Neutrophil Extracellular Traps Profiles in Patients with Incident Systemic Lupus Erythematosus and Lupus Nephritis.

OBJECTIVE: Neutrophil extracellular traps (NET) expose modified antigens for autoantibodies in vasculitis. Little is known about levels and removal pathways of NET in systemic lupus erythematosus (SLE), especially in lupus nephritis (LN). We determined circulating levels and defined NET removal in large subsets of patients with incident SLE (iSLE), some of whom had new-onset nephritis. METHODS: Serum levels of NET (ELISA), DNase1/DNase1L3 (ELISA), and DNase activity (functional assay) were determined in 216 patients with iSLE [103 had incident LN (iLN)], in 50 patients with other primary glomerulonephritis, and in healthy controls. Ex vivo NET production by neutrophils purified from a random selection of patients was quantified as elastase/DNA release and by immunofluorescence techniques. RESULTS: Serum NET levels were very high in iSLE/iLN compared to all groups of controls and correlated with anti-dsDNA, C3-C4, and proteinuria; iLN had the highest levels. DNase activity was decreased in iLN compared to SLE (20% had one-half DNase activity) despite similar serum levels of DNase1/DNase1L3. In these cases, pretreatment of serum with protein A restored DNase efficiency; 1 patient was homozygous for a c.289_290delAC variant of DNASE1L3. Ex vivo NET production by neutrophils purified from LN, SLE, and normal controls was similar in all cases. CONCLUSION: Patients with iLN have increased circulating NET and reduced DNase activity, the latter being explained by the presence of inhibitory substances in circulation and/or by rare DNase1L3 mutations. Accumulation of NET derives from a multifactorial mechanism, and is associated and may contribute to disease severity in SLE, in particular to renal lesions. (Clinical trial registration: The Zeus study was registered at ClinicalTrials.gov, study number NCT02403115).

Homocysteine and A2A-D2 Receptor-Receptor Interaction at Striatal Astrocyte Processes.

The interaction between adenosine A2A and dopamine D2 receptors in striatal neurons is a well-established phenomenon and has opened up new perspectives on the molecular mechanisms involved in Parkinson's disease. However, it has barely been investigated in astrocytes. Here, we show by immunofluorescence that both A2A and D2 receptors are expressed in adult rat striatal astrocytes in situ, and investigate on presence, function, and interactions of the receptors in the astrocyte processes-acutely prepared from the adult rat striatum-and on the effects of homocysteine on the A2A-D2 receptor-receptor interaction. We found that A2A and D2 receptors were co-expressed on vesicular glutamate transporter-1-positive astrocyte processes, and confirmed that A2A-D2 receptor-receptor interaction controlled glutamate release-assessed by measuring the [3H]D-aspartate release-from the processes. The complexity of A2A-D2 receptor-receptor interaction is suggested by the effect of intracellular homocysteine, which reduced D2-mediated inhibition of glutamate release (homocysteine allosteric action on D2), without interfering with the A2A-mediated antagonism of the D2 effect (maintained A2A-D2 interaction). Our findings indicate the crucial integrative role of A2A-D2 molecular circuits at the plasma membrane of striatal astrocyte processes. The fact that homocysteine reduced D2-mediated inhibition of glutamate release could provide new insights into striatal astrocyte-neuron intercellular communications. As striatal astrocytes are recognized to be involved in Parkinson's pathophysiology, these findings may shed light on the pathogenic mechanisms of the disease and contribute to the development of new drugs for its treatment.

The HMGA gene family in chordates: evolutionary perspectives from amphioxus.

High mobility group A proteins of vertebrates, HMGA1 and 2, are chromatin architectural factors involved in development, cell differentiation, and neoplastic transformation. Here, we characterize an amphioxus HMGA gene ortholog and analyze its expression. As a basal chordate, amphioxus is well placed to provide insights into the evolution of the HMGA gene family, particularly in the transition from invertebrates to vertebrates. Our phylogenetic analysis supports the basal position of amphioxus, echinoderm, and hemichordate HMGA sequences to those of vertebrate HMGA1 and HMGA2. Consistent with this, the genomic landscape around amphioxus HMGA shares features with both. Whole mount in situ hybridization shows that amphioxus HMGA mRNA is detectable from neurula stage onwards in both nervous and non-nervous tissues. This correlates with protein expression monitored immunocytochemically using antibodies against human HMGA2 protein, revealing especially high levels of expression in cells of the lamellar body, the amphioxus homolog of the pineal, suggesting that the gene may have, among its many functions, an evolutionarily conserved role in photoreceptor differentiation.

