Deficiency of vasodilator-stimulated phosphoprotein (VASP) increases blood-brain-barrier damage and edema formation after ischemic stroke in mice

Background

Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification.

Methodology/Principal Findings

Focal cerebral ischemia was induced in Vasp^−/− mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evan''s Blue tracer was applied to visualize the extent of blood-brain-barrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p<0.05) and edema volumes (1.7 mm³±0.5 mm³ versus 0.8 mm³±0.4 mm³; p<0.0001) were significantly enhanced in Vasp^−/− mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm³±17.3 mm³ versus 39.3 mm³±10.7 mm³, respectively; p<0.01) and a non significant trend (p>0.05) towards worse neurological outcomes.

Conclusion

Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage.     

Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation

Aplidin is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin-induced JNK activation and cytotoxicity. Our results show that Aplidin induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.     

Formation of phosphatidic acid, ceramide, and diglyceride on radiolysis of lipids: identification by MALDI-TOF mass spectrometry

By use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, phosphatidic acid was found to be the main product of γ radiolysis of cardiolipin, phosphatidylinositol, and phosphatidylglycerol. It has been shown that γ irradiation of such glycolipids as cerebroside and galactosyl diglyceride leads to formation of ceramide and diglyceride, respectively. These findings, combined with those obtained earlier, allowed an assumption to be made that, owing to radiation-induced free radical fragmentation of lipids in their polar moiety, formation of signaling molecules can occur.     

Molecular and genetic characterization of propionicin F, a bacteriocin from Propionibacterium freudenreichii

This work describes the purification and characterization of propionicin F, the first bacteriocin isolated from Propionibacterium freudenreichii. The bacteriocin has a bactericidal activity and is only active against strains of P. freudenreichii. Propionicin F appears to be formed through a processing pathway new to bacteriocins. The mass of the purified bacteriocin was determined by mass spectrometry, and the N-terminal amino acid sequence was determined by Edman degradation. Sequencing of pcfA, the bacteriocin structural gene, revealed that propionicin F corresponds to a 43-amino-acid peptide in the central part of a 255-amino-acid open reading frame, suggesting that mature propionicin F is excised from the probacteriocin by N- and C-terminal proteolytic modifications. DNA sequencing and Northern blot hybridizations revealed that pcfA is cotranscribed with genes encoding a putative proline peptidase and a protein from the radical S-adenosylmethionine family. A gene encoding an ABC transporter was also identified in close proximity to the bacteriocin structural gene. The potential role of these genes in propionicin F maturation and secretion is discussed.     

Inhibition of THP-1 cell-mediated low-density lipoprotein oxidation by the macrophage-synthesised pterin, 7,8-dihydroneopterin

Human macrophages release the pterin, 7,8-dihydroneopterin when exposed to the immune stimulant gamma-interferon (IFN-gamma). Previous in vitro studies have shown 7,8-dihydroneopterin is a potent antioxidant, which inhibits copper- and peroxyl-radical mediated low-density lipoprotein (LDL) oxidation. Using THP-1 cells, a human derived monocyte-like cell line, we have found that low micromolar concentrations of 7,8-dihydroneopterin inhibit cell mediated oxidation of LDL, as measured by electrophoretic mobility, alpha-tocopherol loss, and lipid oxidation. Stimulation of the THP-1 cells with IFN-gamma caused a significant reduction in the cells' ability to oxidise LDL. The extracellular pterin concentration increased from 0 to 16 nM with IFN-gamma stimulation, while the intracellular concentration increased from 0.21 to 1.69 nmol/mg cell protein.     

PSKH1, a novel splice factor compartment-associated serine kinase

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in splicing factor compartments (SFCs) within the nucleus of interphase cells. Nuclear SFCs are considered mainly as storage sites for splicing factors, supplying splicing factors to active genes. The mechanisms controlling the interaction of the various spliceosome constituents, and the dynamic nature of the SFCs, are still poorly understood. We show here that endogenous PSKH1, a previously cloned kinase, is located in SFCs. Migration of PSKH1-FLAG into SFCs is enhanced during co-expression of T7-tagged ASF/SF2 as well as other members of the SR protein family, but not by two other non-SR nuclear proteins serving as controls. Similar to the SR protein kinase family, overexpression of PSKH1 led to reorganization of co-expressed T7-SC35 and T7-ASF/SF2 into a more diffuse nuclear pattern. This redistribution was not dependent on PSKH1 kinase activity. Different from the SR protein kinases, the SFC-associating features of PSKH1 were located within its catalytic kinase domain and within its C-terminus. Although no direct interaction was observed between PSKH1 and any of the SR proteins tested in pull-down or yeast two-hybrid assays, forced expression of PSKH1-FLAG was shown to stimulate distal splicing of an E1A minigene in HeLa cells. Moreover, a GST-ASF/SF2 fusion was not phosphorylated by PSKH1, suggesting an indirect mechanism of action on SR proteins. Our data suggest a mutual relationship between PSKH1 and SR proteins, as they are able to target PSKH1 into SFCs, while forced PSKH1 expression modulates nuclear dynamics and the function of co-expressed splicing factors.     

