action might be guaranteed further by a physical interaction between mtNOS-I and CcOX. This possibility has never been investigated. Here we demonstrate that mtNOS-I is associated with CcOX, as proven by electron microscopic immunolocalization and co-immunoprecipitation studies. By affinity chromatography, we found that association is due to physical interaction of mtNOS-I with the C-terminal peptide of the Va subunit of CcOX, which displays a consensus sequence for binding to the PDZ domain of mtNOS-I previously unreported for CcOX. The molecular details of the interaction have been analyzed by means of molecular modeling and molecular dynamics simulations. This is the first evidence of a protein-protein interaction mediated by PDZ domains involving CcOX.

Previous results have indicated that lipopolysaccharide (LPS) plus interferon-γ (IFNγ) inhibits nitric-oxide synthase (NOS)-I activity in glial cells. We report here that arachidonic acid (AA) plays a pivotal role in this response, which was consistently reproduced in different glial cell lines and in primary rat astrocytes. This notion was established using pharmacological inhibitors of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2) antisense oligonucleotides, and AA add-back experiments. This approach not only allowed the demonstration that AA promotes inhibition of NOS-I activity but also produced novel experimental evidence that LPS/IFNγ itself is a potential stimulus for NOS-I. Indeed, LPS/IFNγ fails to generate nitric oxide (NO) via NOS-I activation simply because it activates the AA-dependent signal that impedes NOS-I activity. Otherwise, LPS/IFNγ promotes NO formation, sensitive to exogenous AA, in cells in which cPLA2 is pharmacologically inhibited or genetically depleted. Because NO suppresses the NFκB-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFNγ-induced AA pathway provides optimal conditions for NFκB activation and subsequent NOS-II expression. Inhibition of cPLA2 activity, while reducing the availability of AA, consistently inhibited NFκB activation and NOS-II mRNA induction and delayed NO formation. These responses were promptly reestablished by addition of exogenous AA. Finally, we have demonstrated that the LPS/IFNγ-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.

A number of pathologies, including neurodegeneration and inflammation, have been associated with iron dysmetabolism in the brain. Hence, systems involved in iron homeostasis at the cellular level have aroused considerable interest in recent years. The iron exporter ferroportin-1 (FP) and the multicopper oxidase ceruloplasmin (CP) are essential for iron efflux from cells. By using RT-PCR, we demonstrate that FP and CP gene expression is up-regulated by treatment with the pro-inflammatory cytokine IL-1beta in rat C6 cells, taken as a glial cellular model. Following stimulation with IL-1beta, a higher expression level of CP and FP was also confirmed by Western blotting. Moreover, IL-1beta has been found to increase iron efflux from C6 cells, suggesting that both proteins may play a crucial role in iron homeostasis in pathological brain conditions, such as inflammatory and/or neurodegenerative diseases.

In the present study, we have analyzed the expression of nitric oxide synthase (NOS) in the preoptic-hypothalamo-hypophyseal system of the teleost Oreochromis niloticus. The assay for enzyme activity demonstrated that a constitutive NOS activity is present both in soluble and particulate fractions of the homogenates of diencephalons. Western blot analysis using an antibody against the N-terminus of human nNOS revealed two bands both in the supernatant and in the pellet. One band co-migrates at approximately 150 kDa with that detected in the rat cerebellum homogenates and presumably corresponds to neuronal NOS (nNOS) of mammals. The additional band, which migrates at approximately 180 kDa, might be attributed to an alternatively spliced nNOS isoform. Using NADPH diaphorase (NADPHd) histochemistry in combination with NOS immunohistochemistry, nNOS expression has been detected in preoptic nuclei, hypophysiotrophic nuclei of the ventral hypothalamus, and the pituitary gland. Various degrees of dissociation of NADPHd activity and nNOS immunoreactivity have been detected that could be attributed to the expression of different subtypes of nNOS in the preoptic/hypothalamo/hypophysial system of tilapia. In this paper, we also investigated the colocalization of nNOS with arginine-vasotocin (AVT) by means of immunolabeling of consecutive sections. Results suggest that NO may be colocalized with AVT in a subpopulation of neurosecretory neurons. Present findings suggest that nitric oxide (NO) is implicated in the modulation of hormone release in teleosts in a similar way to mammals.