Evaluation of the Acquisition of the Aerobic Metabolic Capacity by Myelin, during its Development.

Our previous reports indicate that the electron transfer chain and FoF1-ATP synthase are functionally expressed in myelin sheath, performing an extra-mitochondrial oxidative phosphorylation (OXPHOS), which would provide energy to the nerve axon. This supports the idea that myelin plays a trophic role for the axon. Although the four ETC complexes and ATP synthase are considered exquisite mitochondrial proteins, they are found ectopically expressed in several membranous structures. This study was designed to understand when and how the mitochondrial OXPHOS machinery is embedded in myelin, following myelinogenesis in the rat, which starts at birth and continues until the first month of age. Rats were sacrificed at different time points (from day 5 to 90 post birth). Western blot, immunofluorescence microscopy, luminometric, and oximetric analyses show that the isolated myelin starts to show OXPHOS components around the 11th day after birth and increases proportionally to the rat age, becoming similar to those of adult rat around the 30-third day. Interestingly, WB data show the same temporal relationship between myelinogenesis and appearance of proteins involved in mitochondrial fusion and cellular trafficking. It may be speculated that the OXPHOS complexes may be transferred to the endoplasmic reticulum membrane (known to interact with mitochondria) and from there through the Golgi apparatus to the forming myelin membrane.

Review: Morphofunctional and biochemical markers of stress in sea urchin life stages exposed to engineered nanoparticles.

We describe the use of different life stages of the Mediterranean sea urchin Paracentrotus lividus for the assessment of the possible risk posed by nanoparticles (NPs) in the coastal water. A first screening for the presence of NPs in sea water may be obtained by checking their presence inside tissues of organisms taken from the wild. The ability of NPs to pass from gut to the coelomic fluid is demonstrated by accumulation in sea urchin coelomocytes; the toxicity on sperms can be measured by embryotoxicity markers after sperm exposure, whereas the transfer through the food chain can be observed by developmental anomalies in larvae fed with microalgae exposed to NPs. The most used spermiotoxicity and embryotoxicity tests are described, as well as the biochemical and histochemical analyses of cholinesterase (ChE) activities, which are used to verify toxicity parameters such as inflammation, neurotoxicity, and interference in cell-to-cell communication. Morphological markers of toxicity, in particular skeletal anomalies, are described and classified. In addition, NPs may impair viability of the immune cells of adult specimens. Molecular similarity between echinoderm and human immune cells is shown and discussed. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1552-1562, 2016.

Support of Nerve Conduction by Respiring Myelin Sheath: Role of Connexons.

Recently, we have demonstrated that myelin conducts an extramitochondrial oxidative phosphorylation, hypothesizing a novel supportive role for myelin in favor of the axon. We have also hypothesized that the ATP produced in myelin could be transferred thought gap junctions. In this work, by biochemical, immunohistochemical, and electrophysiological techniques, the existence of a connection among myelin to the axon was evaluated, to understand how ATP could be transferred from sheath to the axoplasm. Data confirm a functional expression of oxidative phosphorylation in isolated myelin. Moreover, WB and immunohistochemistry on optic nerve slices show that connexins 32 and 43 are present in myelin and colocalize with myelin basic protein. Interestingly, addition of carbenoxolone or oleamide, two gap junction blockers, causes a decrease in oxidative metabolism in purified myelin, but not in mitochondria. Similar effects were observed on conduction speed in hippocampal Schaffer collateral, in the presence of oleamide. Confocal analysis of optic nerve slices showed that lucifer yellow (that only passes through aqueous pores) signal was found in both the sheath layers and the axoplasma. In the presence of oleamide, but not with oleic acid, signal significantly decreased in the sheath and was lost inside the axon. This suggests the existence of a link among myelin and axons. These results, while supporting the idea that ATP aerobically synthesized in myelin sheath could be transferred to the axoplasm through gap junctions, shed new light on the function of the sheath.

Functional Expression of Electron Transport Chain and FoF1-ATP Synthase in Optic Nerve Myelin Sheath.

Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and ß subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration.

Multidisciplinary screening of toxicity induced by silica nanoparticles during sea urchin development.