Physiological significance of alpha(2)-adrenergic receptor subtype diversity: one receptor is not enough

Alpha(2)-adrenergic receptors mediate part of the diverse biological effects of the endogenous catecholamines epinephrine and norepinephrine. Three distinct subtypes of alpha(2)-adrenergic receptors, alpha(2A), alpha(2B), alpha(2C), have been identified from multiple species. Because of the lack of sufficiently subtype-selective ligands, the specific biological functions of these receptor subtypes were largely unknown until recently. Gene-targeted mice carrying deletions in the genes encoding for individual alpha(2)-receptor subtypes have added important new insight into the physiological significance of adrenergic receptor diversity. Two different strategies have emerged to regulate adrenergic signal transduction. Some biological functions are controlled by two counteracting alpha(2)-receptor subtypes, e.g., alpha(2A)-receptors decrease sympathetic outflow and blood pressure, whereas the alpha(2B)-subtype increases blood pressure. Other biological functions are regulated by synergistic alpha(2)-receptor subtypes. The inhibitory presynaptic feedback loop that tightly regulates neurotransmitter release from adrenergic nerves also requires two receptor subtypes, alpha(2A) and alpha(2C). Similarly, nociception is controlled at several levels by one of the three alpha(2)-receptor subtypes. Further investigation of the specific function of alpha(2)-subtypes will greatly enhance our understanding of the relevance of closely related receptor proteins and point out novel therapeutic strategies for subtype-selective drug development.     

Inhibition of p38 MAPK by glucocorticoids via induction of MAPK phosphatase-1 enhances nontypeable Haemophilus influenzae-induced expression of toll-like receptor 2

Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. We have demonstrated recently that glucocorticoids synergistically enhance nontypeable Haemophilus influenzae (NTHi)-induced expression of Toll-like receptor 2 (TLR2), an important TLR family member that has been shown to play a critical role in host immune and defense response. However, the molecular mechanisms underlying the glucocorticoid-mediated enhancement of TLR2 induction still remain unknown. Here we show that glucocorticoids synergistically enhance NTHi-induced TLR2 expression via specific up-regulation of the MAPK phosphatase-1 (MKP-1) that, in turn, leads to dephosphorylation and inactivation of p38 MAPK, the negative regulator for TLR2 expression. Moreover, increased expression of TLR2 in epithelial cells greatly enhances the NTHi-induced expression of several key cytokines, including tumor necrosis factor-alpha and interleukins 1beta and 8, thereby contributing significantly to host immune and defense response. These studies may bring new insights into the novel role of glucocorticoids in orchestrating and optimizing host immune and defense responses during bacterial infections and enhance our understanding of the signaling mechanisms underlying the glucocorticoid-mediated attenuation of MAPKs.     

Reactivity of Cartilage and Selected Carbohydrates with Hydroxyl Radicals: An NMR Study to Detect Degradation Products

It was investigated to what extent isolated, monomeric and polymeric carbohydrates as well as cartilage specimens are affected by hydroxyl radicals generated by γ-irradiation or Fenton reaction and what products can be detected by means of NMR spectroscopy. Resonances of all protons in glucose and other monosac-charides as well as carbon resonances in ¹³C-enriched glucose were continuously diminished upon γ-irradiation. Formate and malondialdehyde were found as NMR detectable products in irradiated glucose solutions under physiologically relevant (aerated) conditions. In polysaccharide solutions (e.g. hyaluronic acid) γ-irradiation and also treatment with the Fenton reagent caused first an enhancement of resonances according to mobile N-acetyl groups at 2.02 ppm. This indicates a breakdown of glycosidic bonds in polysac-charides. Using higher radiation doses or higher concentrations of the Fenton reagent formate was also detected. The same sequence of events was observed upon treatment of bovine nasal cartilage with the Fenton reagent. First, glycosidic linkages in cartilage polysaccharides were cleaved and subsequently formate was formed. In contrast, collagen of cartilage was affected only to a very low extent. Thus, HO-radicals caused the same action on cartilage as on isolated polymer solutions, inducing a fragmentation of polysaccharides and the formation of formate.