We have investigated whether increase in the oxidation rate of exogenous cytochrome c (cyto-c), induced by long-chain ceramides, might be due to an increased rate of cytosolic NADH/cyto-c electron transport pathway. This process was identified in isolated liver mitochondria and has been studied in our laboratory for many years. Data from highly specific test of sulfite oxidase prove that exogenous cyto-c both in the absence and presence of ceramide cannot permeate through the mitochondrial outer membrane. However, the oxidation of added NADH, mediated by exogenous cyto-c and coupled to the generation of a membrane potential supporting the ATP synthesis, can also be stimulated by ceramide. The results obtained suggest that ceramide molecules, by increasing mitochondrial permeability, with the generation of either raft-like platforms or channels, may have a dual function. They can promote the release of endogenous cyto-c and activate, with an energy conserving process, the oxidation of cytosolic NADH either inducing the formation of new respiratory contact sites or increasing the frequency of the pre-existing porin contact sites. In agreement with the data in the literature, an increase of mitochondrial ceramide molecules level may represent an efficient strategy to activate and support the correct execution of apoptotic program

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation ?F508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na(+)/H(+) exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o- rescues the functional expression of ?F508CFTR by promoting F-actin organization and formation of the NHERF1-ezrin-actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o- cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o- cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ?F508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o- cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity.

The deposition of as-received nanodiamond (ND) particles on silicon substrate was performed by the pulsed spray technique, using a dispersion of 250nm ND in 1,2-dichloroethane. A set of samples was sprayed by varying the number of pulses from 1 to 500. The morphology of the samples was characterized and monitored by means of optical, atomic force, and confocal microscopies. At a low number of pulses, sparse diamond particles were observed, whereas at a high number of pulses dense/quasi-continuous ND layers were formed. The electrical conductivity measurements of surface silicon substrate evidenced a remarkable change for the presence of ND particles. This behavior is also found by theoretical simulations (finite element method). Finally, a comparison between the electrical resistances measured on these samples versus the pulse number and the inverse current density calculated as a function of the number of ND particles, showed a good agreement. The experimental results highlighted an increase of the electrical current by using a number of pulses <100, whereas the simulation results proved the enhancement of current density and its surface rectification by employing a specific number of particles. The current increased by increasing the temperature and during the heating-cooling cycles hysteresis was observed. (a) Scheme of the sprayed ND particles on silicon substrate, (b) 3D AFM image 5×5?m² of 10 pulses sample, (c) trends of measured R and calculated 1/J.

Oxygen uptake, respiratory complexes, and metabolic activities have been studied in mitochondria isolated from thehepatopancreata of Mytilus galloprovincialis collected in the "Mar Grande" of Taranto (Ionian Sea, Italy). Although exposure to 5.0 ?gZn2+/L resulted in a significant increase of states 3 and 4 respiration with glutamate + pyruvate as respiratory substrate, it was foundthat the exposure of mussels to different concentrations of Zn2+ (2.5-7.5 ?g/L) neither inhibited mitochondrial respiration nor exertedany inhibitory effect on representative mitochondrial dehydrogenases. It rather stimulates these activities, producing an extra synthesisof adenosine triphosphate by hepatopancreas mitochondria and possibly increasing its availability in the cytoplasmic compartment.This might be considered as a specific strategy utilized by the mussel to cope with variations in the heavy metals content of the marineenvironment, and it could be used to detect toxic effects.

Functional and structural damages to mitochondria have been critically associated with the pathogenesis of Down syndrome (DS), a human multifactorial disease caused by trisomy of chromosome 21 and associated with neurodevelopmental delay, intellectual disability and early neurodegeneration. Recently, we demonstrated in neural progenitor cells (NPCs) isolated from the hippocampus of Ts65Dn mice -a widely used model of DS - a severe impairment of mitochondrial bioenergetics and biogenesis and reduced NPC proliferation. Here we further investigated the origin of mitochondrial dysfunction in DS and explored a possible mechanistic link among alteration of mitochondrial dynamics, mitochondrial dysfunctions and defective neurogenesis in DS. We first analyzed mitochondrial network and structure by both confocal and transmission electron microscopy as well as by evaluating the levels of key proteins involved in the fission and fusion machinery. We found a fragmentation of mitochondria due to an increase in mitochondrial fission associated with an up-regulation of dynamin-related protein 1 (Drpl), and a decrease in mitochondrial fusion associated with a down-regulation of mitofusin 2 (Mnf2) and increased proteolysis of optic atrophy 1 (Opal). Next, using the well-known neuroprotective agent mitochondrial division inhibitor 1 (Mdivi-1), we assessed whether the inhibition of mitochondrial fission might reverse alteration of mitochondrial dynamics and mitochondrial dysfunctions in DS neural progenitors cells. We demonstrate here for the first time, that Mdivi-1 restores mitochondrial network organization, mitochondrial energy production and ultimately improves proliferation and neuronal differentiation of NPCs. This research paves the way for the discovery of new therapeutic tools in managing some DS-associated clinical manifestations.

