Room-temperature single-photon emitters in titanium dioxide optical defects.

Fluorescence properties of crystallographic point defects within different morphologies of titanium dioxide were investigated. For the first time, room-temperature single-photon emission in titanium dioxide optical defects was discovered in thin films and commercial nanoparticles. Three-level defects were identified because the g(2) correlation data featured prominent shoulders around the antibunching dip. Stable and blinking photodynamics were observed for the single-photon emitters. These results reveal a new room-temperature single-photon source within a wide bandgap semiconductor.

Platelet membrane phospholipid asymmetry: from the characterization of a scramblase activity to the identification of an essential protein mutated in Scott syndrome.

Like all eukaryotic cells, platelets maintain plasma membrane phospholipid asymmetry in normal blood circulation via lipid transporters, which control transbilayer movement. Upon platelet activation, the asymmetric orientation of membrane phospholipids is rapidly disrupted, resulting in a calcium-dependent exposure of the anionic phospholipid, phosphatidylserine (PS), at the outer platelet surface. This newly-exposed PS surface is a major component of normal hemostasis because it supports platelet procoagulant function. Binding of blood clotting enzyme complexes to this negatively-charged membrane surface allows a dramatic increase in the rate of conversion of zymogens to active serine proteases, which in turn produce a burst of thrombin leading to the formation of a fibrin clot and further platelet activation. Cells have the capacity to catalyze transbilayer phospholipid exchange via ATP-requiring translocase enzymes (flippases and floppases), which control unidirectional phospholipid transport against a concentration gradient. They also use an energy-independent, calcium-dependent scramblase activity to govern the bidirectional exchange of phospholipids between the two leaflets of the bilayer; this activity is essential for PS exposure during platelet activation. Scramblase activity, biochemically characterized in the 1980s, is deficient in patients with Scott syndrome, a rare inherited bleeding disorder with defective platelet procoagulant activity. Despite considerable efforts, the platelet scramblase protein remained elusive for years but a significant advance has recently been made with the identification of TMEM16F, a membrane protein essential for calcium-dependent PS exposure whose loss of function mutations are found in Scott syndrome. This review recalls historical aspects of platelet membrane asymmetry characterization, summarizes the mechanisms and roles of PS exposure following platelet activation and discusses the recent identification of TMEM16F and its significance in the scrambling process.

A simple and reliable method for rapid production and purification of Pseudomonas aeruginosa haemolytic phospholipase C.

AIMS: To design a simple method to produce active recombinant Pseudomonas aeruginosa haemolytic phospholipase C (PLC). METHOD AND RESULTS: Pseudomonas aeruginosa PLC is a virulence factor mainly involved in inflammatory and cytotoxic responses. While ammonium sulphate purification requires large amounts of bacterial suspensions and leads to low yields, production of recombinant protein in Escherichia coli is no more successful because of frequent inclusion bodies and accumulation of inactive PLC in the periplasmic space. Using an inducible system based on the glucose-repressed inv1 promoter in the yeast Schizosaccharomyces pombe, we were able to produce up to 10 IU ml(-1) of pure toxin within 24 h. CONCLUSIONS: This work describes the first method to easily get recombinant haemolytic PLC. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides a powerful tool to study the mechanisms leading to its cellular toxicity.

[Lipids and skin inflammation: role of phospholipases A2]. / Lipides et inflammation cutanée: place des phospholipases A2.

Phospholipases A2 (PLA2) are enzymes that catalyse the hydrolysis of glycerophospholipids at the sn-2 position, generating free fatty acids and lysophospholipids. At present, PLA2 family consists of 12 groups. PLA2 are involved in many pathophysiological processes such as barrier function, eicosanoid production, and inflammation. They are implicated in inflammatory diseases of the skin: psoriasis, eczema, atopy. The presence of PLA2 activity has been demonstrated several years ago, however the precise localization of all these PLA2 in the epidermis and its appendages has to be determined. Further studies have shown that these enzymes are expressed in various layers of epidermis. This differential localization suggests different roles for each PLA2 in skin physiology and during inflammation.

Production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidic acid in platelet rafts: evidence for a critical role of cholesterol-enriched domains in human platelet activation.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.

