The Role of Kinin Receptors in Preventing Neuroinflammation and Its Clinical Severity during Experimental Autoimmune Encephalomyelitis in Mice

Background: Multiple sclerosis (MS) is a demyelinating and neuroinflammatory disease of the human central nervous system (CNS). the expression of kinins is increased in MS patients, but the underlying mechanisms by which the kinin receptor regulates MS development have not been elucidated.Methodology/Principal Findings: Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice by immunization with MOG(35-55) peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Here, we report that blockade of the B(1)R in the induction phase of EAE markedly suppressed its progression by interfering with the onset of the immune response. Furthermore, B(1)R antagonist suppressed the production/expression of antigen-specific T(H)1 and T(H)17 cytokines and transcription factors, both in the periphery and in the CNS. in the chronic phase of EAE, the blockade of B(1)R consistently impaired the clinical progression of EAE. Conversely, administration of the B(1)R agonist in the acute phase of EAE suppressed disease progression and inhibited the increase in permeability of the blood-brain barrier (BBB) and any further CNS inflammation. of note, blockade of the B(2)R only showed a moderate impact on all of the studied parameters of EAE progression.Conclusions/Significance: Our results strongly suggest that kinin receptors, mainly the B(1)R subtype, play a dual role in EAE progression depending on the phase of treatment through the lymphocytes and glial cell-dependent pathways.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Programa de Apoio aos Nucleos de Excelencia (PRONEX), BrazilFundacaode Apoio a Pesquisa Cientifica Tecnologica do Estado de Santa Catarina (FAPESC), BrazilUniv Fed Santa Catarina, Dept Pharmacol, Ctr Biol Sci, Florianopolis, SC, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilWeb of Scienc

RNA and DNA aptamers in cytomics analysis

Background: the systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. in contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised.Methods: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays.Results: We demonstrate the in vitro selection of antikinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane.Conclusions: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics. (C) 2004 Wiley-Liss, Inc

Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides

Somatic angiotensin I-converting enzyme (ACE) has two homologous active sites (N and C domains) that show differences in various biochemical properties. in a previous study, we described the use of positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain (Bersanetti et al., 2004). in the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. the peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P-3 to P-1' positions. the fluorogenic analog Abz-DVAK(Dnp)-OH (Abz=ortho-aminobenzoic acid; Dnp=2,4-dinitrophenyl) showed the highest selectivity for ACE N-domain (k(cat)/K-m = 1.76 mu M-1.s(-1)). Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. the binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Research FoundationUCTUniversidade Federal de São Paulo, Dept Biophys, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Hlth Informat Dept, BR-04023062 São Paulo, BrazilUCT Fac Hlth Sci, Div Med Biochem, Inst Infect Dis & Mol Med, ZA-7925 Cape Town, South AfricaUniv Cape Town, Dept Chem, ZA-7700 Cape Town, South AfricaUniversidade Federal de São Paulo, Dept Biophys, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Hlth Informat Dept, BR-04023062 São Paulo, BrazilWeb of Scienc

Cyclopalladated compounds as chemotherapeutic agents: Antitumor activity against a murine melanoma cell line

Palladacycle compounds obtained from N, N-dimethyl- I phenethylamine (dmpa), phenyl-2-pyridinyl-acetylene and 1-phenyl-3-N, N-dimethylamine-propine, respectively, were complexed to 1, 2 ethanebis (diphenylphosphine) (dppe) ligand to synthesize antitumor cyclopallaclated complexes that were tested in vitro and in vivo against syngeneic B16F10-Nex2 murine melanoma cells of low immunogenicity implanted subcutaneously in mice. Complexes were not toxic to mice injected 3 times i.p. with as much as 60 muM/animal/week. of 3 cyclopallaclated complexes that were inhibitory in vitro at low concentrations (< 1.25 muM), complex 7a was the most active in vivo, delaying tumor growth and prolonging animal survival. in vitro, binucleate complex 7a caused a collapse of respiratory activity with an abrupt decrease of extracellular acidification on short incubation (up to 100 min), followed by DNA degradation after 24 hr. the apoptosis-like reaction to this Pd-complex was not accompanied by increased levels of caspases I and 3. Complex 7a bound to a bacterial plasmid DNA, causing late conformational changes after 24 hr. Two other complexes with different C, N-cycles were also apoptotic and 2 binucleated ones were inactive. These results introduce the palladacycle-dppe complexes as promising antitumor drugs with exquisite structural specificities and for action in vivo and in vitro. (C) 2003 Wiley-Liss, Inc

