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1.
Int J Mol Sci ; 20(16)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426491

RESUMO

TASK-3 is a two-pore domain potassium (K2P) channel highly expressed in the hippocampus, cerebellum, and cortex. TASK-3 has been identified as an oncogenic potassium channel and it is overexpressed in different cancer types. For this reason, the development of new TASK-3 blockers could influence the pharmacological treatment of cancer and several neurological conditions. In the present work, we searched for novel TASK-3 blockers by using a virtual screening protocol that includes pharmacophore modeling, molecular docking, and free energy calculations. With this protocol, 19 potential TASK-3 blockers were identified. These molecules were tested in TASK-3 using patch clamp, and one blocker (DR16) was identified with an IC50 = 56.8 ± 3.9 µM. Using DR16 as a scaffold, we designed DR16.1, a novel TASK-3 inhibitor, with an IC50 = 14.2 ± 3.4 µM. Our finding takes on greater relevance considering that not many inhibitory TASK-3 modulators have been reported in the scientific literature until today. These two novel TASK-3 channel inhibitors (DR16 and DR16.1) are the first compounds found using a pharmacophore-based virtual screening and rational drug design protocol.

2.
J Med Chem ; 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260312

RESUMO

Rational drug design targeting ion channels is an exciting and always evolving research field. New medicinal chemistry strategies are being implemented to explore the wild chemical space and unravel the molecular basis of the ion channels modulators binding mechanisms. TASK channels belong to the two-pore domain potassium channel family and are modulated by extracellular acidosis. They are extensively distributed along the cardiovascular and central nervous systems, and their expression is up- and downregulated in different cancer types, which makes them an attractive therapeutic target. However, TASK channels remain unexplored, and drugs designed to target these channels are poorly selective. Here, we review TASK channels properties and their known blockers and activators, considering the new challenges in ion channels drug design and focusing on the implementation of computational methodologies in the drug discovery process.

3.
BMC Plant Biol ; 19(1): 316, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307394

RESUMO

BACKGROUND: HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species. RESULTS: By analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results. CONCLUSION: We identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cátions/metabolismo , Clonagem Molecular , Eletrofisiologia , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Relação Estrutura-Atividade , Xenopus laevis
4.
Hum Immunol ; 80(10): 842-847, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31320124

RESUMO

The natural killer group 2 (NKG2) family of receptors, encoded within the NK complex gene region (NKC), modulate the cytotoxic activity of NK cells. Two haplotype blocks throughout the NKC, hb-1 and hb-2 have been associated with different levels of overall natural cytotoxicity. Here, we evaluated allelic and genotype frequencies at rs1049174, rs2617160, rs2617170, rs2617171, rs1983526 (hb-1 haplotype), and rs2255336 and rs2246809 (hb-2 haplotype) in 928 subjects examined from Mexico City. The most frequent alleles and genotypes were as follows: C, CG to rs1049174; G, GG to rs2255336; T, AT to rs2617160; G, GG to rs2246809; C, CT to rs2617170; G, CG to rs2617171; and G, CG to rs1983526. Linkage disequilibrium analysis revealed that rs1049174, rs2617160, rs2617170, and rs2617171 constituted the haplotype block-1 variant (hb-1v) (r2 ≥ 0.89). Two predominant haplotypes of hb-1v were identified based on the allele content and included CTCG and GATC. This study is the first to evaluate the allelic and genotype frequency distribution of rs1049174, rs2255336, rs2617160, rs2246809, rs2617170, rs2617171, and rs1983526 in the population of Mexico City.