P2×7R is a member of the ionotropic family of purinergic receptors activated by millimolar concentrations of extracellular ATP such as induced by inflammatory stimuli. The receptor is widely expressed in cells of haematopoietic origin such as monocytes, macrophages and microglia. There is growing interest in anta-gonist compounds of the P2×7R since it has been demonstrated to be a viable therapeutic target for inflammatory diseases. Here, we tested the possible P2×7 antagonist effect of MED1101, a newly synthesised dialdehydic compound on U937 monocyte cells.

Diamond powders of various size ranging from few nanometers to tens of micrometers arecommonly used to treat the silicon substrate in order to enhance the nucleation process beforethe growth of thin diamond films by chemical vapor deposition (CVD) techniques[1 and refs.therein]. Recently a great attention is paid to nanodiamond (ND) particles which include stablenitrogen-vacancy (N-V) color centers [2]. In this work we propose the spray technique [3] todirectly deposit natural ND layers on silicon substrate using particles of 250 nm. ND particles weredispersed in the apolar solvent 1, 2 - dichloroethano (DCE) by sonication for 30 minutes. Then thedispersion was sprayed on the Si substrate, obtaining the highest ND density in the middle of it.The obtained ND films were analyzed by Raman spectroscopy, atomic force microscopy (AFM) and3D confocal microscopy. The first technique allows the measurement of the chemical andstructural composition and the photoluminescent properties, whereas the other ones measure thetopography and morphology of the layers.A careful morphological analysis showed the existence of pillar-like self-assembled structuresdistributed in an irregular way. The highest pillar density was found far from the center of thesample, where the ND layer is non-uniform. The evolution of the structures were well observed bythe 3D image analysis performed by confocal microscopy and AFM. The studyon the formationmechanisms of ND self-assembled structures will be presented and discussed.

A valuable analog of the K+-ionophore valinomycin (1), bearing a pentafluorophenyl ester moiety, has been obtained by selective reaction between the tertiary hydroxyl moiety of analog 2 (available from valinomycin hydroxylation) and the isocyanate group of pentafluorophenyl N-carbonyl glycinate (3) catalyzed by bis(N,N-dimethylformamide)dichlorodioxomolybdenum(VI). LC-HRMS studies show that analog 4 undergoes easy derivatization under mild conditions by reaction with OH- and NH2-containing compounds. Mitochondrial depolarization assays suggest that 4 acts as a K+-ionophore, provided that the glycine carboxyl group is appropriately masked.

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM. The rate of cytosolic NADH/cyto-c electron transport pathway is also greatly stimulated. The test on the permeability of mitochondrial outer membrane to exogenous cyto-c rules out the possibility that the increased rate of exogenous NADH oxidation could be ascribed either to extensively damaged or broken mitochondria. Accumulation of potassium inside the mitochondria, mediated by the highly specific ionophore valinomycin, promotes an increase in the volume of matrix (evidenced by swelling) and the interaction points between the two mitochondrial membranes are expected to increase. The data reported and those previously published are consistent with the view that ''respiratory contact sites'' are involved in the transfer of reducing equivalents from cytosol to inside the mitochondria both in the absence and the presence of valinomycin. Magnesium ions prevent at least in part the valinomycin effects. Rather than to the dissipation of membrane potential, the pro-apoptotic property of valinomycin can be ascribed to both the release of cyto-c from mitochondria to cytosol and the increased rate of cytosolic NADH coupled with an increased availability of energy in the form of glycolytic ATP, useful for the correct execution of apoptotic program

In order to investigate whether and how a modification of mitochondrialmetabolism can affect yeast sensitivityto programmed cell death (PCD) induced by acetic acid (AA-PCD), yeast cells were grown on raffinose, as a sole carbon source, which, differently from glucose, favours mitochondrial respiration. We found that, differently from glucose-grown cells, raffinose-grown cells were mostly resistant to AA-PCD and that this was due to the activation of mitochondrial retrograde (RTG) response, which increased with time, as revealed by the upregulationof the peroxisomal isoform of citrate synthase and isocitrate dehydrogenase isoform 1, RTG pathwaytarget genes. Accordingly, the deletion of RTG2 and RTG3, a positive regulator and a transcription factor of the RTG pathway, resulted in AA-PCD, as shown by TUNEL assay. Neither deletion in raffinose-grown cells of HAP4,encoding the positive regulatory subunit of the Hap2,3,4,5 complex nor constitutive activation of the RTG pathway in glucose-grown cells due to deletion of MKS1, a negative regulator of RTG pathway, had effect on yeast AA-PCD. The RTG pathway was found to be activated in yeast cells containing mitochondria, in which membrane potential wasmeasured, capable to consume oxygen in amanner stimulated by the uncoupler CCCP and inhibited by the respiratory chain inhibitor antimycin A. AA-PCD resistance in raffinose-grown cells occurs with a decrease in both ROS production and cytochrome c release as compared to glucose-grown cells en route to AA-PCD.