Inhibition of Wnt/Axin/beta-catenin pathway activity promotes ventral CNS midline tissue to adopt hypothalamic rather than floorplate identity

Ventral midline cells in the neural tube form floorplate throughout most of the central nervous system (CNS) but in the anterior forebrain, they differentiate with hypothalamic identity. The signalling pathways responsible for subdivision of midline neural tissue into hypothalamic and floorplate domains are uncertain, and in this study, we have explored the role of the Wnt/Axin/beta-catenin pathway in this process. This pathway has been implicated in anteroposterior regionalisation of the dorsal neural tube but its role in patterning ventral midline tissue has not been rigorously assessed. We find that masterblind zebrafish embryos that carry a mutation in Axin1, an intracellular negative regulator of Wnt pathway activity, show an expansion of prospective floorplate coupled with a reduction of prospective hypothalamic tissue. Complementing this observation, transplantation of cells overexpressing axin1 into the prospective floorplate leads to induction of hypothalamic gene expression and suppression of floorplate marker gene expression. Axin1 is more efficient at inducing hypothalamic markers than several other Wnt pathway antagonists, and we present data suggesting that this may be due to an ability to promote Nodal signalling in addition to suppressing Wnt activity. Indeed, extracellular Wnt antagonists can promote hypothalamic gene expression when co-expressed with a modified form of Madh2 that activates Nodal signalling. These results suggest that Nodal signalling promotes the ability of cells to incorporate into ventral midline tissue, and within this tissue, antagonism of Wnt signalling promotes the acquisition of hypothalamic identity. Wnt signalling also affects patterning within the hypothalamus, suggesting that this pathway is involved in both the initial anteroposterior subdivision of ventral CNS midline fates and in the subsequent regionalisation of the hypothalamus. We suggest that by regulating the response of midline cells to signals that induce ventral fates, Axin1 and other modulators of Wnt pathway activity provide a mechanism by which cells can integrate dorsoventral and anteroposterior patterning information.     

Euchromatic and heterochromatic domains at Drosophila telomeres.

Noncoding repetitive sequences make up a large portion of eukaryotic genomes, but their function is not well understood. Large blocks of repetitive DNA-forming heterochromatin around the centromeres are required for this region to function properly, but are difficult to analyze. The smaller regions of heterochromatin at the telomeres provide an opportunity to study their DNA and protein composition. Drosophila telomere length is maintained through the targeted transposition of specific non-long terminal repeat retrotransposons to chromosome ends, where they form long tandem arrays. A subterminal telomere-associated sequence (TAS) lies immediately proximal to the terminal-retrotransposon array. Here, we review the experimental support for the heterochromatic features of Drosophila telomeres, and provide evidence that telomeric regions contain 2 distinct chromatin subdomains: TAS, which exhibits features that resemble beta heterochromatin; and the terminal array of retrotransposons, which appears euchromatic. This organization is significantly different from the telomeric organization of other eukaryotes, where the terminal telomerase-generated repeats are often folded in a t-loop structure and become part of the heterochromatin protein complex.     

Visceral Adipose Tissue Immune Homeostasis Is Regulated by the Crosstalk between Adipocytes and Dendritic Cell Subsets

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Curcumin Modulates the NMDA Receptor Subunit Composition Through a Mechanism Involving CaMKII and Ser/Thr Protein Phosphatases