An interaction between apo C-III variants and protease inhibitors contributes to high triglyceride/low HDL levels in treated HIV patients.

BACKGROUND: Long-term therapy with protease inhibitors (PI) is associated with hypertriglyceridaemia, low high-density lipoprotein (HDL) levels and accumulation of apolipoprotein (apo) E- and apo C-III-containing lipoproteins. OBJECTIVES: To evaluate the impact, on this dyslipaemic phenotype, of three polymorphisms of the apo C-III gene: two on an insulin response element and one in the 3'-region. Apo E genotypes were evaluated also. DESIGN: Sixty consecutive male patients attending the HIV follow-up consultation were included during a 3-month period. All patients received at least one PI. Apo C-III and apo E genotypes were determined. Besides routine bio-clinical examination, a detailed exploration of lipoproteins and of insulin secretion markers was carried out. METHODS: Plasma lipoparticles, insulin, proinsulin and C-peptide were measured by specific immuno-assays. Determination of apo C-III genotypes (-455C/T, -482C/T and SstI) and of apo E alleles (epsilon2, epsilon3 and epsilon4) were performed by amplification and endonuclease digestion and were confirmed by allele-specific oligonucleotide hybridization. RESULTS: Distribution of apo C-III alleles defined four major haplotypes. Carriers of the -455C variant had 30% lower levels of HDL-cholesterol than non-carriers. Plasma triglycerides increased according to the number of variant alleles. In multivariate analysis, a model including age, body mass index, clinical stage and treatment length, plasma insulin and apo C-III haplotypes explained around 43% of the HDL-cholesterol and triglycerides variability. Measurements of lipids before and after the use of PI demonstrated synergistic effects of the treatment and apo C-III variants on triglyceride levels. CONCLUSIONS: Apo C-III polymorphisms might identify a genetic predisposition to develop dyslipidaemia under PI therapy.

Vascular smooth muscle cell spreading onto fibrinogen is regulated by calpains and phospholipase C.

Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of (32)P-labeled phosphatidic acid and phosphatidylinositol-3,4-bisphosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4-bisphosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains.

Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

Rho-kinase is involved in the sustained phosphorylation of myosin and the irreversible platelet aggregation induced by PAR1 activating peptide.

We have addressed the role of Rho-kinase in the different steps of thrombin receptor agonist peptide (TRAP)-induced platelet activation. Interestingly, under physiological conditions, incubation of platelets with increasing concentrations of the specific Rho-kinase inhibitor Y-27632 resulted in a dose-dependent reversion of the aggregation induced by 10 microM TRAP, without affecting serotonin secretion. Addition of Y-27632 after three minutes of TRAP stimulation, when the maximal aggregation was reached, resulted in a rapid disaggregation of platelets. Accordingly, the early peak of myosin light chain (MLC) phosphorylation induced by TRAP was not affected by Y-27632 but its sustained phosphorylation, observed during the irreversible phase of aggregation, was dependent of Rho-kinase activity. The rapid decrease in MLC phosphorylation upon Y-27632 treatment correlated well with the specific disappearance of myosin heavy chain from the cytoskeleton and preceded platelet disaggregation. Finally, we provide evidence that secreted ADP, known to play a key role in TRAP-induced irreversible phase of aggregation, was involved in the sustained MLC phosphorylation through Rho-kinase and could be replaced by epinephrine.

Characterization of a G protein-activated phosphoinositide 3-kinase in vascular smooth muscle cell nuclei.

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), whereas guanosine 5'-(beta-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgammaS-induced PtdIns(3,4,5)P(3) synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P(3) accumulation, suggesting that nuclear G(i)/G(0) proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha(0) proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the control of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.

Enterophilins, a new family of leucine zipper proteins bearing a b30.2 domain and associated with enterocyte differentiation.

