Wnt secretion and gradient formation.

Concentration gradients formed by the lipid-modified morphogens of the Wnt family are known for their pivotal roles during embryogenesis and adult tissue homeostasis. Wnt morphogens are also implicated in a variety of human diseases, especially cancer. Therefore, the signaling cascades triggered by Wnts have received considerable attention during recent decades. However, how Wnts are secreted and how concentration gradients are formed remains poorly understood. The use of model organisms such as Drosophila melanogaster has provided important advances in this area. For instance, we have previously shown that the lipid raft-associated reggie/flotillin proteins influence Wnt secretion and spreading in Drosophila. Our work supports the notion that producing cells secrete Wnt molecules in at least two pools: a poorly diffusible one and a reggie/flotillin-dependent highly diffusible pool which allows morphogen spreading over long distances away from its source of production. Here we revise the current views of Wnt secretion and spreading, and propose two models for the role of the reggie/flotillin proteins in these processes: (i) reggies/flotillins regulate the basolateral endocytosis of the poorly diffusible, membrane-bound Wnt pool, which is then sorted and secreted to apical compartments for long-range diffusion, and (ii) lipid rafts organized by reggies/flotillins serve as "dating points" where extracellular Wnt transiently interacts with lipoprotein receptors to allow its capture and further spreading via lipoprotein particles. We further discuss these processes in the context of human breast cancer. A better understanding of these phenomena may be relevant for identification of novel drug targets and therapeutic strategies.

Endothelial nitric oxide synthase gene transfer restores endothelium-dependent relaxations and attenuates lesion formation in carotid arteries in apolipoprotein E-deficient mice.

Nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP-1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP-1 expression. We have evaluated adenovirus-mediated gene transfer of human endothelial NO synthase (eNOS) and of a N-terminal deletion (8ND) mutant of the MCP-1 gene that acts as a MCP-1 inhibitor in arteriosclerosis-prone, apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-dependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5-week follow-up (p < 0.05). In contrast, 8ND-MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium-dependent vasodilation and inhibits lesion formation in ApoE(-/-) mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long-term efficacy of eNOS gene therapy for primary arteriosclerosis.

Role of cytokines in the formation and downregulation of hepatic circumoval granulomas and hepatic fibrosis in Schistosoma mansoni-infected mice

Schistosoma mansoni infections are associated with a strong Th2 cytokine response. Treatment of mice with IL-12 or anti-IL-2 or anti-IL-4 before i.v. injection of eggs increased IFN-gamma production and downregulated Th2 responses and pulmonary granuloma size. Conversely, anti-IFN-gamma antibody treatment increased Th2 responses and granuloma size. Similar manipulation produced less dramatic results in infected mice. However, sensitization of mice with eggs + IL-12 before infection augmented the Th1 response and decreased Th2 cytokines, granuloma size and fibrosis. Antisera to IFN-gamma, TNF-alpha or IL-12 during IL-12-egg immunization partly restored granuloma size and fibrosis following infection. Variations in the size of granulomas in acute (8 week) infections may be influenced primarily by the number and state of activation of T cells. In chronic (12-16 week) infections immunologic downmodulation proceeded normally in mice without functional CD8+ cells and in IFN-gamma KO mice but not in B cell KO (muMT) mice or in mice deficient in FcR expression in spite of the fact that these mice downregulated their T cell and cytokine responses. It is evident that the participation of cytokines in granuloma formation and regulation is complicated and that the mechanisms controlling both these phenomena are likely to involve both T cells and antibody/FcR interactions.

Profile of organic acid concentrations in the digestive gland and hemolymph of Biomphalaria glabrata under estivation

Using high performance liquid chromatography (HPLC) analysis it was possible to determine simultaneously the concentration of organic acids (pyruvate, lactate, succinate, fumarate, malate, acetate, propionate, acetoacetate, and ß-hydroxybutyrate) in the digestive gland and the extracellular concentration of these same acids in the hemolymph of estivating Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. After a 7 day period of estivation, there was a significant increase in the tissue levels of lactate, succinate, malate and acetate compared to non-estivating snails. After 14 days of estivation, the levels of lactate and acetate were also significantly elevated. The hemolymph concentrations of pyruvate and acetate increased significantly after 7 days and acetate concentrations continued to be significantly increased up to 14 days of estivation. The other organic acids studied, such as ketone body acetoacetate and ß-hydroxybutyrate or the volatile acid propionate, did not accumulate. Their tissue concentrations, however, increased on the 7th day of estivation and reached normal levels within two weeks of estivation for some of them. One should take into consideration how the reduction in metabolism can be handled under aerobic conditions, and what role anaerobic pathways may play in both energy formation and redox balance processes.