Curcumin is one of the major compounds contained in turmeric, the powdered rhizome of Curcuma longa. Results obtained in various experimental models indicate that curcumin has the potential to treat a large variety of neuronal diseases. Excitotoxicity, the toxicity due to pathological glutamate receptors stimulation, has been considered to be involved in several ocular pathologies including ischemia, glaucoma, and diabetic retinopathy. The NMDA receptor (NMDAR), a heteromeric ligand-gated ion channel, is composed of GluN1 and GluN2 subunits. There are four GluN2 subunits (GluN2A-D), which are major determinants of the functional properties of NMDARs. It is widely accepted that GluN2B has a pivotal role in excitotoxicity while the role of GluN2A remains controversial. We previously demonstrated that curcumin is neuroprotective against NMDA-induced excitotoxicity with a mechanism involving an increase of GluN2A subunit activity. In this paper, we investigate the mechanisms involved in curcumin-induced GluN2A increase in retinal cultures. Our results show that curcumin treatment activated CaMKII with a time-course that paralleled those of GluN2A increase. Moreover, KN-93, a CaMKII inhibitor, was able to block the effect of curcumin on GluN2A expression. Finally, in our experimental model, curcumin reduced ser/thr phosphatases activity. Using okadaic acid, a specific PP1 and PP2A blocker, we observed an increase in GluN2A levels in cultures. The ability of okadaic acid to mimic the effect of curcumin on GluN2A expression suggests that curcumin might regulate GluN2A expression through a phosphatase-dependent mechanism. In conclusion, our findings indicate curcumin modulation of CaMKII and/or ser/thr phosphatases activities as a mechanism involved in GluN2A expression and neuroprotection against excitotoxicity.     

Influence of local and landscape factors on bumblebees in semi-natural meadows: a multiple-scale study in a forested landscape

Understanding the effects of local and landscape factors on bumblebees is relevant for the conservation of this group of pollinators. Bumblebees have been well-studied in agricultural landscapes of Western Europe, Asia and North America, but few studies have been developed on bumblebees in forest-dominated landscapes of Eastern Europe. We developed this study in 22 semi-natural meadows located in a patchy forested landscape of Estonia. We investigated the influence of habitat characteristics and landscape factors (calculated at four spatial scales: 250, 500, 1,000 and 2,000 m radius) on the total species richness and abundance of bumblebees. Correlation analysis, partial least squares (PLS) and stepwise forward-selection multiple regression analysis were applied in this study. The presence of a high diversity of flowering plants in semi-natural meadows may benefit the abundance of bumblebees. At the local level, patch area and shape seem to have positive and negative influences, respectively, on bumblebee species richness. At the landscape level, human settlements with the presence of gardens may favour bumblebee richness and abundance. Also, bumblebee species may increase with a high presence of meadows in the landscape, and may decrease with high percentages of forest and young forest. Overall, forested landscapes with a strong presence of edges and a diverse matrix may support a higher species richness and abundance of bumblebees. Both local and landscape factors should be considered when designing conservation strategies and agri-environmental measures.     

Influence of a cyanobacterial crude extract containing microcystin-LR on the physiology and antioxidative defence systems of different spinach variants

Plants can be contaminated with cyanobacterial toxins during spray irrigation of lake water containing toxic cyanobacteria. Here, long-term effects of cyanobacterial crude extract (containing microcystin-LR) on the growth and physiology of different spinach (Spinacia oleracea) variants under semifield conditions were investigated. Changes in antioxidative enzyme activities, and in glutathione, ascorbate and tocopherol contents were investigated to assess the reaction of the antioxidative defence systems in spinach to toxin exposure. In addition to severe morphological effects, such as growth inhibition and chlorosis, the generation of oxidative stress was observed at the cellular level. In response to the negative effects of oxidative stress, plants stimulated an antioxidative system consisting of an enzyme network with superoxide dismutases, peroxidases, catalases, glutathione S-transferases and glutathione reductases, as well as a set of low-molecular-weight antioxidants, including glutathione, ascorbate and tocopherols. Exposure of spinach to cyanobacterial crude extract affected germination, growth and morphology, as well as antioxidative response parameters. Different variants of the same plant reacted in different ways to certain toxicants.     

Homocysteine induces VCAM-1 gene expression through NF-kappaB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants

