ANGPTL3 stimulates endothelial cell adhesion and migration via integrin alpha vbeta 3 and induces blood vessel formation in vivo

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to alpha(v)beta(3) and induced integrin alpha(v)beta(3)-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin alpha(v)beta(3) and suggest a possible role in the regulation of angiogenesis.     

Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits tau's ability to promote microtubule assembly

In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615-622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitro results suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules.     

Metabolic Dysfunction in Familial, but Not Sporadic, Amyotrophic Lateral Sclerosis

Abstract: Autosomal dominant familial amyotrophic lateral sclerosis (FALS) is associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Previous studies have implicated the involvement of metabolic dysfunction in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined SOD activity and mitochondrial oxidative phosphorylation enzyme activities in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Cytosolic SOD activity, predominantly Cu/Zn SOD, was decreased ∼50% in all regions in FALS patients with SOD mutations but was not significantly altered in other patient groups. Marked increases in complex I and II–III activities were seen in FALS patients with SOD mutations but not in SALS patients. We also measured electron transport chain enzyme activities in a transgenic mouse model of FALS. Complex I activity was significantly increased in the forebrain of 60-day-old G93A transgenic mice overexpressing human mutant SOD1, relative to levels in transgenic wild-type animals, supporting the hypothesis that the motor neuron disorder associated with SOD1 mutations involves a defect in mitochondrial energy metabolism.     

1,7-Disubstituted oxyindoles are potent and selective EP3 receptor antagonists

A series of novel 1,7-disubstituted oxyindoles were shown to be potent and selective EP₃ receptor antagonists. Variation of substitution pattern at the C-3 position of indole enhanced in vitro metabolic stability of the resulting derivatives. Series 27a–c showed >1000-fold selectivity over a panel of prostanoid receptors including IP, FP, EP₁, EP₂ and EP₄. These agents also featured low CYP inhibition and good activity in the functional rat platelet aggregation assay.     

Exenatide once weekly treatment maintained improvements in glycemic control and weight loss over 2 years

Background

Latinos in the United States have a higher prevalence of type 2 diabetes than non-Latino whites, even after controlling for adiposity. Decreased adiponectin is associated with insulin resistance and predicts T2DM, and therefore may mediate this ethnic difference. We compared total and high-molecular-weight (HMW) adiponectin in Latino versus white individuals, identified factors associated with adiponectin in each ethnic group, and measured the contribution of adiponectin to ethnic differences in insulin resistance.

Methods

We utilized cross-sectional data from subjects in the Latinos Using Cardio Health Actions to reduce Risk study. Participants were Latino (n = 119) and non-Latino white (n = 60) men and women with hypertension and at least one other risk factor for CVD (age 61 ± 10 yrs, 49% with T2DM), seen at an integrated community health and hospital system in Denver, Colorado. Total and HMW adiponectin was measured by RIA and ELISA respectively. Fasting glucose and insulin were used to calculate the homeostasis model insulin resistance index (HOMA-IR). Variables independently associated with adiponectin levels were identified by linear regression analyses. Adiponectin's contribution to ethnic differences in insulin resistance was assessed in multivariate linear regression models of Latino ethnicity, with logHOMA-IR as a dependent variable, adjusting for possible confounders including age, gender, adiposity, and renal function.

Results

Mean adiponectin levels were lower in Latino than white patients (beta estimates: -4.5 (-6.4, -2.5), p < 0.001 and -1.6 (-2.7, -0.5), p < 0.005 for total and HMW adiponectin), independent of age, gender, BMI/waist circumference, thiazolidinedione use, diabetes status, and renal function. An expected negative association between adiponectin and waist circumference was seen among women and non-Latino white men, but no relationship between these two variables was observed among Latino men. Ethnic differences in logHOMA-IR were no longer observed after controlling for adiponectin levels.

Conclusions

Among patients with CVD risk, total and HMW adiponectin is lower in Latinos, independent of adiposity and other known regulators of adiponectin. Ethnic differences in adiponectin regulation may exist and future research in this area is warranted. Adiponectin levels accounted for the observed variability in insulin resistance, suggesting a contribution of decreased adiponectin to insulin resistance in Latino populations.     

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs

Background

Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.

Results

Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca²⁺)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca²⁺ concentration [Ca²⁺] difference between bulk cytosolic and the sub-plasma membrane rim.

Conclusion

This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.     

Compromised humoral and delayed-type hypersensitivity responses in IL-23-deficient mice

The heterodimeric cytokine IL-23 consists of a private cytokine-like p19 subunit and a cytokine receptor-like subunit, p40, which is shared with IL-12. Previously reported IL-12p40-deficient mice have profound immune defects resulting from combined deficiency in both IL-12 and IL-23. To address the effects of specific IL-23 deficiency, we generated mice lacking p19 by gene targeting. These mice display no overt abnormalities but mount severely compromised T-dependent humoral immune responses. IL-23p19(-/-) mice produce strongly reduced levels of Ag-specific Igs of all isotypes, but mount normal T-independent B cell responses. In addition, delayed type hypersensitivity responses are strongly impaired in the absence of IL-23, indicating a defect at the level of memory T cells. T cells stimulated with IL-23-deficient APCs secrete significantly reduced amounts of the proinflammatory cytokine IL-17, and IL-23-deficient mice phenotypically resemble IL-17-deficient animals. Thus, IL-23 plays a critical role in T cell-dependent immune responses, and our data provide further support for the existence of an IL-23/IL-17 axis of communication between the adaptive and innate parts of the immune system.     

Endogenous IFN-alpha production by plasmacytoid dendritic cells exerts an antiviral effect on thymic HIV-1 infection

Plasmacytoid dendritic cells (pDC) are the principal producers of IFN-alpha in response to viral infection. Because pDC are present in the thymus, we investigated the consequences of HIV-1-induced IFN-alpha production by thymic pDC. We observed that thymic pDC as well as thymocytes express intracellular IFN-alpha upon infection with HIV-1. However, only the pDC could suppress HIV-1 replication, because depletion of pDC resulted in enhancement of HIV-1 replication in thymocytes. Thymic pDC could also produce IFN-alpha in response to CpG oligonucleotides, consistent with the observations of others that peripheral pDC produce IFN-alpha upon engagement of TLR-9. Importantly, CpG considerably increased IFN-alpha production induced by HIV-1, and addition of CpG during HIV-1 infection enhanced expression of the IFN response protein MxA in thymocytes and strongly reduced HIV-replication. Our data indicate that thymic pDC modulate HIV-1 replication through secretion of IFN-alpha. The degree of inhibition depends on the level of IFN-alpha produced by the thymic pDC.