In mutant superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS), accumulation of misfolded mutant SOD1 in spinal cord mitochondria is thought to cause mitochondrial dysfunction. Whether mutant SOD1 is toxic per se or whether it damages the mitochondria through interactions with other mitochondrial proteins is not known. We previously identified Bcl-2 as an interacting partner of mutant SOD1 specifically in spinal cord, but not in liver, mitochondria of SOD1 mice and patients. We now show that mutant SOD1 toxicity relies on this interaction. Mutant SOD1 induces mitochondrial morphological changes and compromises mitochondrial membrane integrity leading to release of Cytochrome C only in the presence of Bcl-2. In cells, mouse and human spinal cord with SOD1 mutations, the binding to mutant SOD1 triggers a conformational change in Bcl-2 that results in the uncovering of its toxic BH3 domain and conversion of Bcl-2 into a toxic protein. Bcl-2 carrying a mutagenized, non-toxic BH3 domain fails to support mutant SOD1 mitochondrial toxicity. The identification of Bcl-2 as a specific target and active partner in mutant SOD1 mitochondrial toxicity suggests new therapeutic strategies to inhibit the formation of the toxic mutant SOD1/Bcl-2 complex and to prevent mitochondrial damage in ALS.

The goal of this study was to evaluate annexin V-positive microparticles (MPs) and neutrophil activation in humans following decompression from open-water SCUBA diving with the hypothesis that changes are related to intravascular bubble formation. Sixteen male volunteer divers followed a uniform profile of four daily SCUBA dives to 18 m of sea water for 47 min. Blood was obtained prior to and at 80 min following the first and fourth dives to evaluate the impact of repetitive diving, and intravascular bubbles were quantified by trans-thoracic echocardiography carried out at 20-min intervals for 2 h after each dive. MPs increased by 3.4-fold after each dive, neutrophil activation occurred as assessed by surface expression of myeloperoxidase and the CD18 component of β2-integrins, and there was an increased presence of the platelet-derived CD41 protein on the neutrophil surface indicating interactions with platelet membranes. Intravascular bubbles were detected in all divers. Surprisingly, significant inverse correlations were found among postdiving bubble scores and MPs, most consistently at 80 min or more after the dive on the fourth day. There were significant positive correlations between MPs and platelet-neutrophil interactions after the first dive and between platelet-neutrophil interactions and neutrophil activation documented as an elevation in β2-integrin expression after the fourth dive. We conclude that MPs- and neutrophil-related events in humans are consistent with findings in an animal decompression model. Whether there are causal relationships among bubbles, MPs, platelet-neutrophil interactions, and neutrophil activation remains obscure and requires additional study.

Yang M, Milovanova TN, Bogush M, Uzun G, Bhopale VM, Thom SR. Microparticle enlargement and altered surface proteins after air decompression are associated with inflammatory vascular injuries. J Appl Physiol 112: 204-211, 2012. First published September 29, 2011 doi:10.1152/japplphysiol.00953.2011.-Studies in a murine model have shown that decompression stress triggers a progressive elevation in the number of circulating annexin V-coated microparticles derived from leukocytes, erythrocytes, platelets, and endothelial cells. We noted that some particles appeared to be larger than anticipated, and size continued to increase for Ն24 h postdecompression. These observations led to the hypothesis that inert gas bubbles caused the enlargement and particle size could be reduced by hydrostatic pressure. After demonstrating pressure-induced particle size reduction, we hypothesized that annexin V-positive particle changes associated with decompression contributed to their proinflammatory potential. Intravenous injection of naive mice with particles isolated from decompressed mice, but not control mice, caused intravascular neutrophil activation; perivascular neutrophil sequestration and tissue injuries were documented as elevations of vascular permeability and activated caspase-3. These changes were not observed if mice were injected with particles that had been subjected to hydrostatic recompression or particles that had been emulsified by incubation with polyethylene glycol telomere B surfactant. Hydrostatic pressure and surfactant incubation also altered the pattern of proteins expressed on the surface of particles. We conclude that proinflammatory events and vascular damage are due to enlargement of annexin V-coated particles and/or changes in surface marker protein pattern associated with provocative decompression. Injection of annexin V-coated particles from decompressed mice will recapitulate the pathophysiological vascular changes observed following decompression stress.

