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The article aims to identify the key chemokine receptors associated with macrophage infiltration into the vascular wall during deoxycortisosterone acetate (DOCA)/salt-induced hypertension in mice. This evaluates the impact of pharmacological inhibition of receptors on blood pressure and lukocyte accumulation. The findings highlight CCR2 as a promising therapeutic target to reduce both macrophage accumulation in the vascular wall and blood pressure in hypertension.

Nox2 oxidase is one isoform in a family of seven NADPH oxidases that generate reactive oxygen species (ROS) and thereby contribute to physiological and pathological processes including host defense, redox signaling and oxidative tissue damage. While alternative mRNA splicing has been shown to influence the activity of several Nox-family proteins, functionally relevant splice variants of Nox2 have not previously been identified. We immunoscreened several mouse tissues and cells for the presence of truncated Nox2 proteins and identified a 30 kDa protein in lung, spleen and macrophages. RT-PCR analysis of mRNA from primary and immortalised (RAW264.7) mouse macrophages, and from human alveolar macrophages, identified a truncated Nox2 transcript which, upon sequence analysis, was found to be a product of the 'exon skipping' mode of alternative splicing, lacking exons 4-10 of the Nox2 gene. The predicted protein is comparable in size to that identified by immunoscreening and contains two transmembrane helices and an extended cytosolic C-terminus with binding sites for NADPH and the Nox organiser protein p47phox. Importantly, selective siRNA-mediated knockdown of the transcript reduced expression of the 30 kDa protein in macrophages, and suppressed phorbol ester-stimulated ROS production by 50%. We thus provide the first evidence that Nox2 undergoes alternative mRNA splicing to yield a 30 kDa protein -- herein termed Nox2β -- that regulates NADPH oxidase activity in macrophages from mice and humans. The discovery of Nox2β paves the way for future examination of its role in physiological and pathological processes. [ABSTRACT FROM AUTHOR]

We have investigated the glycosylation, disulfide bonding, and subunit structure of mouse TRPM8. To do this, amino-terminal c-myc or hemagglutinin epitope-tagged proteins were incorporated and expressed in Chinese hamster ovary cells. These modifications had no obvious effects on channel function in intracellular calcium imaging assays upon application of agonists, icilin or menthol, and cold temperatures. Unmodified TRPM8 migrates with an apparent mass of 129 kDa and can be glycosylated in Chinese hamster ovary cells to give glycoproteins with apparent masses of 136 and 147 kDa. We identified two potential N-linked glycosylation sites in TRPM8 (Asn-821 and Asn-934) and mutated them to show that only the site in the putative pore region at position 934 is modified and that glycosylation of this site is not absolutely necessary for cell surface expression or responsiveness to icilin, menthol, and cool temperatures. Enzymatic cleavage of the carbohydrate chains indicated that they are complex carbohydrate. The glycosylation site is flanked in the pore by two cysteine residues that we mutated, to prove that they are involved in a conserved double cysteine motif, which is essential for channel function. Mutation of either of these cysteines abolishes function and forces the formation of a non-functional complex of the size of a homodimer. The double cysteine mutant is also non-functional. Finally, we showed in Perfluoro-octanoic acid-polyacrylamide gels that TRPM8 can form a tetramer (in addition to dimer and trimer forms), consistent with current thinking that functional TRP ion channels are tetrameric. [ABSTRACT FROM AUTHOR]

This study investigated the effects of the endocannabinoid, anandamide, on M1 muscarinic acetylcholine receptors (mAChRs) expressed in Chinese hamster ovary (CHO) cells. In the presence of anandamide, [3H]N-methylscopolamine ([3H]NMS) inhibition binding was characterized by Hill coefficients greater than 1 while saturation binding isotherms were characterized by a reduction in radioligand Bmax. Anandamide did not affect the potency of classic agonists, antagonists or allosteric modulators to inhibit [3H]NMS binding, indicating that the site of anandamide action did not involve receptor regions recognized by these compounds. Although the mode of binding of anandamide was reversible, the order of ligand addition was important; the inhibitory effect was greatest when anandamide was equilibrated with the receptor prior to radioligand addition, and weakest in the converse situation. Interestingly, the inhibitory potency of anandamide was reduced on pre-equilibration with non-transfected CHO cell membranes, prior to addition of M1 mAChR-transfected membranes. In phosphoinositide (PI) hydrolysis assays, anandamide significantly reduced the maximal response to acetylcholine, but at higher concentrations than those needed to fully inhibit radioligand binding. Studies utilizing a range of agonists with varying intrinsic activities showed that the inhibitory effects of anandamide on agonist function were most pronounced with the lowest efficacy agonists. These findings suggest that the mechanism of action of anandamide at the M1 mAChR involves perturbation of the receptor via the membrane in a manner that is sensitive to the conformation of the receptor (occupied versus vacant). [Copyright &y& Elsevier]

The article establishes a contrast between approaches in ethical and political theory from vulnerability and approaches which require an account of the perfect conditions of morality or of the flourishing of human life. The stage is set by offering various example of approaches from vulnerability, then the Wittgensteinian perspective - especially elaborated by Stanley Cavell - is presented as one fruitful approach within this family of views. Human life in its naturalness, easiness and happiness is seen as the overcoming and reparation of episodes of crisis and blocks: it is achieved as a condition inherently open to vulnerability. This is also connected to the notion of forms of life as the work involved in such a reparation requires a redefinition of who we are from the point of view of other beings, and of various sorts of others. [ABSTRACT FROM AUTHOR]

The effect of ligand pretreatment on human 5-hydroxytryptamine2C (5-HT2C) receptors was examined in CHO cells expressing high (CHO-1C7; 67±3 pmol/mg) or low (CHO-1C19; 72±10 fmol/mg) levels of the receptor. Seventy-two hours pretreatment of CHO-1C7 cells with various ligands did not affect receptor expression. Pretreatment with inverse agonists enhanced 5-HT-mediated inositol phosphate accumulation with no change in constitutive receptor activity. The enhanced agonist responsiveness was inversely correlated with the intrinsic activity of the pretreatment ligand. Seventy-two hours of pretreatment with the weak agonist, 5-methoxygramine, caused an elevation in constitutive activity but no alteration in 5-HT-mediated signaling. In CHO-1C19 cells, 24 but not 72 h of pretreatment with the inverse agonist mianserin enhanced 5-HT-mediated signaling, with no effect on basal signaling; pretreatment with 5-methoxygramine had no significant effect. These findings highlight differences in the pattern of chronic regulation of 5HT2C receptor signaling between high and low receptor expression levels in a common cellular background. [Copyright &y& Elsevier]