5.
FEMS Yeast Res ; 19(5)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247642

RESUMO

Finding new potential antagonists of potassium channels is a continuing task. TASK potassium channels operate over a large physiological range of membrane voltages, why they are thought to contribute to the excitability and resting potential of mammalian membrane potentials. Additionally, they are regulated by extracellular stimuli like changes in pH and K+ concentrations. TASK malfunctions are associated with diseases, which makes them popular targets for the search of new antagonists. Identification of channel inhibitors can be a time-consuming and expensive project. Here, we present an easy-to-use and inexpensive yeast system for the expression of the two-pore domain K+ channel TASK-3, and for the characterization of TASK-3 antagonists. The Saccharomyces cerevisiae strain BYT45 was used to express guinea pig TASK-3. The system allowed the expression and characterization of TASK-3 at variable pH values and K+ concentrations. Three known TASK-3 antagonists have been tested in the BYT45 yeast system: PK-THPP, ZnCl2 and Bupivacaine. Their inhibitory effect on TASK-3 was tested in solid and liquid media assays, and half maximal inhibitory concentrations were estimated. Although the system is less sensitive than more refined systems, the antagonistic activity could be confirmed for all three inhibitors.

6.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067753

RESUMO

TASK-3 potassium (K+) channels are highly expressed in the central nervous system, regulating the membrane potential of excitable cells. TASK-3 is involved in neurotransmitter action and has been identified as an oncogenic K+ channel. For this reason, the understanding of the action mechanism of pharmacological modulators of these channels is essential to obtain new therapeutic strategies. In this study we describe the binding mode of the potent antagonist PK-THPP into the TASK-3 channel. PK-THPP blocks TASK-1, the closest relative channel of TASK-3, with almost nine-times less potency. Our results confirm that the binding is influenced by the fenestrations state of TASK-3 channels and occurs when they are open. The binding is mainly governed by hydrophobic contacts between the blocker and the residues of the binding site. These interactions occur not only for PK-THPP, but also for the antagonist series based on 5,6,7,8 tetrahydropyrido[4,3-d]pyrimidine scaffold (THPP series). However, the marked difference in the potency of THPP series compounds such as 20b, 21, 22 and 23 (PK-THPP) respect to compounds such as 17b, inhibiting TASK-3 channels in the micromolar range is due to the presence of a hydrogen bond acceptor group that can establish interactions with the threonines of the selectivity filter.


Assuntos
Simulação de Acoplamento Molecular , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/química , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Sítios de Ligação , Humanos , Bloqueadores dos Canais de Potássio/química , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Ligação Proteica , Piridinas/química , Pirimidinas/química , Xenopus
7.
FASEB J ; 33(8): 9434-9452, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31112396

RESUMO

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective cationic channel involved in a wide variety of physiologic and pathophysiological processes. Bioinformatics analyses of the primary sequence of TRPM4 allowed us to identify a putative motif for interaction with end-binding (EB) proteins, which are microtubule plus-end tracking proteins. Here, we provide novel data suggesting that TRPM4 interacts with EB proteins. We show that mutations of the putative EB binding motif abolish the TRPM4-EB interaction, leading to a reduced expression of the mature population of the plasma membrane channel and instead display an endoplasmic reticulum-associated distribution. Furthermore, we demonstrate that EB1 and EB2 proteins are required for TRPM4 trafficking and functional activity. Finally, we demonstrated that the expression of a soluble fragment containing the EB binding motif of TRPM4 reduces the plasma membrane expression of the channel and affects TRPM4-dependent focal adhesion disassembly and cell invasion processes.-Blanco, C., Morales, D., Mogollones, I., Vergara-Jaque, A., Vargas, C., Álvarez, A., Riquelme, D., Leiva-Salcedo, E., González, W., Morales, D., Maureira, D., Aldunate, I., Cáceres, M., Varela, D., Cerda, O. EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.

8.
Int J Biochem Cell Biol ; 112: 18-23, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31026506

RESUMO

Transient receptor potential vanilloid 1 (TRPV1) is a polymodal cation channel activated by heat, voltage, and ligands. Also known as the capsaicin receptor, TRPV1 is expressed in numerous tissues by different cell types, including peripheral sensory fibers where acts as a thermal and chemical detector in nociceptive pathways. TRPV1 channels are able to bind a wide range of ligands, including a number of vanilloid derivatives all modulating channel's activity. When expressed by sensory neurons, activation of TRPV1 channels by heat (>40 °C), capsaicin (sub-micromolar), or acid environment (pH < 6), causes depolarization leading to burning pain sensation in mammals. Naturally occurring chalcones (1,3-diaryl-2-propen-1-ones) have been reported as effective inhibitors of TRPV1. Their relatively simple chemical structure and the possibility for handy chemical modification make them attractive ligands for the treatment of peripheral pain. By taking advantage of the structural information available, here we discuss pharmacological properties of chalcones and their putative mechanism of binding to TRPV1 channels.