Hyperhomocysteinemia is a recognized risk factor for vascular disease, but pathogenetic mechanisms involved in its vascular actions are largely unknown. Because VCAM-1 expression is crucial in monocyte adhesion and early atherogenesis, we evaluated the NF-kappaB-related induction of VCAM-1 by homocysteine (Hcy) and the possible inhibitory effect of dietary polyphenolic antioxidants, such as trans-resveratrol (RSV) and hydroxytyrosol (HT), which are known inhibitors of NF-kappaB-mediated VCAM-1 induction. In human umbilical vein endothelial cells (HUVEC), Hcy, at 100 micromol/l, but not cysteine, induced VCAM-1 expression at the protein and mRNA levels, as shown by enzyme immunoassay and Northern analysis, respectively. Transfection studies with deletional VCAM-1 promoter constructs demonstrated that the two tandem NF-kappaB motifs in the VCAM-1 promoter are necessary for Hcy-induced VCAM-1 gene expression. Hcy-induced NF-kappaB activation was confirmed by EMSA, as shown by the nuclear translocation of its p65 (RelA) subunit and the degradation of the inhibitors IkappaB-alpha and IkappaB-beta by Western analysis. Hcy also increased intracellular reactive oxygen species by NAD(P)H oxidase activation, as shown by the membrane translocation of its p47(phox) subunit. NF-kappaB inhibitors decreased Hcy-induced intracellular reactive oxygen species and VCAM-1 expression. Finally, we found that nutritionally relevant concentrations of RSV and HT, but not folate and vitamin B6, reduce (by >60% at 10(-6) mol/l) Hcy-induced VCAM-1 expression and monocytoid cell adhesion to the endothelium. These data indicate that pathophysiologically relevant Hcy concentrations induce VCAM-1 expression through a prooxidant mechanism involving NF-kappaB. Natural Mediterranean diet antioxidants can inhibit such activation, suggesting their possible therapeutic role in Hcy-induced vascular damage.     

Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life

The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Delta2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Delta2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Delta2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.     

Anti-angiogenic gene therapy of cancer: current status and future prospects

The discovery of endogenous inhibitors of angiogenesis has made it possible to test the hypothesis that blocking the angiogenic switch may keep tumor growth in check, and has added a new investigational arm to the field of cancer gene therapy. Angiogenesis inhibitors are heterogeneous in origin and potency, and their growing list includes proteolysis products of larger molecules with a different function, such as angiostatin, endostatin and vasostatin, modulators of vascular endothelial growth factor activity, such as sFLT-1, and some cytokines/chemokines with marked anti-endothelial activity, such as IL-12, IFN-alpha, and CXCL10. Pre-clinical studies have clearly indicated that these factors are essentially cytostatic and that they need long-term administration in order to obtain prolonged anti-tumor effects, representing a rational basis for their delivery by a gene therapy approach. The experimental approaches attempted to date, reviewed herein, indicate overall that anti-angiogenic gene therapy has efficacy mainly as an early intervention strategy and that a better understanding of the biological mechanisms underlying resistance to angiogenesis inhibition, as well as appropriate combined treatments, are required to generate a conceptual advancement which could drive the field towards successful management of established tumors.     

Functional characterization of the three mitogen‐activated protein kinase kinases (MAP2Ks) present in the Cryphonectria parasitica genome reveals the necessity of Cpkk1 and Cpkk2, but not Cpkk3, for pathogenesis on chestnut (Castanea spp.)

The biological function(s) of the cpkk1, cpkk2 and cpkk3 genes, encoding the three mitogen‐activated protein kinase kinases (MAP2Ks) of Cryphonectria parasitica, the causal agent of chestnut blight, were examined through knockout strains. Cpkk1, the Mkk1 orthologue, acts in a phosphorylation cascade essential for cell integrity; Cpkk2 is the Ste7 orthologue involved in the pheromone response pathway; Cpkk3 is the Pbs2 orthologue, the MAP2K activated during the high‐osmolarity response. Our analysis confirmed the role of each MAP2K in its respective signalling cascade with some peculiarities: abnormal hyphae with a reduced number of septa and thinner cell walls were observed in Δcpkk1 mutants, and a strong growth defect on solid media was evident in Δcpkk2 mutants, when compared with the controls. Virulence on chestnut was affected in both the Δcpkk1 and Δcpkk2 strains, which were also unable to complete the developmental steps essential for mating. No alterations were reported in Δcpkk3, except under hyperosmotic conditions and in the presence of fludioxonil. Δcpkk2 mutants, however, showed higher sensitivity during growth in medium containing the antibiotic G418 (Geneticin).