HIV-associated neurocognitive disorder (HAND) is a common cognitive impairment in AIDS that affects 15 to 50% of adults infected with human immunodeficiency virus (HIV). Excessive amounts of nitric oxide (NO), as produced by inducible NO synthase (iNOS) upon exposure of activated microglia and astrocytes to cytokines and/or viral proteins (e.g., HIV tat and gp120), are assumed to contribute to neuronal abnormalities in HAND. Evidence exists supporting the notion that iNOS induction takes place after an early decline in physiological NO levels (i.e., NO released by constitutive NOS). Here, we demonstrate that HIV-1 gp120 is able to inhibit neuronal NOS through a cytosolic phospholipase A(2) (cPLA(2))-dependent arachidonic acid (AA) production, this response being critical for allowing activation of the transcriptional factor NF-kappa B and subsequent iNOS and interleukin-1 beta transcription in astroglial cells. In this context, AA seems to act as an upstream proinflammatory effector. In view of the pathogenic role of cPLA(2) in HAND, a deeper insight into the molecular and cellular mechanisms of its modulation may be helpful in finding new drugs to manage cognitive impairment in HIV-1 patients.

Nitric oxide (NO), produced in different cell types through the conversion of Larginine into Lcitrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NOreactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acidbase catalysed Cys?93-nitrosylation and Cys?93NOdenitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites 1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for CysNO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the 2 position has been observed in some NOreactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri or tetrapeptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691 696]. Here, we analyse the threedimensional structure of several proteins containing NOreactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the CysS? atomic structural microenvironment rather than on the tri or tetrapeptide sequence consensus motif.

Nitric oxide (NO) has been reported to regulate NF-kappaB, one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli. NO has been suggested to induce or inhibit the activation of NF-kappaB, its effect depending, among others, on the cell type considered. In this review, the inhibitory effect of NO on NF-kappaB (and subsequent suppression of NF-kappaB-dependent gene expression) in glial cells is reported. In particular, exogenous and endogenous NO has been observed to keep NF-kappaB suppressed, thus preventing the expression of NF-kappaB-induced genes, such as inducible NO synthase itself or HIV-1 long terminal repeat. Furthermore, the possible molecular mechanisms of NO-mediated NF-kappaB inhibition are discussed. More specifically, NO has been reported to suppress NF-kappaB activation inducing and stabilizing the NF-kappaB inhibitor, IkappaB-alpha. On the other hand, NO may inhibit NF-kappaB DNA binding through S-nitrosylation of cysteine residue (i. e., Cys62) of the p50 subunit. As a whole, a novel concept that the balance of intracellular NO levels may control the induction of NF-kappaB in glial cells has been hypothesized.

Gabexate mesylate (GM; commercialized under the brand name FOY) is a nonantigenic synthetic inhibitor of plasmatic and pancreatic serine proteinases that is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. The inhibitory effect of GM on nitric oxide synthase as well as serine proteinases and swine kidney copper amine oxidase, all acting on cationic substrates, has been investigated. On the basis of the available X-ray crystal structures of the enzymes considered, the possible binding mode(s) of GM has(have) been analyzed. The enzyme cross-inhibition by GM suggests that the use of this drug should be under careful control. With the aim to improve the scarce plasma stability of GM, the positively charged drug has been complexed to the surface of preformed anionic liposomes. The liposome-complexed GM half-life increases about five-fold, indicating the protective effect of liposomes on GM degradation. Moreover, the GM complexation with liposomes does not alter its inhibitory activity on NOS-I and porcine pancreatic trypsin.

The low density lipoprotein receptor-related protein (LRP) has been localized in human brain at the level of neurons, astrocytes and along capillary membranes. It is a multifunctional receptor responsible for binding and internalization of lipoproteins enriched with apoliprotein E, lipoprotein lipase, protease-alpha 2 macroglobulin complexes and plasminogen activator-inhibitor complexes. LRP expression is observed in cells involved in Alzheimer's disease, neoplastic transformation and tissue repair. Moreover, its synthesis is modulated during brain development. In this study we used the SK-N-AS human neuroblastoma cell line as a model system to study LRP expression during cellular differentiation induced by phorbol esters, retinoic acid and interferon gamma. Since LRP plays a major role in the regulation of lipid metabolism, the decreased levels of LRP measured by immunofluorescence, western blot and PCR on differentiated neuroblastoma cells may be the consequence of the lower requirements of cholesterol and lipids of differentiated cells in relation to their reduced mitotic index.