9.
Cell Physiol Biochem ; 52(5): 1223-1235, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31001961

RESUMO

BACKGROUND/AIMS: The two-pore-domain potassium channel TASK-1 regulates atrial action potential duration. Due to the atrium-specific expression of TASK-1 in the human heart and the functional upregulation of TASK-1 currents in atrial fibrillation (AF), TASK-1 represents a promising target for the treatment of AF. Therefore, detailed knowledge of the molecular determinants of TASK-1 inhibition may help to identify new drugs for the future therapy of AF. In the current study, the molecular determinants of TASK-1 inhibition by the potent and antiarrhythmic compound A293 (AVE1231) were studied in detail. METHODS: Alanine-scanning mutagenesis together with two-electrode voltage-clamp recordings were combined with in silico docking experiments. RESULTS: Here, we have identified Q126 located in the M2 segment together with L239 and N240 of the M4 segment as amino acids essential for the A293-mediated inhibition of TASK-1. These data indicate a binding site which is different to that of A1899 for which also residues of the pore signature sequence and the late M4 segments are essential. Using in silico docking experiments, we propose a binding site at the lower end of the cytosolic pore, located at the entry to lateral side fenestrations of TASK-1. Strikingly, TASK-1 inhibition by the low affinity antiarrhythmic TASK-1 blockers propafenone, amiodarone and carvedilol was also strongly diminished by mutations at this novel binding site. CONCLUSION: We have identified the A293 binding site in the central cavity of TASK-1 and propose that this site might represent a conserved site of action for many low affinity antiarrhythmic TASK-1 blockers.


Assuntos
Antiarrítmicos/química , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Xenopus laevis
10.
Sci Total Environ ; 668: 1055-1063, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31018447

RESUMO

New adjuvant formulations, based on proteoliposomes <40 nm and cochleates <100 nm, without Al(OH)3 adjuvant, were evaluated regarding their ability to generate Th1 immune response through a Delayed -Type Hypersensitivity Test, at the mouse model, by using a Neisseria meningitidis B protein complex as antigen. The formulations were administered by intramuscular (IM) (2 inoculations - at baseline and after 14 days) and intranasal (IN) (3 inoculations at 7 days) immunization pathways. All IM immunized groups were able to induce similar response to these formulations as well as to VA-MENGOC-BC® vaccine - containing Al(OH)3 adjuvant (used as positive control of the trial). In all groups, the induced inflammation (IP) rate was statistically higher than in the negative control group (CN) (p < 0.05). Immunogenicity, measured by HSR and CD4+ lymphocyte increase was equivalent to the control vaccine and most important, granuloma reactogenicity at the site of injection was eliminated, fact demonstrated by histological study. All groups of animals immunized by IN route showed HSR reactions and statistically significant differences with respect to the CN group. However, IP values were lower, with statistical differences (p < 0.05) for the same adjuvant formulation IM administered, except the AIF2-nCh formulation that generated statistically similar induction (p > 0.05) by both immunization pathways, suggesting it to be the best candidate for the next IN trial. Proteoliposome and cochleate formulations tested were able to mount potent Th-1 immune response, equivalent to the original vaccine formulation, with the advantage of less reactogenicity in the site of the injection, caused by the toxicity of Al(OH)3 adjuvant gel.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Antígenos de Bactérias/imunologia , Imunidade Celular , Meningite Meningocócica/prevenção & controle , Vacinas Meningocócicas , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Injeções Intramusculares , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis , Proteolipídeos
11.
Science ; 363(6429): 875-880, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30792303

RESUMO

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.