The aim of this study was to investigate the potential toxicity of Silica nanoparticles (SiO2 NPs) in seawater by using the sea urchin Paracentrotus lividus as biological model. SiO2 NPs exposure effects were identified on the sperm of the sea urchin through a multidisciplinary approach, combining developmental biology, ecotoxicology, biochemistry, and microscopy analyses. The following responses were measured: (i) percentage of eggs fertilized by exposed sperm; (ii) percentage of anomalies and undeveloped embryos and larvae; (iii) enzyme activity alterations (acetylcholinesterase, AChE) in the early developmental stages, namely gastrula and pluteus. Sperms were exposed to seawater containing SiO2 NPs suspensions ranging from 0.0001mg/L to 50mg/L. Fertilization ability was not affected at any concentration, whereas a significant percentage of anomalies in the offspring were observed and quantified by means of EC50 at gastrula stage, including undeveloped and anomalous embryos (EC50=0.06mg/L), and at pluteus stage, including skeletal anomalies and delayed larvae (EC50=0.27mg/L). Moreover, morphological anomalies were observed in larvae at pluteus stage, by immunolocalizing molecules involved in larval development and neurotoxicity effects - such as acetylated tubulin and choline acetyltransferase (ChAT) - and measuring AChE activity. Exposure of sea urchins to SiO2 NPs caused neurotoxic damage and a decrease of AChE expression in a non-dose-dependent manner. In conclusion, through the multidisciplinary approach used in this study SiO2 NPs toxicity in sea urchin offspring could be assessed. Therefore, the measured responses are suitable for detecting embryo- and larval- toxicity induced by these NPs.

Exposure of Paracentrotus lividus male gametes to engineered nanoparticles affects skeletal bio-mineralization processes and larval plasticity.

The aim of this study is to contribute to the understanding of the mechanisms underlying nanoparticle (NP)-induced embryotoxicity in aquatic organisms. We previously demonstrated that exposure of male gametes to NPs causes non-dose-dependent skeletal damage in sea urchin (Paracentrotus lividus) larvae. In the present study, the molecular mechanisms responsible for these anomalies in sea urchin development from male gametes exposed to cobalt (Co), titanium dioxide (TiO2) and silver (Ag) NPs were investigated by histochemical, immunohistochemical and Western blot analyses. P. lividus sperm were exposed to different NP concentrations (from 0.0001 to 1 mg/L). The distribution of molecules related to skeletogenic cell identification, including ID5 immunoreactivity (IR), wheat germ agglutinin (WGA) affinity and fibronectin (FN) IR, were investigated by confocal laser scanning microscopy at the gastrula (24 h) and pluteus (72 h) stages. Our results identified a spatial correspondence among PMCs, ID5 IR and WGA affinity sites. The altered FN pattern suggests that it is responsible for the altered skeletogenic cell migration, while the Golgi apparatus of the skeletogenic cells, denoted by their WGA affinity, shows different aspects according to the degree of anomalies caused by NP concentrations. The ID5 IR, a specific marker of skeletogenic cells in sea urchin embryos (in particular of the msp130 protein responsible for Ca(2+) and Mg(2+) mineralization), localized in the cellular strands prefiguring the skeletal rods in the gastrula stage and, in the pluteus stage, was visible according to the degree of mineralization of the skeleton. In conclusion, the present study suggests that the investigated NPs suspended in seawater interfere with the bio-mineralization processes in marine organisms, and the results of this study offer a new series of specific endpoints for the mechanistic understanding of NP toxicity.

Pharmacological characterization of NMDA-like receptors in the single-celled organism Paramecium primaurelia.

Paramecium primaurelia is a unicellular eukaryote that moves in freshwater by ciliary beating and responds to environmental stimuli by altering motile behaviour. The movements of the cilia are controlled by the electrical changes of the cell membrane: when the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, the ciliary beating reverses its direction, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. Here, we evaluated the effects due to the activation or blockade of N-methyl-d-aspartic acid (NMDA) receptors on swimming behaviour in Paramecium. Paramecia normally swim forward, drawing almost linear tracks. We observed that the simultaneous administration of NMDA and glycine induced a partial ciliary reversal (PaCR) leading to a continuous spiral-like swim. Furthermore, the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, was longer in NMDA+glycine-treated cells. NMDA action required the presence of Ca(2+), as the normal forward swimming was restored when the ion was omitted from the extracellular milieu. The PaCR and the enhancement of CCR duration significantly decreased when the antagonists of the glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK-801 or the glycine site antagonist DCKA was added. The action of NMDA+glycine was also abolished by Zn(2+) or ifenprodil, the GluN2A and the GluN2B NMDA-containing subunit blockers, respectively. Searches of the Paramecium genome database currently available indicate that the NMDA-like receptor with ligand-binding characteristics of an NMDA receptor-like complex, purified from rat brain synaptic membranes and found in some metazoan genomes, is also present in Paramecium. These results provide evidence that functional NMDA receptors similar to those typical of mammalian neuronal cells are present in the single-celled organism Paramecium and thus suggest that the glutamatergic NMDA system is a phylogenetically old behaviour-controlling mechanism.