Chemical and carbon isotopic evolution of hydrocarbons during prograde metamorphism from 100°C to 550°C: Case study in the Liassic black shale formation of Central Swiss Alps

Hydrocarbon distributions and stable isotope ratios of carbonates (delta(13)C(car), delta(18)O(car)), kerogen (delta(13)C(ker)), extractable organic matter (delta(13)C(EOM)) and individual hydrocarbons of Liassic black shale samples from a prograde metamorphic sequence in the Swiss Alps were used to identify the major organic reactions with increasing metamorphic grade. The studied samples range from the diagenetic zone (< 100 degrees C) to amphibolite facies (similar to 550 degrees C). The samples within the diagenetic zones (< 100 and 150 degrees C) are characterized by the dominance of C-< 20 n-alkanes, suggesting an origin related with marine and/or bacterial inputs. The metamorphic samples (200 to 550 degrees C) have distributions significantly dominated by C-12 and C-13 n-alkanes, C-14, C-16 and C-18 n-alkylcyclopentanes and to a lesser extend C-15, C-17 and C-21 n-alkylcyclohexanes. The progressive C-13-enrichment (up to 3.9 parts per thousand) with metamorphism of the C-> 17 n-alkanes suggests the occurrence of cracking reactions of high molecular weight compounds. The isotopically heavier (up to 5.6 parts per thousand) C-< 17 n-alkanes in metamorphic samples are likely originated by thermal degradation of long-chain homologous with preferential release of isotopically light C-1 and C-2 radicals. The dominance of specific even C-number n-alkylcyclopentanes suggests an origin related to direct cyclization mechanism (without decarboxylation step) of algal or bacterial fatty acids occurring in reducing aqueous metamorphic fluid conditions. The regular increase of the concentrations of n-alkylcycloalkanes vs. C-> 13 n-alkanes with metamorphism suggests progressive thermal release of kerogen-linked fatty acid precursors and degradation of n-alkanes. Changes of the steroid and terpenoid distributions are clearly related to increasing metamorphic temperatures. The absence of 18 alpha(H)-22,29,30-trisnorneohopane (Ts), the occurrence of 17 beta(H)-trisnorhopane, 17 beta(H), 21 alpha(H)-hopanes in the C-29 to C-31 range and 5 alpha(H),14 alpha(H),17 alpha(H)-20R C-27, C-29 steranes in the low diagenetic samples (< 100 degrees C) are characteristic of immature bitumens. The higher thermal stress within the upper diagenetic zone (150 degrees C) is marked by the presence of Ts, the disappearance of 17 beta(H)-trisnorhopane and thermodynamic equilibrium of the 22S/(22S + 22R) homohopane ratios. The increase of the alpha alpha alpha-sterane 20S/(20S + 20R) and 20R beta beta/(beta beta + alpha alpha) ratios (from 0.0 to 0.55 and from 0.0 to 0.40, respectively) in the upper diagenetic zone indicates the occurrence of isomerization reactions already at < 150 degrees C. However, the isomerization at C-20 (R -> S) reaches thermodynamic equilibrium values already at the upper diagenesis (similar to 150 degrees C) whereas the epimerisation at C-14 and C-17 (alpha alpha ->beta beta) arrives to constant values in the lower anchizone (similar to 200 degrees C). The ratios Ts vs. 17 alpha(H)-22,29,30-trisnorneohopane [(Ts/(Ts + Tm)] and 18 alpha(H)-30-norneohopane (C29Ts) vs. 17 alpha(H),21 beta(H)-30-norhopane [C29Ts/(C29Ts + C-29)] increase until the medium anchizone (200 to 250 degrees C) from 0.0 to 0.96 and from 0.0 to 0.44, respectively. An opposite trend owards lower values is observed in the higher metamorphic samples. The occurrence of specific hydrocarbons (e.g., n-alkylcyclopentanes, cadalene, hydrogenated aromatic compounds) in metamorphic samples points to kerogen degradation reactions most probably occurring in the presence of water and under reducing conditions. The changes of hydrocarbon distributions and carbon isotopic compositions of n-alkanes related to metamorphism suggest that the organic geochemistry may help to evaluate the lowest grades of prograde metamorphism. Copyright (c) 2005 Elsevier Ltd.

