Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation

Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid.     

Extra-nuclear signaling of progesterone receptor to breast cancer cell movement and invasion through the actin cytoskeleton

Progesterone plays a role in breast cancer development and progression but the effects on breast cancer cell movement or invasion have not been fully explored. In this study, we investigate the actions of natural progesterone and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin cytoskeleton remodeling and on breast cancer cell movement and invasion. In particular, we characterize the nongenomic signaling cascades implicated in these actions. T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices in the presence of both progestins. Exposure to the hormones triggers a rapid remodeling of the actin cytoskeleton and the formation of membrane ruffles required for cell movement, which are dependent on the rapid phosphorylation of the actin-regulatory protein moesin. The extra-cellular small GTPase RhoA/Rho-associated kinase (ROCK-2) cascade plays central role in progesterone- and MPA-induced moesin activation, cell migration and invasion. In the presence of progesterone, progesterone receptor A (PRA) interacts with the G protein G alpha(13), while MPA drives PR to interact with tyrosine kinase c-Src and to activate phosphatidylinositol-3 kinase, leading to the activation of RhoA/ROCK-2. In conclusion, our findings manifest that progesterone and MPA promote breast cancer cell movement via rapid actin cytoskeleton remodeling, which are mediated by moesin activation. These events are triggered by RhoA/ROCK-2 cascade through partially differing pathways by the two compounds. These results provide original mechanistic explanations for the effects of progestins on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.     

Cardiopulmonary resuscitation in the mouse.

We sought to develop a model of cardiac arrest and resuscitation on mice that would be comparable to that of large mammals and would allow for more fundamental investigations on cardiopulmonary arrest and cardiac resuscitation. A model of cardiopulmonary resuscitation previously developed by our group on rats was adapted to anesthetized, mechanically ventilated adult male Institute of Cancer Research mice that weighed 46 +/- 3 g. The trachea was intubated through the mouth, and end-tidal PCO(2) (PET(CO(2))) was measured with a microcapnometer. Catheters were advanced into the aorta and into the right atrium, and coronary perfusion pressure (CPP) was computed. A 1.5-mA alternating current was delivered to the right ventricular endocardium, which produced ventricular fibrillation or a pulseless rhythm. Precordial compression was begun 4 min later. Ten sequential studies were performed, during which five animals were successfully resuscitated and five failed resuscitation efforts. Successful resuscitation was contingent on the restoration of threshold levels of CPP and PET(CO(2)) during chest compression. As in rats, swine, and human patients, threshold levels of mean aortic pressure, CPP, and PET(CO(2)) were critical determinates of resuscitability in this murine model of threshold level of cardiac arrest and resuscitation.     

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases

Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1^+/+), hemizygous (Pin1^+/?) or knock-out (Pin1^?/?) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.     

Structures of the tumor necrosis factor α inducing protein Tipα: A novel virulence factor from Helicobacter pylori

Helicobacter pylori secretes a unique virulence factor, Tipα, that enters gastric cells and both stimulates the production of the TNF-α and activates the NF-κB pathway. The structures of a truncated version of Tipα (TipαN34) in two crystal forms are presented here. Tipα adopts a novel β₁α₁α₂β₂β₃α₃α₄ topology that can be described as a combination of three domains. A first region consists in a short flexible extension, a second displays a dodecin-like fold and a third is a helical bundle domain similar to the sterile alpha motif (SAM). Analysis of the oligomerisation states of TipαN34 in the crystals and in solution suggests that the disulfide bridges could hold together Tipα monomers during their secretion in the gastric environment.

Structured summary

MINT-7033680:TIP alpha (uniprotkb:B2UTN0) and TIP alpha (uniprotkb:B2UTN0) bind (MI:0407) by cosedimentation (MI:0027)     

Expression and Developmental Regulation of the Cystine/Glutamate Exchanger (x<Stack><Subscript>c</Subscript><Superscript>−</Superscript></Stack>) in the Rat

The cystine/glutamate exchanger (antiporter x_c⁻) is a membrane transporter involved in the uptake of cystine, the rate-limiting amino acid in the synthesis of glutathione. Recent studies suggest that the antiporter plays a role in the slow oxidative excitotoxity and in the pathological effects of β-N-oxalylamino-l-alanine, the molecule responsible for neurolathyrism, a neurotoxic upper motor neuron disease. The mouse cystine/glutamate exchanger has been cloned and showed to be composed of two distinct proteins, one of which being a novel protein, named xCT, of 502 amino acids and 12 putative trans-membrane domains. We have generated and purified a polyclonal antibody to mouse xCT and studied its expression in rat brain and in different cultured cells (astrocytes, fibroblasts and neurons) using Western blot and immunocytochemical techniques. Expression of xCT was also studied in rat brain and muscle at different developmental stages. Parallel experiments were carried out with antibodies to the heavy chain of 4F2 surface antigen, the non-specific subunit of the antiporter x_c⁻. xCT antibody detected in all cell and tissue extracts a specific band of about 40 kDa. Subcellular fractionation demonstrated that xCT is concentrated mainly in the microsomal-mitochondrial fraction, in accord with its structure as transmembrane protein. Immunocytochemical analysis showed a strong staining in all cells examined, included neurons. Furthermore, both xCT and the heavy chain of 4F2 surface antigen increased in the brain during development, reaching the highest expression in adulthood. The study of the expression and developmental profile of xCT represents a first step towards a better characterization of its biochemical properties and function, which in turn may help to understand the relative contribution of the x_c⁻ antiporter in the pathogenesis of certain neurodegenerative diseases.