THE aim of this study was to investigate whether Mycobacterium tuberculosis (MTB) infection affects human immunodeficiency virus (HIV) replication in T67 human astrocytoma cells and whether HIV and MTB infections induce iNOS mRNA expression in T67 cells. T67 cells were susceptible to both HIV Lai and MTB infections, and MTB was able to increase HIV infection in T67 cells, as demonstrated by a marked increase in p24 release. Furthermore, both HIV and MTB infections strongly induced inducible nitric oxide synthase (iNOS) mRNA expression, as verified by RT-PCR. These findings suggest that HIV and MTB-induced iNOS expression of astroglial cells may be involved in the neuronal damage associated with HIV infection, particularly in the presence of opportunistic infections such as tuberculosis.

Chronic oxidative stress, which occurs in brain tissues of HIV-infected patients, is involved in the pathogenesis of HIV-associated dementia. Oxidative stress can be induced by HIV-1-secreted proteins, either directly or indirectly through the release of cytotoxic factors. In particular, HIV-1 Tat is able to induce neuronal death by interacting with and activating the polyamine-sensitive subtype of the NMDA receptor (NMDAR). Here, we focused on the role of polyamine catabolism in Tat-induced oxidative stress in human neuroblastoma (SH-SY5Y) cells. First, Tat was found to induce reactive oxygen species production and to affect cell viability in SH-SY5Y cells, these effects being mediated by spermine oxidase (SMO). Second, Tat was observed to increase SMO activity as well as decreasing the intracellular spermine levels. Third, Tat-induced SMO activation was completely prevented by the NMDAR antagonist MK-801, clearly indicating an involvement of NMDAR stimulation. Finally, pretreatment of cells with N -acetylcysteine, a scavenger of H 2 O 2 , and with MK-801 was able to completely inhibit reactive oxygen species formation and to restore cell viability. Altogether, these data strongly suggest a role for polyamine catabolism-derived H 2 O 2 in neurotoxicity as elicited by Tat-stimulated NMDAR.

Noninvasive methods in shell shape variation may help to understand evolution, ecology, stress and role of molluscan in aquatic ecosystems. Imaging analysis is a suitable diagnostic tool in morphological studies to (1) evaluate the health status of investigated animals, and (2) monitor sea coastal habitats. We introduce the feasibility of the cone-beam computed tomography as an optimal technique for 3D surface scanning to obtain virtual valve surfaces of Mytilus galloprovincialis , and analyze them exploiting the geometric morphometric facilities. Statistical output revealed morphological difference between mussels coming from different extensive rearing systems highlighting how the entire valve surface contributed to discriminate between groups when we compared 2- and 3D analyses. Many factors drive the morphological differences observed in the valve shape variation between the two sites, such as geographical genetic differentiation, natural environmental effects and culture conditions. The simplicity of the proposed methodology avoids damage and handling of individuals, makes this approach useful for morphological data collection, and helps to detect detrimental agents for sea ecosystems by using molluscans.

The ability of mammals to feel noxious stimuli lies in a heterogeneous group of primary somatosensory neurons termed nociceptors, which express specific membrane receptors, such as the Transient Receptor Potential (TRP) family. Here, we show that one of the most important nociceptive-like pathways is conserved in the freshwater coelenterate Hydra vulgaris, the most primitive organism possessing a nervous system. In particular, we found that H. vulgaris expresses TRPM3, a nociceptor calcium channel involved in the detection of noxious heat in mammals. Furthermore, we detected that both heat shock and TRPM3 specific agonist (i.e., pregnenolone sulfate) induce the modulation of the heat shock protein 70 (HSP70) and the nitric oxide synthase (NOS), two genes activated by TRP-mediated heat painful stimuli in mammals. As expected, these effects are inhibited by a TRPM3 antagonist (i.e., mefenamic acid). Interestingly, the TRPM3 agonist and heat shock also induce the expression of nuclear transcription erythroid 2-related factor (Nrf2) and superoxide dismutase (SOD), known markers of oxidative stress; noteworthy gene expression was also inhibited by the TRPM3 antagonist. As a whole, our results demonstrate the presence of conserved molecular oxidative/nociceptive-like pathways at the primordial level of the animal kingdom.