Enterocyte terminal differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, many of which code for proteins of the brush border membrane such as intestinal alkaline phosphatase, sucrase-isomaltase, or the microvillar structural protein villin. Several studies have shown that this sharp increase in specific mRNA levels is intimately associated with arrest of cell proliferation. We isolated several clones from a guinea pig intestine cDNA library. They encode new proteins characterized by an original structure associating a carboxyl-terminal B30.2/RFP-like domain and a long leucine zipper at the amino terminus. The first member of this novel gene family codes for a 65-kDa protein termed enterophilin-1, which is specifically expressed in enterocytes before their final differentiation. Enterophilin-1 is the most abundant in the small intestine but is still present in significant amounts in colonic enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized by a specific anti-enterophilin-1 antibody, and its expression was positively correlated with cell differentiation status. In addition, transfection of HT-29 cells with enterophilin-1 full-length cDNA slightly inhibited cell growth and promoted an increase in alkaline phosphatase activity. Taken together, these data identify enterophilins as a new family of proteins associated with enterocyte differentiation.

A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor.

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.

FcgammaRIIA requires a Gi-dependent pathway for an efficient stimulation of phosphoinositide 3-kinase, calcium mobilization, and platelet aggregation.

FcgammaRIIA, the only Fcgamma receptor present in platelets, is involved in heparin-associated thrombocytopenia (HIT). Recently, adenosine diphosphate (ADP) has been shown to play a major role in platelet activation and aggregation induced by FcgammaRIIA cross-linking or by sera from HIT patients. Herein, we investigated the mechanism of action of ADP as a cofactor in FcgammaRIIA-dependent platelet activation, which is classically known to involve tyrosine kinases. We first got pharmacologic evidence that the ADP receptor coupled to Gi was required for HIT sera or FcgammaRIIA clustering-induced platelet secretion and aggregation. Interestingly, the signaling from this ADP receptor could be replaced by triggering another Gi-coupled receptor, the alpha(2A)-adrenergic receptor. ADP scavengers did not significantly affect the tyrosine phosphorylation cascade initiated by FcgammaRIIA cross-linking. Conversely, the Gi-dependent signaling pathway, initiated either by ADP or epinephrine, was required for FcgammaRIIA-mediated phospholipase C activation and calcium mobilization. Indeed, concomitant signaling from Gi and FcgammaRIIA itself was necessary for an efficient synthesis of phosphatidylinositol 3,4,5-trisphosphate, a second messenger playing a critical role in the process of phospholipase Cgamma2 activation. Altogether, our data demonstrate that converging signaling pathways from Gi and tyrosine kinases are required for platelet secretion and aggregation induced by FcgammaRIIA.

Phosphoinositide 3-kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing alpha(IIb)beta(3) integrin activation.

Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.

Toxoplasma gondii secretes a calcium-independent phospholipase A(2).

Phospholipases A(2) (PLA(2)) play an important role in Toxoplasma gondii host cell penetration. They are also key enzymes in the host cell response to the parasite invasion. PLA(2) hydrolyse cellular phospholipids, releasing multiple inflammatory lipidic mediators. We have investigated the biochemical characterisation of T. gondii PLA(2) activity in a mouse-cultured tachyzoite homogenate and in the peritoneal exudate from infected mice, using the hydrolysis of a fluorescent phosphatidylglycerol labelled at the sn-2 position. Spectrofluorimetry and thin-layer chromatography showed a PLA(2) activity (about 0.5-2 nmol/min per mg), calcium-independent, secreted into infected mice peritoneal exudate, with a broad pH activity ranging between 6.5 and 9.5 and resistant to a great number of potential PLA(2) inhibitors except dithio-nitrobenzoic acid (1 mM). An associated phospholipase A(1) activity was also displayed. These results suggest that Toxoplasma gondii displays specific phospholipases different from host enzymes and probably involved at critical steps of infectious cycle.

The integrin alpha IIb/beta 3 in human platelet signal transduction.

Platelets are critical for the maintenance of the integrity of the vascular system and are the first line of defence against haemorrhage. When they encounter a subendothelial matrix exposed by injury to a vessel, platelets adhere, are activated, and become adhesive for other platelets so that they aggregate. alpha IIb/beta 3, a platelet-specific integrin, is largely prominent amongst the adhesion receptors and is essential for platelet aggregation. The ligands for alpha IIb/beta 3 are the multivalent adhesive proteins fibrinogen and von Willebrand factor. In resting platelets, alpha IIb/beta 3 is normally in a low activation state, unable to interact with soluble fibrinogen. Stimulation of platelets with various agonists will induce a conformational change in alpha IIb/beta 3 (inside-out signalling), which is then able to bind soluble fibrinogen resulting in the onset of platelet aggregation. However, fibrinogen binding to its membrane receptor is not simply a passive event allowing the formation of intercellular bridges between platelets. Indeed, a complex signalling pathway triggered by integrin ligation and clustering (outside-in signalling) will regulate the extent of irreversible platelet aggregation and clot retraction. Amongst the signalling enzymes activated downstream of alpha IIb/beta 3 engagement, phosphoinositide 3-kinase plays an important role in the control of the irreversible phase of aggregation.