Effects of nanosilver exposure on cholinesterase activities, CD41, and CDF/LIF-like expression in zebrafish (Danio rerio) larvae.

Metal nanosolicoparticles are suspected to cause diseases in a number of organisms, including man. In this paper, we report the effects of nanosilver (Ag, 1-20 nm particles) on the early development of the zebrafish, a well-established vertebrate model. Embryos at the midgastrula stage were exposed to concentrations ranging from 100 to 0.001 mg/L to verify the effects on different endpoints: lethality, morphology, expression of cholinergic molecules, and development of the immune system. (1) Relative risk of mortality was exponential in the range between 0.001 and 10 mg/L. Exposure to 100 mg/L caused 100% death of embryos before reaching the tail-bud stage. (2) Developmental anomalies were present in the 72 h larvae obtained from embryos exposed to nanosilver: whole body length, decreased eye dimension, and slow response to solicitation by gentle touch with a needle tip, with a significant threshold at 0.1 mg/L. (3) Dose-dependent inhibition of acetylcholinesterase activity was significant among the exposures, except between 1 mg/L and 10 mg/L. (4) The distribution of CD41+ cells and of CDF/LIF-like immunoreactivity was altered according to the Ag concentration. The possible effect of nanosilver in impairing immune system differentiation through the inhibition of molecules related to the cholinergic system is discussed.

Tricarboxylic acid cycle-sustained oxidative phosphorylation in isolated myelin vesicles.

The Central Nervous System (CNS) function was shown to be fueled exclusively by oxidative phosphorylation (OXPHOS). This is in line with the sensitivity of brain to hypoxia, but less with the scarcity of the mitochondria in CNS. Consistently with the ectopic expression of FoF1-ATP synthase and the electron transfer chain in myelin, we have reported data demonstrating that isolated myelin vesicles (IMV) conduct OXPHOS. It may suggest that myelin sheath could be a site for the whole aerobic degradation of glucose. In this paper, we assayed the functionality of glycolysis and of TCA cycle enzymes in IMV purified from bovine forebrain. We found the presence and activity of all of the glycolytic and TCA cycle enzymes, comparable to those in mitochondria-enriched fractions, in the same experimental conditions. IMV also contain consistent carbonic anhydrase activity. These data suggest that myelin may be a contributor in energy supply for the axon, performing an extra-mitochondrial aerobic OXPHOS. The vision of myelin as the site of aerobic metabolism may shed a new light on many demyelinating pathologies, that cause an a yet unresolved axonal degeneration and whose clinical onset coincides with myelin development completion.

Developmental abnormalities and changes in cholinesterase activity in sea urchin embryos and larvae from sperm exposed to engineered nanoparticles.

The objective of this study is to examine the toxicity of engineered nanoparticles (NPs) that are dispersed in sea water by using an in vivo model. Because many products of nanotechnology contain NPs and are commonly used and well-established in the market, the accidental release of NPs into the air and water is quite possible. Indeed, at the end of their life cycle, some NPs are inevitably released into waste water and can reach marine ecosystem and affect the organisms there. Although there are few data on the presence of NPs in the marine environment, our awareness of their potential impact on environmental and organismal health is growing. Shallow-water benthonic organisms such as sea urchins provide planktonic larvae as a trophic base for finfish juveniles and are exposed to water from estuaries and precipitation. Such organisms can therefore be directly affected by NPs that are dispersed into those media. We evaluated the effects of exposure to different concentrations of nanosilver, titanium oxide and cobalt NPs on the sperm of the sea urchin Paracentrotus lividus by analyzing the functionality and the morphology and biochemistry of the first developmental stages of the sea urchin. Sperm were exposed to sea water containing suspensions of NPs ranging from 0.0001 mg/L to 1 mg/L. Fertilization ability was not affected, but developmental anomalies were identified in embryos from the gastrula to pluteus stages, including morphological alterations of the skeletal rods. In addition, the enzymatic activity (cholinesterase, ChE) of the larvae was measured. Acetylcholinesterase (AChE) and propionylcholinesterase activity (PrChE) was affected in all of the exposed samples. The results did not vary consistently with the concentration of NP, but controls were significantly different from exposed samples. Exposure of sea urchin to these NPs may cause neurotoxic damage, and the altered ChE activity may be involved in skeletogenic aberrations. In conclusion, the sea urchin represents a suitable and sensitive model for testing the toxicity and effects of engineered NPs that are dispersed in sea water.

