Long-term nitric oxide deficiency causes muscarinic supersensitivity and reduces beta(3)-adrenoceptor-mediated relaxation, causing rat detrusor overactivity

Background and purpose: Overactive bladder is a complex and widely prevalent condition, but little is known about its physiopathology. We have carried out morphological, biochemical and functional assays to investigate the effects of long-term nitric oxide (NO) deficiency on muscarinic receptor and beta-adrenoceptor modulation leading to overactivity of rat detrusor muscle. Experimental approach: Male Wistar rats received No-nitro-L-arginine methyl ester (L-NAME) in drinking water for 7-30 days. Functional responses to muscarinic and b-adrenoceptor agonists were measured in detrusor smooth muscle (DSM) strips in Krebs-Henseleit solution. Measurements of [H-3] inositol phosphate, NO synthase (NOS) activity, [H-3] quinuclidinyl benzilate ([H-3]QNB) binding and bladder morphology were also performed. Key results: Long-term L-NAME treatment significantly increased carbachol-induced DSM contractile responses after 15 and 30 days; relaxing responses to the beta(3)-adrenoceptor agonist BRL 37-344 were significantly reduced at 30 days. Constitutive NOS activity in bladder was reduced by 86% after 7 days and maintained up to 30 days of L-NAME treatment. Carbachol increased sixfold the [H-3] inositol phosphate in bladder tissue from rats treated with L-NAME. [H-3] QNB was bound with an apparent KD twofold higher in bladder membranes after L-NAME treatment compared with that in control. No morphological alterations in DSM were found. Conclusions and implications: Long-term NO deficiency increased rat DSM contractile responses to a muscarinic agonist, accompanied by significantly enhanced KD values for muscarinic receptors and [H-3] inositol phosphate accumulation in bladder. This supersensitivity for muscarinic agonists along with reductions of beta(3)-adrenoceptor-mediated relaxations indicated that overactive DSM resulted from chronic NO deficiency.

S100 beta Protein Expression: Gender- and Age-Related Daily Changes

S100 beta is a soluble protein released by glial cells mainly under the activation of the 5-HT1A receptor. It has been reported as a neuro-trophic and -tropic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and the plasticity underlying long-term potentiation in adult brains. The ability of S100 beta to rapidly regulate neuronal morphology raises the interesting point of whether there are daily rhythm or gender differences in S100 beta level in the brain. To answer this question, the S100 beta expression in adult female and male rats, as well as in adult female CD-21 and S100 beta -/- female mice, were investigated. Scintillation counting and morphometric analysis of the immunoreactivity of S100 beta, showed rhythmic daily expression. The female and male rats showed opposite cycles. Females presented the highest value at the beginning of the rest phase (5:00 h), while in males the maximum value appeared in the beginning of the motor activity period (21:00 h). These results confirm previous S100 beta evaluations in human serum and cerebrospinal fluid reporting the protein`s function as a biomarker for brain damage (Gazzolo et al. in Clin Chem 49:967-970, 2003; Clin Chim Acta 330:131-133, 2003; Pediatr Res 58:1170-1174, 2005), similar behavior was also observed for GFAP in relation to Alzheimer Disease (Fukuyama et al. in Eur Neurol 46:35-38, 2001). The data should be taken into account when considering S100 beta as a biomarker of health condition. In addition, the results raise questions on which structure or condition imposes these rhythms as well as on the physiological meaning of the observed gender differences.

Beyond the average: Detecting global singular nodes from local features in complex networks

Deviations from the average can provide valuable insights about the organization of natural systems. The present article extends this important principle to the systematic identification and analysis of singular motifs in complex networks. Six measurements quantifying different and complementary features of the connectivity around each node of a network were calculated, and multivariate statistical methods applied to identify singular nodes. The potential of the presented concepts and methodology was illustrated with respect to different types of complex real-world networks, namely the US air transportation network, the protein-protein interactions of the yeast Saccharomyces cerevisiae and the Roget thesaurus networks. The obtained singular motifs possessed unique functional roles in the networks. Three classic theoretical network models were also investigated, with the Barabasi-Albert model resulting in singular motifs corresponding to hubs, confirming the potential of the approach. Interestingly, the number of different types of singular node motifs as well as the number of their instances were found to be considerably higher in the real-world networks than in any of the benchmark networks. Copyright (C) EPLA, 2009

Structural stability and reversible unfolding of recombinant porcine S100A12

Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca(2+) homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn(2+) or Ca(2+) with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses. (c) 2008 Elsevier B.V. All rights reserved.

Novel Zn(2+)-binding Sites in Human Transthyretin IMPLICATIONS FOR AMYLOIDOGENESIS AND RETINOL-BINDING PROTEIN RECOGNITION

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn(2+) enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn(2+) at pH 4.6-7.5. All four structures reveal three tetra-coordinated Zn(2+)-binding sites (ZBS 1-3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR.Zn(2+) binding perturbs loop E-alpha-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases similar to 5-fold in the presence of Zn(2+). Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn(2+). HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn(2+) binding, although the alpha-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn(2+), which is consistent with the tertiary structural perturbation provoked by Zn(2+) binding, tetramer stability is only marginally affected by Zn(2+). These data highlight structural and functional roles of Zn(2+) in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.