Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO

<div><h3>Background</h3><p>Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.</p> <h3>Methods</h3><p>Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.</p> <h3>Results</h3><p>Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.</p> <h3>Conclusion</h3><p>Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.</p> </div

Petrogenesis of Early Permian olivine-bearing cumulates and associated basalt dykes from Bocca di Tenda (Northern Corsica): Implications for post-collisional Variscan evolution

The Early Permian gabbroic sequence of Bocca di Tenda (Northem Corsica) intrudes a plutonic suite made of hornblende-granitoids and is crosscut by basalt dykes with chilled margins. The gabbroic sequence includes olivine-gabbronorite cumulates that consist mainly of euhedral olivine (Fo78-72) and plagioclase (An68-61), and poikilitic clinopyroxene (Mg#=0.85-0.80), orthopyroxene and Ti-pargasite. Pyroxenes from the olivine-gabbronorites have delta(18)O values of +6.3 parts per thousand to +6.7 parts per thousand, which are evidence for the involvement of a crustal component in the petrogenesis of these rocks. The basalts have Mg# values of 57 to 53 and chondrite-normalised patterns characterised by a steady decrease from LREE to HREE. Relative to modern N-MORB. they show also moderate enrichment of Nb and Ta over Zr and Hf, which are in turn slightly enriched over Y and HREF The olivine-gabbronorites and basalts show overlapping Nd isotopic compositions (initial epsilon(Nd) of +2.4 to +1.4 and of +3.0 to +1.5, respectively) and have clinopyroxenes with nearly parallel incompatible trace element patterns, thus revealing that these two rock types formed by melts with similar compositions. Most likely, the primary melts of Bocca di Tenda gabbroic sequence were slightly enriched in most incompatible elements and Nd isotopic compositions relative to modem N-MORB. (C) 2008 Elsevier B.V. All rights reserved

The magmatic-hydrothermal transition in the lower oceanic crust. Clues from the Ligurian ophiolites, Italy

The gabbroic bodies from the Jurassic Ligurian ophiolites are structurally and compositionally similar to the gabbroic sequences from the oceanic core complexes of the Mid Atlantic Ridge. Initial cooling of the Ligurian gabbros is associated with local development of hornblende-bearing felsic dykes and hornblende vein networks. The hornblende veining is correlated with the widespread development of hornblende as coronas/pseudomorphs after the clinopyroxene in the host gabbros. In addition, the studied gabbroic body includes a mantle sliver locally containing hornblende gabbros and hornblendite veins. The hornblendes from the felsic dykes and the hornblende-rich rocks within the mantle sliver show a similar geochemical signature, characterized by low Mg#, CaO and Al2O3, negligible Cl, and high TiO2, K2O, REE, Y, Zr and Nb concentrations. The whole-rock Sm-Nd isotopic compositions of the felsic dykes and the hornblende-rich rocks define a Sm-Nd isochron corresponding to an age of 154 + 20Ma and an initial epsilon(Nd) of 9.2 +/- 0.5. The delta O-18 of the hornblendes and coexisting zircons from these rocks (about +4.5 parts per thousand and +5.8 parts per thousand, respectively) do not indicate the presence of a seawater component in these melts. The formation of the felsic dykes and of the hornblende-rich rocks within the mantle sliver involved SiO2-rich silicate melts with negligible seawater component, which presumably were derived from high degree fractional crystallization of MOR-type basalts. The vein and the coronitic/pseudomorphic hornblendes show high Mg# and CaO, significant Cl (0.02-0.17 wt%) and low TiO2 and K2O concentrations. The coronitic/pseudomorphic hornblendes have trace element compositions similar to those of the clinopyroxenes from the gabbros and delta O-18 values (+1.0 parts per thousand to 0.7 parts per thousand) close to seawater, suggesting an origin by reaction between migrating seawater-derived fluids and the host gabbros. The vein hornblendes commonly show slight LREE enrichment, relatively high concentrations of Nb (up to 2.5 ppm) and delta O-18 ranging from +3.7 parts per thousand to +0.8 parts per thousand. The crystallization of these hornblendes most likely involved both seawater and magmatic components. (c) 2014 Elsevier Ltd. All rights reserved