Diabetes mellitus (DM) is a disorder due to the inability properly to metabolize glucose associated with dysregulation of metabolic pathways of lipids and proteins resulting in structural and functional changes of various organ systems. DM has detrimental effects on the vasculature, resulting in the development of various cardiovascular diseases and stemming from microvascular injury. The blood brain barrier (BBB) is a highly specialized structure protecting the unique microenvironment of the brain. Endothelial cells, connected by junctional complexes and expressing numerous transporters, constitute the main cell type in the BBB. Other components, including pericytes, basement membrane, astrocytes and perivascular macrophages, join endothelial cells to form the neurovascular unit (NVU) and contribute to the proper function and integrity of the BBB. The role of the BBB in the pathogenesis of diabetic encephalopathy and other diabetes-related complications in the central nervous system is apparent. However, the mechanisms, timing and consequences of BBB injury in diabetes are not well understood. The importance of further studies related to barrier dysfunction in diabetes is dictated by its potential involvement in the cognitive demise associated with DM. This review summarizes the impact of DM on BBB/NVU integrity and function leading to neurological and cognitive complications.

The study goal was to evaluate responses in humans following decompression from open-water SCUBA diving with the hypothesis that exertion underwater and use of a breathing mixture containing more oxygen and less nitrogen (enriched air nitrox) would alter annexin V-positive microparticle (MP) production and size changes and neutrophil activation, as well as their relationships to intravascular bubble formation. Twenty-four divers followed a uniform dive profile to 18 m of sea water breathing air or 22.5 m breathing 32% oxygen/68% nitrogen for 47 min, either swimming with moderately heavy exertion underwater or remaining stationary at depth. Blood was obtained pre- and at 15 and 120 min postdive. Intravascular bubbles were quantified by transthoracic echocardiography postdive at 20-min intervals for 2 h. There were no significant differences in maximum bubble scores among the dives. MP number increased 2.7-fold, on average, within 15 min after each dive; only the air-exertion dive resulted in a significant further increase to 5-fold over baseline at 2 h postdive. Neutrophil activation occurred after all dives. For the enriched air nitrox stationary at depth dive, but not for other conditions, the numbers of postdive annexin V-positive particles above 1 μm in diameter were correlated with intravascular bubble scores (correlation coefficients ∼0.9, P < 0.05). We conclude that postdecompression relationships among bubbles, MPs, platelet-neutrophil interactions, and neutrophil activation appear to exist, but more study is required to improve confidence in the associations.

The lymphoid protein T-cell ubiquitin ligand (TULA)/suppressor of T-cell receptor signaling (Sts)-2 is associated with c-Cbl and ubiquitylated proteins and has been implicated in the regulation of signaling mediated by protein-tyrosine kinases. The results presented in this report indicate that TULA facilitates T-cell apoptosis independent of either T-cell receptor/ CD3-mediated signaling or caspase activity. Mass spectrometry-based analysis of protein-protein interactions of TULA demonstrates that TULA binds to the apoptosis-inducing protein AIF, which has previously been shown to function as a key factor of caspase-independent apoptosis. Using RNA interference, we demonstrate that AIF is essential for the apoptotic effect of TULA. Analysis of the subcellular localization of TULA and AIF together with the functional analysis of TULA mutants is consistent with the idea that TULA enhances the apoptotic effect of AIF by facilitating the interactions of AIF with its apoptotic co-factors, which remain to be identified. Overall, our results shed new light on the biological functions of TULA, a recently discovered protein, describing its role as one of very few known functional interactors of AIF.We recently identified TULA among multiple proteins that co-purified with c-Cbl from T-lymphoblastoid cells (1). TULA contains an N-terminal UBA domain, a centrally positioned SH3 2 domain, and a region of homology to phosphoglyceromutases, which was initially termed HCD ( Fig. 1) (1, 2). TULA binds to c-Cbl through its SH3 domain and to ubiquitin and ubiquitylated proteins through its UBA domain (1, 3). Dimerization of TULA through its phosphoglyceromutase domain has also been shown (3). Analysis of cell and tissue expression of TULA demonstrates that this protein is expressed primarily in T and B lymphocytes and is localized both in the cytoplasm and nucleus (1, 4). A mouse orthologue of TULA (Sts-2) was recently identified (4), as was a second member of the family, Sts-1 (5). Unlike TULA, Sts-1 is expressed ubiquitously (4, 5). (In this report we will use the term TULA for consistency.)TULA has been implicated in the regulation of cell signaling mediated by protein-tyrosine kinases. On the one hand, TULA was reported to increase activity of receptor protein-tyrosine kinases by inhibiting c-Cbl-driven down-regulation of their activated forms. This appears to be mediated by preventing interactions between ubiquitylated forms of activated proteintyrosine kinases and proteins recruiting them to the degradation pathway and, possibly, by decreasing the level of c-Cbl (1, 3). On the other, the lack of both proteins of the TULA/Sts family resulted in hyper-reactivity of T lymphocytes correlated with an increase in the activity of Zap-70, the molecular basis of which remained unclear (4). These results implied that the effect of TULA on protein-tyrosine kinases might not be the only mechanism through which TULA exerts its biological effect. Indeed, the presence in TULA of multiple functional domains and extensive stretches of amino...