Assuntos
Clorobenzenos/farmacologia , Canal de Potássio ERG1/agonistas , Canal de Potássio ERG1/química , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Tetra-Hidronaftalenos/farmacologia , Tetrazóis/farmacologia , Tioureia/análogos & derivados , ortoaminobenzoatos/farmacologia , Animais , Células CHO , Clorobenzenos/química , Cricetulus , Cristalografia por Raios X , Desenho de Drogas , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Domínios Proteicos , Tetra-Hidronaftalenos/química , Tetrazóis/química , Tioureia/química , Tioureia/farmacologia , Xenopus , ortoaminobenzoatos/química
12.
Nitric Oxide ; 86: 54-62, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30797972

RESUMO

Under normal conditions, connexin (Cx) hemichannels have a low open probability, which can increase under pathological conditions. Since hemichannels are permeable to relatively large molecules, their exacerbated activity has been linked to cell damage. Cx46 is highly expressed in the lens and its mutations have been associated to cataract formation, but it is unknown whether Cx46 has a role in non-genetic cataract formation (i.e. aging and diabetes). Nitric oxide (NO) is a key element in non-genetic cataract formation and Cx46 hemichannels have been shown to be sensitive to NO. The molecular mechanisms of the effects of NO on Cx46 are unknown, but are likely to result from Cx46 S-nitrosation (also known as S-nitrosylation). In this work, we found that lens opacity was correlated with Cx46 S-nitrosation in an animal model of cataract. Consistent with this result, a NO donor increased Cx46 S-nitrosation and hemichannel opening in HLE-B3 cells (cell line derived from human lens epithelial cells). Mutagenesis studies point to the cysteine located in the fourth transmembrane helix (TM4; human C212, rat C218) as the NO sensor. Electrophysiological studies performed in Xenopus oocytes revealed that rat Cx46 hemichannels are sensitive to different NO donors, and that the presence of C218 is necessary to observe the NO donors' effects. Unexpectedly, gap junctions formed by Cx46 were insensitive to NO or the reducing agent dithiothreitol. We propose that increased hemichannel opening and/or changes in their electrophysiological properties of human Cx46 due to S-nitrosation of the cysteine in TM4 could be an important factor in cataract formation.


Assuntos
Catarata/etiologia , Conexinas/metabolismo , Cisteína/química , Óxido Nítrico/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Conexinas/química , Cricetulus , Junções Comunicantes/metabolismo , Humanos , Masculino , Potenciais da Membrana/fisiologia , Mesocricetus , Camundongos , Nitrosação , Conformação Proteica em alfa-Hélice , Processamento de Proteína Pós-Traducional , Ratos Sprague-Dawley , Alinhamento de Sequência , Xenopus laevis , Peixe-Zebra
13.
Elife ; 82019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30803485

RESUMO

Two-pore-domain potassium (K2P) channels are key regulators of many physiological and pathophysiological processes and thus emerged as promising drug targets. As for other potassium channels, there is a lack of selective blockers, since drugs preferentially bind to a conserved binding site located in the central cavity. Thus, there is a high medical need to identify novel drug-binding sites outside the conserved lipophilic central cavity and to identify new allosteric mechanisms of channel inhibition. Here, we identified a novel binding site and allosteric inhibition mechanism, disrupting the recently proposed K+-flux gating mechanism of K2P channels, which results in an unusual voltage-dependent block of leak channels belonging to the TASK subfamily. The new binding site and allosteric mechanism of inhibition provide structural and mechanistic insights into the gating of TASK channels and the basis for the drug design of a new class of potent blockers targeting specific types of K2P channels.