A neurochemical map of the developing amphioxus nervous system.

BACKGROUND: Amphioxus, representing the most basal group of living chordates, is the best available proxy for the last invertebrate ancestor of the chordates. Although the central nervous system (CNS) of amphioxus comprises only about 20,000 neurons (as compared to billions in vertebrates), the developmental genetics and neuroanatomy of amphioxus are strikingly vertebrate-like. In the present study, we mapped the distribution of amphioxus CNS cells producing distinctive neurochemicals. To this end, we cloned genes encoding biosynthetic enzymes and/or transporters of the most common neurotransmitters and assayed their developmental expression in the embryo and early larva. RESULTS: By single and double in situ hybridization experiments, we identified glutamatergic, GABAergic/glycinergic, serotonergic and cholinergic neurons in developing amphioxus. In addition to characterizing the distribution of excitatory and inhibitory neurons in the developing amphioxus CNS, we observed that cholinergic and GABAergic/glycinergic neurons are segmentally arranged in the hindbrain, whereas serotonergic, glutamatergic and dopaminergic neurons are restricted to specific regions of the cerebral vesicle and the hindbrain. We were further able to identify discrete groups of GABAergic and glutamatergic interneurons and cholinergic motoneurons at the level of the primary motor center (PMC), the major integrative center of sensory and motor stimuli of the amphioxus nerve cord. CONCLUSIONS: In this study, we assessed neuronal differentiation in the developing amphioxus nervous system and compiled the first neurochemical map of the amphioxus CNS. This map is a first step towards a full characterization of the neurotransmitter signature of previously described nerve cell types in the amphioxus CNS, such as motoneurons and interneurons.

The sea urchin, Paracentrotus lividus, as a model to investigate the onset of molecules immunologically related to the α-7 subunit of nicotinic receptors during embryonic and larval development.

Nicotinic acetylcholine receptors play a major role in the regulation of electrochemical synapses at neuromuscular junctions. During the early stages of Paracentrotus lividus development, the nicotinic receptor-like molecules are found and localized by use of the specific blocker, -bungarotoxin, and by α-7 subunit immunoreactivity. Both the methods identify and localize the nicotinic receptor-like molecules at the sites where active changes in ionic intracellular concentration take place. These are well known to lead either fertilization, sperm propulsion or co-ordinated ciliary movement. After neural differentiation, immunoreactivity for the α-7 subunit is localized mainly in ganglia, ectoderm ciliary bands and in the motile cells forming the gut wall. Both α-bungarotoxin binding sites and α-7 subunits are also localized at the cells linked to the skeletal rods, performing the small movements which drive the swimming direction in the water column. The localization of these molecules paves the way to a speculation on their function and possible role in neurogenesis as well as neurodegeneration.

Role of GbpA protein, an important virulence-related colonization factor, for Vibrio cholerae's survival in the aquatic environment.

Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin binding protein and a virulence factor involved in the colonization of human intestine. We investigated the distribution and genetic variations of gbpA in 488 V. cholerae strains of environmental and clinical origin, belonging to different serogroups and biotypes. We found that the gene is consistently present and highly conserved including an environmental V. cholerae-related strain of ancestral origin. The gene was also consistently expressed in a number of representative V. cholerae strains cultured in laboratory aquatic microcosms under conditions simulating those found in temperate marine environments. Functional analysis carried out on V. cholerae O1 El Tor N16961 showed that GbpA is not involved in adhesion to inorganic surfaces but promotes interaction with environmental biotic substrates (plankton and bivalve hepatopancreas cells) representing known marine reservoir or host for the bacterium. It is suggested that the ability of GbpA to colonize human intestinal cells most probably originated from its primary function in the aquatic environment.

Metabotropic γ-aminobutyric acid (GABAB) receptors modulate feeding behavior in the calcisponge Leucandra aspera.