Changes in peripheral nervous system activity produced in rats by prenatal exposure to carbon monoxide

The present experiments were designed to investigate whether alterations of peripheral nervous system activity may be produced in male Wistar rats by prenatal exposure (from day 0 to day 20 of pregnancy) to relatively low levels of CO (75 and 150 ppm). The voltage clamp analysis of ionic currents recorded from sciatic nerve fibres showed that prenatal exposure to CO produced modifications of sodium current properties. In particular, in 40-day-old rats exposed to CO (75 and 150 ppm) during gestation, the inactivation kinetics of transient sodium current were significantly slowed. Analysis of the potential dependence of steady-state Na inactivation, h(infinity) (V), showed that the percentage of the maximum number of activatable Na channels at the normal resting potential (-80 mV) was increased to almost-equal-to 85% in CO-exposed rats. Moreover, the voltage-current relationship showed a negative shift of sodium equilibrium potential in CO treated animals. In 270-day-old CO-exposed rats, parameters of sodium inactivation were not significantly modified; the reversal potential was still lower with respect to controls. The results indicate that prenatal exposure to mild CO concentrations produces reversible changes in sodium inactivation kinetics and on irreversible change in sodium equilibrium potential. These alterations could reflect CO influence on the rate of ion channel developmen

Abnormal H-Tfam in a patient harboring a single mtDNA deletion.

We report on a patient suffering from a progressive mitochondrial disorder characterized by ocular myopathy, exercise intolerance, and muscle wasting. Morphological examination of muscle biopsy showed increased variability in fiber size and scattered ragged-red fibers. Analysis of muscle mitochondrial DNA by Southern blot and PCR revealed a heteroplasmic single deletion of 4100 base pairs, located between nucleotide positions 8300 and 12,400. Western blot analysis showed high levels of the human mitochondrial transcription factor A (Tfam). Interestingly, we also detected an additional Tfam product, of approximately 22 kDa. This is the first case in which a qualitatively abnormal Tfam has been found to be associated with a mitochondrial disorder in humans

Neurobehavioral changes produced by developmental exposure to environmental pollutants