14.
Horiz. sanitario (en linea) ; 17(3): 209-216, sep.-dic. 2018. tab, graf
Artigo em Espanhol | LILACS-Express | ID: biblio-1002104

RESUMO

Resumen Objetivo: Comparar la prevalencia de caries dental y necesidades de tratamiento según criterios International Caries Detection and Assessment System (ICDAS) y CPO-D, en niños de 7-8 años de Centros Escolares públicos rurales pertenecientes a 16 municipios de El Salvador. Materiales y métodos: El diseño corresponde a una investigación epidemiológica descriptiva transversal realizada a partir de las historias clínicas de 420 escolares de 7 - 8 años de zonas rurales de El Salvador; el diagnóstico de caries se estableció con base en criterios ICDAS. Las variables analizadas fueron: prevalencia de caries y necesidades de tratamientos. El CPO-D/ceo-d se estableció excluyendo las fases precavitacionales de la enfermedad, se empleó T de Student para probar las diferencias de las medias entre índices y la Prueba de Levene para evaluar la igualdad de las varianzas. La significación estadística fue fijada en p <0,05. Resultados: Según ICDAS, cada niño presentó un promedio de 9.52 dientes afectados por caries y 6.24 según CPO. La diferencia encontrada entre índices es significativa en dientes permanentes y en primarios (p<0,05). Los diagnósticos según criterios ICDAS, generaron 4,269 necesidades de tratamientos curativos y 4,475 preventivos; mientras que con CPO, 2,997 tratamientos curativos y ningún preventivo. Conclusiones: ICDAS reflejó una mayor afectación por caries al considerar los estadios precavitacionales; por tanto, permite establecer una diversidad de tratamientos preventivos y de limitación del daño, que, en su mayoría, no es posible indicar con CPO.


Abstract Objective: To compare the prevalence of dental cavity and treatment needs according to International Cavity Detection and Assessment System (ICDAS) and DMF, in 7-8 years old children of rural public school centers belonging to 16 municipal towns of El Salvador. Materials and methods: The design corresponds to a transversal descriptive epidemiological investigation made from clinical histories of 7-8 year- old schoolchildren in rural areas of El Salvador; the diagnosis of dental cavities was established based on ICDAS criteria. The variables analyzed were: caries prevalence and treatment needs. The DMF-T/ dmf-t was established, excluding the precavitation phases of the disease, Student's T was used to test the differences of the means between indexes and the Levene test to evaluate the equality of the variances. The statistical significance was set at p <0.05. Results: According to ICDAS, each child presented an average of 9.52 teeth affected by caries and, according to DMF, 6.24. The difference found between indexes is significant in permanent teeth and in primary teeth (p<0,05). Diagnoses according to ICDAS criteria, generated 4269 needs for curative treatments and 4475 preventive ones; while with DMF, 2997 curative treatments and no preventive ones. Conclusions: ICDAS reflected a higher affectation by dental cavities when considering the precavitation stages; therefore, it allows establishing a diversity of preventive treatments and damage limitation; which, mostly, it is not possible to indicate with DMF.


Resumo Objetivo: Comparar a prevalência de cáries dentárias e necessidade de tratamento de acordo com os critérios do International Caries Detection and Assessment System (ICDAS) e da CPO, em criabas de 7 a 8 años de escolas públicas em áreas rurais pertencentes a 16 municipios de El Salvador. Materiais e métodos: O projeto corresponde a uma investigação epidemiológica descritiva transversal, realizada a partir da história clínicas de 420 alunos de 7 a 8 anos de áreas rurais de El Salvador. O diagnóstico de cárie foi estabelecido com base nos critérios do ICDAS. As variáveis analisadas foram: a prevalencia de cárie e a necessidade de tratamento. O CPO-D/ceo-d foi estabelecido excluindo as fases pré-cavitacionais da doença. Utilizou- se o teste t de student para comparar as médias dos índices e o teste de levene teste de Levene para a igualdade de variançias. A significancia estatística foi definida em p <0,05. Resultados: De acordo com o ICDAS, cada criaba apresentou em média de 9,52 dentes afetados por cáries e 6,24 de acordo com CPO. A diferen9a encontrada entre os índices é significativa nos dentes definitivos e nos primeiros dentes (p<0,05). Os diagnósticos, de acordo com os critérios do ICDAS, criaram 4269 necessidades para tratamentos curativos e 4475 preventivos; mas com os de CPO, apenas 2997 tratamentos curativos e nenhum preventivo. Conclusões: O ICDAS demostrou maior quantidade de dentes afetados por cárie ao considerar as etapas pré-cavitacionais; no entanto, permite estabelecer uma diversidade de tratamentos preventivos e de limitação de danos que, na maior parte das vezes, náo é possível estabelecer com CPO.