Here, we report the presence of the γ-aminobutyric acid (GABA)-ergic system in the calcisponge Leucandra aspera and examine the cellular localization of the components of this system, including GABA-like receptors using immunofluorescence and confocal microscopy. Furthermore, we demonstrate for the first time that GABA plays a functional role as a messenger in regulating sponge-feeding behavior. We found that both GABA(B) R1 and R2 subunits are present in the choanocytes of sponges as well as in the eso- and endopinacocytes. The functional role of GABA in the feeding behavior of this sponge was tested. The involvement of GABA receptors in the endocytic processes in L. aspera was demonstrated with dextran conjugated to Texas Red as a marker for material ingestion and by treating isolated sponge cells with a GABA(B) receptor agonist and an antagonist. The amount of dextran that was ingested increased in dissociated sponge cells when the GABA(B) receptor agonist baclofen was used, and this stimulatory effect was prevented by treatment with the GABA(B) receptor antagonist phaclofen. The baclofen effect on uptake was blocked by treatment with pertussis toxin, thus indicating a role for G proteins in modulating feeding behavior in L. aspera. Moreover, we found evidence that GABA receptors are involved in the consumption of dissolved organic matter by sponge cells. These findings suggest that GABA receptors and their functional role are highly conservative traits in the animal kingdom prenervous system evolution.

Evidence for ectopic aerobic ATP production on C6 glioma cell plasma membrane.

Extracellular ATP plays a pivotal role as a signaling molecule in physiological and pathological conditions in the CNS. In several glioma cell lines, ATP is a positive factor for one or more characteristics important for the abnormal growth and survival of these cells. This work presents immunofluorescence and biochemical analyses suggesting that an aerobic metabolism, besides mitochondria, is located also on the plasma membrane of C6 glioma cells. An ATP synthesis coupled to oxygen consumption was measured in plasma membrane isolated from C6 cells, sensitive to common inhibitors of respiratory chain complexes, suggesting the involvement of a putative surface ATP synthase complex. Immunofluorescence imaging showed that Cytochrome c oxydase colocalized with WGA, a typical plasma membrane protein, on the plasma membrane of glioma cells. Cytochrome c oxydase staining pattern appeared punctuate, suggesting the intriguing possibility that the redox chains may be expressed in discrete sites on C6 glioma cell membrane. Data suggest that the whole respiratory chain is localized on C6 glioma cell surface. Moreover, when resveratrol, an ATP synthase inhibitor, was added to culture medium, a cytostatic effect was observed, suggesting a correlation among the ectopic ATP synthesis and the tumor growth. So, a potential direction for the design of new targets for future therapies may arise.

NMDA R1 receptor distribution in the cyprid of Balanus amphitrite (=Amphibalanus amphitrite) (Cirripedia, Crustacea).

The ontogenetic cycle of the barnacle Balanus amphitrite (=Amphibalanus amphitrite) (Darwin, 1854) includes a cyprid that binds submerged surfaces, metamorphosing into a sessile adult. γ-Aminobutyric acid (GABA) and GABA receptors have recently been located in its cyprid with a similar pattern to other crustaceans. Since NMDA R1 ionotropic glutamatergic receptors have been identified in crustacean neuromuscular junctions, we have investigated their presence in the B. amphitrite cyprid. The presence of NMDA R1 receptors might indicate a role for glutamate in neuromuscular control in B. amphitrite cyprids, therefore we studied the presence and distribution of the NMDA R1 by immunohistochemistry. Its distribution was observed in the peripheral nervous system and in non-neuronal elements. Actually, NMDA R1 immunoreactivity was detected in thoracic appendages, at the level of neuromuscular junctions, thus suggesting an involvement in motor control functions, as already demonstrated in other crustaceans. Immunoreactivity was also detected in ommatidia cells of the eye, in antennules, and in epidermal cells. The distribution pattern comparable to that of GABAergic molecules could indicate an interrelated agonistic/antagonistic role for these two systems, which could be considered as potential targets of combined antifouling strategies.

Protection capabilities of nanostructured shells toward cell encapsulation: a Saccharomyces/Paramecium model.

In this work we report the formation of nanostructured hybrid objects, made up of living cells encapsulated in a protective multilayer shell assembly of nanostructured polyelectrolyte. Such constructs can be hosted on nanostructured surfaces or can be installed around living organisms, at the right time. Their construction is based on the layer-by-layer (LbL) self-assembly of two oppositely alternated charged polyelectrolytes (PEs) on cell membranes as earlier done for nanocapsules or fuzzy structured nanoshells. This communication reports the optimal conditions for cell encapsulation in terms of nanoshell design and construction.