Higher education as formation of the self and citizenship: an ethnography of the University Students from Amman, Jordan

My interest in higher education and citizenship in the Middle East at large and in Jordan in particular is fostered by some of the reflections Eickelman proposed (1992). Being a quite recent phenomenon, intimately linked with the more general topic of state formation it seemed to me more suitable to study it in a little country with a recent history (a field study left almost unexplored until now as far as Jordan is concerned, to the best of my knowledge, since Antoun 1994 focuses on the migration as a quest for higher education). The process of state formation in Jordan is quite studied. I thus intended to study the higher education policies as an attempt both to create a national citizenry and more recently as a way of controlling the more problematic part of the population (youth, which constitutes more than the double of the population. See UNDP and Ministry of Planning 2000). How do the young students enter the university system, and in which way does this system work? How is this system designed, in order to retain social control of the students (since they are usually perceived to be a factor of social and political instability, as in Iran or in Egypt)? Is there any significant difference between different faculties? And if so, why? My conclusions at this stage are that the university system is an integral part of the survival of the regime. The system works quite well, and Jordan has one of the best educational position in the region. Yet there are important distinctions to be made: the access to the better faculties is socially selective while the less valued faculties are left to the poorer and less wealthy youth. This results in a different treatment of the students and of the courses that I analysed. In the better faculties the teaching standards are quite high, and the relationship between professors and students is almost on a same-level base, while in the less privileged faculties the opposite is true. Thus we can observe a concrete politics of divide et impera intended to split the youth in two. For the more privileged there are some freedoms, both within and outside classes, designed I guess at forging them as autonomous individuals. On the opposite the less privileged are kept under tight control, even if also these students are a privileged category among youth at large.

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites.

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.

Mechanotransduction, PROX1, and FOXC2 cooperate to control connexin37 and calcineurin during lymphatic-valve formation.

Lymphatic valves are essential for efficient lymphatic transport, but the mechanisms of early lymphatic-valve morphogenesis and the role of biomechanical forces are not well understood. We found that the transcription factors PROX1 and FOXC2, highly expressed from the onset of valve formation, mediate segregation of lymphatic-valve-forming cells and cell mechanosensory responses to shear stress in vitro. Mechanistically, PROX1, FOXC2, and flow coordinately control expression of the gap junction protein connexin37 and activation of calcineurin/NFAT signaling. Connexin37 and calcineurin are required for the assembly and delimitation of lymphatic valve territory during development and for its postnatal maintenance. We propose a model in which regionally increased levels/activation states of transcription factors cooperate with mechanotransduction to induce a discrete cell-signaling pattern and morphogenetic event, such as formation of lymphatic valves. Our results also provide molecular insights into the role of endothelial cell identity in the regulation of vascular mechanotransduction.

Role of topological exclusion in formation and organization of chromosomal territories

Chromosomes of eukaryotic organisms are composed of chromatin loops. Using Monte Carlo simulations we investigate how the topological exclusion between loops belonging to different chromosomes affects chromosome behaviour. We show that in a confined space the topological exclusion limiting catenation between loops belonging to different chromosomes entropically drives the formation of chromosomal territories. The same topological exclusion in a connection with interchromosomal binding via transcription factories explains why actively transcribed genes are found preferentially at the peripheries of their chromosomal territories. This paper is based in part on the results presented in J. Dorier and A. Stasiak, Nucl. Acids Res. 37 (2009), 6316 and 38 (2010), 7410.

Angiogenesis and schistosomal granuloma formation

An increasing amount of evidences points to angiogenesis as playing a paramount role in fibrosis development. However, granulomas in general, and periovular schistosomal granulomas in particular, are considered avascular structures, although they usually result in dense areas of focal fibrosis. In order to clarify this apparent paradox, the presence of blood vessels was systematically searched in hepatic schistosomal granulomas of mice, during different stages of the infection, and at different stages of granuloma evolution, by means of vascular injections of colored masses, demonstration of laminin in vascular basement membranes and by ultra structural analysis. Vascular proliferation appeared evident at the early stages of granuloma formation, gradually decreasing thereafter, older granulomas becoming almost avascular structures, sometimes delimited at the periphery by a rich vascular network.

Mechanisms of leukocyte lipid body formation and function in inflammation

An area of increasingly interest for the understanding of cell signaling are the spatio-temporal aspects of the different enzymes involved in lipid mediator generation (eicosanoid-forming enzymes, phospholipases and their regulatory kinases and phosphatases) and pools of lipid precursors. The compartmentalization of signaling components within discrete and dynamic sites in the cell is critical for specificity and efficiency of enzymatic reactions of phosphorilation, enzyme activation and function. We hypothesized that lipid bodies - inducible non-membrane bound cytoplasmic lipid domains - function as specialized intracellular sites of compartmentalization of signaling with major roles in lipid mediator formation within leukocytes engaged in inflammatory process. Over the past years substantial progresses have been made demonstrating that all enzymes involved in eicosanoid synthesis localize at lipid bodies and lipid bodies are distinct sites for eicosanoid generation. Here we will review our current knowledge on the mechanisms of formation and functions of lipid bodies pertinent to inflammation.

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.