The central nervous system (CNS) of rats reaches functional maturation several weeks after birth. Contrast this to other organs, in which the basic structure and function are completed in utero or shortly after birth. Therefore, the CNS is susceptible to insult for a longer portion of development than other organs. Moreover, the timing of exposure, whether discrete or long term, seems to play a crucial role in determining structural and/or functional alterations.A large body of evidence suggests that behaviour is a sensitive indicator of subtle damages in CNS function caused by exposure to chemicals at dose levels below those associated with overt neurologic signs and that developmental exposure to psychotropic drugs (e.g., neuroleptics, benzodiazepines) and environmental toxicants (e.g., methylmercury, lead) produces neurobehavioral changes which differ markedly from those occurring in response to their administration to mature animals.In this regard, our group has been involved in research programs dealing with the characterization of neurobehavioral effects produced by developmental exposure to environmental pollutants (Di Giovanni et al., 1993, Di Giovanni et al., 1994). In particular, the interest has been recently focused on the assessment of outcomes that follows the developmental exposure to carbon monoxide (CO), a product of incomplete combustion found in elevated concentrations in many urban areas and one of the constituents of cigarette smoke.Several findings have shown that gestational exposure to relatively mild concentrations of CO induces neurobehavioral abnormalities in rats, including lower activity levels through the preweaning, delayed development of homing behavior and negative geotaxis as well as altered ontogeny of emotional reactivity (see for references Annau and Fechter, 1994). However, only few studies have been addressed to the evaluation of long term neurobehavioral changes elicited by prenatal exposure to low concentrations of this gas.Since previous findings have shown alterations in the acquisition of active avoidance schedule (Mactutus and Fechter, 1984; Di Giovanni et al., 1993) and impaired sexual behavior (unpublished data) in young adult rats prenatally exposed to relatively mild concentrations of CO (0, 75 and 150 ppm from day 0 to day 20 of pregnancy), the aim of our recent studies was to evaluate whether these behavioral alterations would dissipate at a later age.The results show that CO (150 ppm)-exposed male rats (18-month old) subjected to an active avoidance task still exhibited notable learning deficit. Moreover, prenatal exposure to this toxicant significantly impaired reacquisition of this schedule in 18-month old animals previously trained at 3-months of age. The impairment in both acquisition and reacquisition of an active avoidance task did not appear to be due to alterations of a nonassociative nature. In fact, changes in emotional reactivity seem to be absent as shown by the analysis of unconditioned responses latencies which were not influenced by CO exposure. Moreover, spontaneous crossings during intertrial intervals were not significantly affected in rats exposed to this gas during gestation (De Salvia et al., 1995).Further studies have also shown that some alterations in sexual performance of CO-exposed male animals were long lasting. In fact, a significant increase in mount intromission latency in 365-day old rats exposed to CO (150 ppm) during pregnancy was found. Furthermore, the significant increase in mount-intromission frequency exhibited by young adult CO-exposed rats lasted up to 200 days of age in both 75 and 150 ppm CO-treated groups.The results described in this paper, as well as results obtained by other investigators, confirm the deleterious and irreversible effects of prenatal carbon monoxide exposure in animals. These data show that behavioral, neurochemical, neuropathological and neurophysiological alterations can be documented following moderate carboxyhemoglobin levels during fetal development, and that the fetal brain is extremely sensitive to the toxic effects of carbon monoxide. To date, very few human studies have been done to verify these findings in children of mothers who smoke cigarettes during pregnancy. Because cigarette smoke is the major source of carbon monoxide exposure in the human population, and epidemiological studies have shown that it increases neonatal mortality, it would seem prudent to scrutinize the potential neurotoxic effects of offspring whose mothers smoke

Coenzyme Q10, exercise lactate and CTG trinucleotide expansion in myotonic dystrophy

Steinert's myotonic dystrophy (DM) is a genetic autosomal dominant disease and the most frequent muscular dystrophy in adulthood. Although causative mutation is recognized as a CTG trinucleotide expansion on 19q13.3, pathogenic mechanisms of multisystem involvement of DM are still under debate. It has been suggested that mitochondrial abnormalities can occur in this disease and deficiency of coenzyme Q 10 (CoQ10) has been considered one possible cause for this. The aim of this investigation was to evaluate, in 35 DM patients, CoQ10 blood levels and relate them to the degree of CTG expansion as well as to the amount of lactate production in exercising muscle as indicator of mitochondrial dysfunction. CoQ10 concentrations appeared significantly reduced with respect to normal controls: 0.85 +/- 0.25 vs. 1.58 +/- 0.28 microg/ml (p < 0.05). Mean values of blood lactate were significantly higher in DM patients than controls (p < 0.05) both in resting conditions (2.9 +/- 0.55 vs. 1.44 +/- 1.11 mmol/L) and at the exercise peak (6.77 +/- 1.79 vs. 4.90 +/- 0.59 mmol/L), while exercise lactate threshold was anticipated (30-50% vs. 60-70% of the predicted normal maximal power output, p < 0.05). Statistical analysis showed that serum CoQ10 levels were significantly (p < 0.05) inversely correlated with both CTG expansion degree and lactate values at exercise lactate threshold level. Our data indicates the occurrence of reduced CoQ10 levels in DM, possibly related to disease pathogenic mechanisms associated with abnormal CTG trinucleotide amplification