Polymorphism of the 5' untranslated region of NHE1 gene associated with type-I diabetes.

The ubiquitous form of the sodium-hydrogen exchanger, NHE1, is devoted to the regulation of intracellular pH and cell volume. In addition, NHE1 activity is stimulated by growth factors and increased NHE rates are found in both circulating and immortalized cells during diabetes or diabetic nephropathy. In this context, we searched for polymorphisms of the 5'-flanking regulatory region of NHE1 gene in subjects with type-I diabetes. We identified a C/T transition 696 bases upstream the translation initiation start site which disrupts a repeated palindromic GC sequence. The TT genotype was significantly more frequent in type-1 diabetics and may have functional importance. Genetic linkage between NHE1 and diabetes has been previously described in NOD mice strains with consequences on NHE rates. Hence, the polymorphism described hereby may act as a predisposition factor to type-I diabetes or to diabetic complications, and may be useful to investigate the genetic involvement of NHE1 in human pathophysiology.

pp60c-src associates with the SH2-containing inositol-5-phosphatase SHIP1 and is involved in its tyrosine phosphorylation downstream of alphaIIbbeta3 integrin in human platelets.

SH2-containing inositol-5-phosphatase 1 (SHIP1) was originally identified as a 145 kDa protein that became tyrosine-phosphorylated in response to multiple cytokines. It is now well established that SHIP1 is specifically expressed in haemopoietic cells and is important as a negative regulator of signalling. We found recently that SHIP1 was present in human blood platelets as an Ins(1,3,4, 5)P(4)-phosphatase and a PtdIns(3,4,5)P(3)-5-phosphatase that became tyrosine-phosphorylated and was relocated to the cytoskeleton in an integrin-dependent manner. Here we report biochemical and pharmacological evidence that the tyrosine kinase pp60(c-src) is constitutively associated with SHIP1 and is involved in its tyrosine phosphorylation downstream of integrin engagement in thrombin-activated human platelets. The use of cytochalasin D allowed us to demonstrate that the actin cytoskeleton reorganization induced on thrombin stimulation was not required for its integrin-mediated phosphorylation. Moreover, the integrin-dependent relocation of SHIP1 to the cytoskeleton did not require its tyrosine phosphorylation. These results suggest that SHIP1 is first recruited to the integrin-linked signalling complexes and then becomes tyrosine-phosphorylated through a Src-kinase-dependent mechanism but independently of the actin cytoskeleton reorganization.

Identification of two secreted phospholipases A2 in human epidermis.

Phospholipases A2 are enzymes that catalyze the release of fatty acids from the sn-2 position of phospholipids. Fatty acids have been suggested to play a key role in the barrier function of the epidermis. The aim of this study was to identify and characterize the type of secretory phospholipase A2 expressed in human epidermis. We report the molecular cloning of two secretory phospholipase A2 in the human epidermis. The first enzyme is identical to human pancreatic type IB phospholipase A2. Western blots revealed a 14 kDa protein localized in the soluble fraction. The second phospholipase A2 is identical to human synovial type IIA enzyme and is localized in the membrane fraction. By semiquantitative reverse transcription-polymerase chain reaction performed on horizontal sections of the epidermis, we found that the mRNAs of both phospholipases A2 were expressed mainly in the basal layers of the epidermis. Our data thus provide evidence for the expression of two secretory phospholipases A2 in human epidermis. The different localization of these two secretory proteins strongly suggests that each enzyme might have a specific role in skin physiology and probably in the barrier function. Taken together, these data validate the reverse transcription-polymerase chain reaction technique performed on thin sections as a first approach to detect gene expression in different layers of the epidermis.