Astrocytes accumulate iron under chronic oxidative stress conditions in ageing and neurological disorders. The soybean isoflavone genistein possesses antioxidant properties and selective estrogen-like activities. Here, a possible role of genistein in modulation of iron transport was explored in glial cells. Genistein significantly increased iron export through estrogen receptor-beta-dependent p38 MAPK activation. Evidence is presented that this effect is associated to a p38 MAPK-triggered up-regulation of the iron export system made by ceruloplasmin and ferroportin-1, a pathway requiring activation of the transcription factor C/EBP.

Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through a coordinate induction of both ferroportin-1 (Fpn) and ceruloplasmin (Cp) synthesis. In this study, we have investigated the signaling pathways that are involved in the transcriptional activation of the Cp and Fpn. Our data show that the expression of Cp and Fpn in response to IL-1β requires the activation of MAP kinase pathways as a consequence of an IL-1β receptor stimulation. Moreover, we have observed that IL-1β regulates the expression of Cp and Fpn genes through (i) p38 MAPK-mediated activation of C/EBP transcription factor, (ii) ERK1/2-, JNK1- and partially p38 MAPK-dependent activation of AP-1, and through (iii) activation of NF-κB partially mediated by p38 MAPK.

The presence of nitric oxide (NO) pathway has been well demonstrated in the main invertebrate groups, showing parallel findings on the role of NO in vertebrates and invertebrates. Noteworthy is the example of the role played by the nitrergic pathway in the sensorial functions, mainly in olfactory-like systems. On the other hand, the emerging molecular information about NOSs from lower metazoans (Porifera, cnidarians up to higher invertebrates) suggests that NO pathways might represent examples of a parallel evolution of the NOS prototypes in different animal lineages. Nevertheless, increasing evidence suggests that NO is one of the earliest and most widespread signaling molecules in living organisms. Here, we attempt to provide a survey of current knowledge of the synthesis and possible roles of NO and the related signaling pathway in lower metazoans (i.e., Porifera and Cnidaria), two phyla forming a crucial bridge spanning the evolutionary gap between the protozoans and higher metazoans. From the literature data here reported, it emerges that future research on the biological roles of NO in basal metazoans is likely to be very important for understanding the evolution of signaling systems.

The multicopper oxidase ceruloplasmin plays a key role in iron homeostasis, and its ferroxidase activity is required to stabilize cell surface ferroportin, the only known mammalian iron exporter. Missense mutations causing the rare autosomal neurodegenerative disease aceruloplasminemia were investigated by testing their ability to prevent ferroportin degradation in rat glioma C6 cells silenced for endogenous ceruloplasmin. Most of the mutants did not complement (i.e. did not stabilize ferroportin) because of the irreversible loss of copper binding ability. Mutant R701W, which was found in a heterozygous very young patient with severe neurological problems, was unable to complement per se but did so in the presence of copper-glutathione or when the yeast copper ATPase Ccc2p was co-expressed, indicating that the protein was structurally able to bind copper but that metal loading involving the mammalian copper ATPase ATP7B was impaired. Notably, R701W exerted a dominant negative effect on wild type, and it induced the subcellular relocalization of ATP7B. Our results constitute the first evidence of "functional silencing" of ATP7B as a novel molecular defect in aceruloplasminemia. The possibility to reverse the deleterious effects of some aceruloplasminemia mutations may disclose new possible therapeutic strategies.

Human astrocytoma T67 cells constitutively express a neuronal NO synthase (NOS-I) and, following administration of lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma), an inducible NOS isoform (NOS-II). Previous results indicated that a treatment of T67 cells with the combination of LPS plus IFNgamma, by affecting NOS-I activity, also inhibited NO production in a very short time. Here, we report that under basal conditions, a NOS-I protein of about 150 kDa was weakly and partially tyrosine-phosphorylated, as verified by immunoprecipitation and Western blotting. Furthermore, LPS plus IFNgamma increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the presence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. On the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduced tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a suboptimal induction of NOS-II mRNA expression in T67 cells was enhanced by vanadate (or L-NAME) and inhibited by genistein. Because exogenous NO has been found to suppress NOS-II expression, the decrease of NO production that we have obtained from the inactivation of NOS-I by LPS/IFNgamma-induced tyrosine phosphorylation provides the best conditions for NOS-II expression in human astrocytoma T67 cells.