Expression of angiotensin I-converting enzymes and bradykinin B-2 receptors in mouse inner medullary-collecting duct cells

We described in mouse inner medullary-collecting duct cells (m1MCD-3) the somatic and the N-domain ACE synthesis and its interaction with the kallikrein-kinin system co-localized in the same cells. We purified two ACE forms from culture medium, M1 (130 kDa) and M2 (N-domain, 60 kDa), and cellular lysate, C1 (130 kDa) and C2 (N-domain, 60 kDa). Captopril. and enalaprilat inhibited the purified enzymes. the immunofluorescence studies indicated that ACE is present in the membrane, cytoplasm and in the cell, nucleus. Kinin B-1 and B-2 receptors were detected by immunofluorescence and showed to be activated by BK and DesR(9) BK, increasing the acidification rate which was enhanced in the presence of enalaprilat. the presence of secreted and intracellular ACE in mIMCD-3 confirmed the hypothesis previously proposed by our group for a new site of ACE secretion in the collecting duct. (c) 2007 Elsevier B.V. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, Depto Med, Disciplina Nefrol, BR-04023900 São Paulo, BrazilDept Biofis, BR-04023900 São Paulo, BrazilDept Microbiol Imunol & Parasitol, Disciplina Parasitol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Depto Med, Disciplina Nefrol, BR-04023900 São Paulo, BrazilWeb of Scienc

Leptin regulates ACE activity in mice

Leptin is a hormone related to metabolism. It also influences blood pressure, but the mechanisms triggered in this process are not yet elucidated. Angiotensin-I converting enzyme (ACE) regulates cardiovascular functions and recently has been associated with metabolism control and obesity. Here, we used ob/ob mice, a model lacking leptin, to answer the question whether ACE and leptin could interact to influence blood pressure, thereby linking the renin-angiotensin system and obesity. These mice are obese and diabetic but have normal 24 h mean arterial pressure. Our results show that plasma and lung ACE activities as well as ACE mRNA expression were significantly decreased in ob/ob mice. in agreement with these findings, the hypotensive effect produced by enalapril administration was attenuated in the obese mice. Plasma renin, angiotensinogen, angiotensin I, bradykinin, and angiotensin 1-7 were increased, whereas plasma angiotensin II concentration was unchanged in obese mice. Chronic infusion of leptin increased renin activity and angiotensin II concentration in both groups and increased ACE activity in ob/ob mice. Acute leptin infusion restored ACE activity in leptin-deficient mice. Moreover, the effect of an ACE inhibitor on blood pressure was not changed in ob/+ mice during leptin treatment but increased four times in obese mice. in summary, our findings show that the renin-angiotensin system is altered in ob/ob mice, with markedly reduced ACE activity, which suggests a possible connection between the renin-angiotensin system and leptin. These results point to an important interplay between the angiotensinergic and the leptinergic systems, which may play a role in the pathogenesis of obesity, hypertension, and metabolic syndrome.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Deutsche Akademische Austauschdienst (DAAD/PROBRAL)Deutsche ForschungsgemeinschaftUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023062 São Paulo, BrazilMax Delbruck Ctr Mol Med MDC, ECRC, Berlin, GermanyUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023062 São Paulo, BrazilDeutsche Forschungsgemeinschaft: BA 1374/16-1Web of Scienc