Inert gases diffuse into tissues in proportion to ambient pressure, and when pressure is reduced, gas efflux forms bubbles due to the presence of gas cavitation nuclei that are predicted based on theory but have never been characterized. Decompression stress triggers elevations in number and diameter of circulating annexin V-coated microparticles (MPs) derived from vascular cells. Here we show that ∼10% MPs from wild-type (WT) but not inflammatory nitric oxide synthase-2 (iNOS) knockout (KO) mice increase in size when exposed to elevated air pressure ex vivo. This response is abrogated by a preceding exposure to hydrostatic pressure, demonstrating the presence of a preformed gas phase. These MPs have lower density than most particles, 10-fold enrichment in iNOS, and generate commensurately more reactive nitrogen species (RNS). Surprisingly, RNS only slowly diffuse from within MPs unless particles are subjected to osmotic stress or membrane cholesterol is removed. WT mice treated with iNOS inhibitor and KO mice exhibit less decompression-induced neutrophil activation and vascular leak. Contrary to injecting naïve mice with MPs from wild-type decompressed mice, injecting KO MPs triggers fewer proinflammatory events. We conclude that nitrogen dioxide is a nascent gas nucleation site synthesized in some MPs and is responsible for initiating postdecompression inflammatory injuries.

Production of reactive species in neutrophils exposed to hyperoxia causes S-nitrosylation of ␤-actin, which increases formation of short actin filaments, leading to alterations in the cytoskeletal network that inhibit ␤ 2 integrin-dependent adherence (Thom, S. R., Bhopale, V. M., Mancini, D. J., and Milovanova, T. N. (2008) J. Biol. Chem. 283, 10822-10834). In this study, we found that vasodilator-stimulated protein (VASP) exhibits high affinity for S-nitrosylated short filamentous actin, which increases actin polymerization. VASP bundles Rac1, Rac2, cyclic AMP-dependent, and cyclic GMP-dependent protein kinases in close proximity to short actin filaments, and subsequent Rac activation increases actin free barbed end formation. Using specific chemical inhibitors or reducing cell concentrations of any of these proteins with small inhibitory RNA abrogates enhanced free barbed end formation, increased actin polymerization, and ␤ 2 integrin inhibition by hyperoxia. Alternatively, incubating neutrophils with formylmethionylleucylphenylalanine or 8-bromo-cyclic GMP activates either cyclic AMP-dependent or cyclic GMP-dependent protein kinase, respectively, outside of the short F-actin pool and phosphorylates VASP on serine 153. Phosphorylated VASP abrogates the augmented polymerization normally observed with S-nitrosylated actin, VASP binding to actin, elevated Rac activity, and elevated formation of actin free barbed ends, thus restoring normal ␤ 2 integrin function.Reactive species generated when neutrophils are exposed to high oxygen partial pressures (hyperbaric oxygen (HBO 2 )2 ) in vivo or in suspensions ex vivo inhibit ␤ 2 integrin-dependent adherence in animals and humans (1-4). This effect ameliorates a variety of ischemia-reperfusion disorders in animal studies, yet HBO 2 does not impair neutrophil immune surveillance processes (5). The unexplained basis for these observations was the impetus for this project.The reversible nature of HBO 2 -mediated neutrophil ␤ 2 integrin inhibition can be shown by incubating cells with chemoattractants, such as fMLP, or with membrane-permeable cyclic GMP (cGMP) analogs such as 8-bromo-cyclic GMP (8-Br-cGMP) (1-4). The goal of this investigation was to evaluate the mechanism for reversal of HBO 2 effects by these agents and improve understanding of how hyperoxia disturbs the neutrophil cytoskeleton.Neutrophils migrate by coordinating ␤ 2 integrin adhesion with turnover of filamentous actin (F-actin). Integrin adherence is controlled by conformational alterations in the extracellular structure to increase affinity and by clustering in the plane of the cell membrane to improve avidity. HBO 2 impedes avidity but not affinity changes by increasing production of reactive species derived from nitric-oxide synthase and myeloperoxidase (MPO), which cause S-nitrosylation of the four cysteine moieties closest to the carboxyl-terminal end of ␤-actin (2). This increases intracellular formation of short actin filaments that appears as a dense mass of intracellular F-actin by confocal mic...