Résumé Objectif: Comparer la prévalence des caries dentaires et des besoins de soins selon les criteres du International Caries Detection and Assessment System (ICDAS) et du CAO-D, chez les enfants de 7 a 8 ans de centres scolaires publiques ruraux de 16 municipalités du Salvador. Matériels et méthodes: L'étude est épidémiologique, transversale et descriptive. Elle a été réalisée à partir de l'histoire clinique de 420 écoliers de 7 a 8 ans, de zones rurales du Salvador. Le diagnostic de caries a été établi sur la base des critères ICDAS. Les variables analysées sont la prévalence de caries et les besoins de soins. Le CAO-D / ceo-d a été établi en excluant les stades pré-cavitaires de la maladie. Le test de Student a été utilisé pour comparer les moyennes obtenues pour les indices et le test de Levene pour évaluer l'égalité des variances. La signification statistique a été fixée à p <0,05. Résultats: Selon l'ICDAS, chaque enfant présentait en moyenne 9,52 dents cariées; et selon le CAO 6,24. La différence entre les indices est significative pour les dents permanentes et les primaires (p <0,05). Les diagnostics selon les criteres ICDAS, ont permis d'identifier 4269 besoins de soins curatifs et 4475 préventifs; avec le CAO, 2997 soins curatifs et aucun soins préventif. Conclusions: L'ICDAS a permis de détecter plus d'affectations par caries en raison de sa prise en compte des stades pré-cavitaires; il permet donc d'établir une grande diversité de soins préventifs pour limiter les dommages, ce qui est généralement impossible avec le CAO.

15.
J Vet Diagn Invest ; 30(6): 807-812, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30284505

RESUMO

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate-phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

16.
Biophys J ; 115(6): 968-980, 2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30195939

RESUMO

Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic α-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results.

17.
Artigo em Inglês | MEDLINE | ID: mdl-30018007

RESUMO

Consecutive treatments with recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) stimulate spermatogenesis and potentiate sperm production in pubescent specimens of the oligospermic Senegalese sole (Solea senegalensis). However, sperm production in response to the hormones is highly variable, and the steroidogenic potential of the testis may be diminished due to sustained hormone supply. Here, we compared the effectiveness of low (9 µg/kg) and high (18 µg/kg) doses of rFsh and rLh to improve sperm production in adult sole during late winter-early spring (onset of the natural spawning period), and in autumn under a controlled temperature of 12 °C (period of testicular recrudescence). Treatment with rFsh over six weeks during spring, followed by a single rLh injection, did not enhance sperm production, possibly because of an advanced stage of sexual maturation of the males, as reflected by high Lh plasma levels (~17 ng/ml) before rFsh treatment. In contrast, in autumn, when the Lh circulating levels were much lower (~3 ng/ml), the low doses of rFsh and rLh generated a four-times increase in sperm production, whereas the high doses of the hormones were ineffective. However, treatment with rLh, regardless of the effect of rFsh, improved the motility of spermatozoa during both spring and autumn. These data confirm that consecutive rFsh and rLh treatments increase sperm production and quality in adult sole males, although they seem to be highly sensitive to the rFsh dose. The efficiency of recombinant gonadotropins also appears to be season-dependent despite the asynchronous nature of the sole testis.

18.
Toxins (Basel) ; 10(1)2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29271884

RESUMO

Most snake venom toxins are proteins, and participate to envenomation through a diverse array of bioactivities, such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic or neurotoxic effects. The venom of a single snake species contains hundreds of toxins, and the venoms of the 725 species of venomous snakes represent a large pool of potentially bioactive proteins. Despite considerable discovery efforts, most of the snake venom toxins are still uncharacterized. Modern bioinformatics tools have been recently developed to mine snake venoms, helping focus experimental research on the most potentially interesting toxins. Some computational techniques predict toxin molecular targets, and the binding mode to these targets. This review gives an overview of current knowledge on the ~2200 sequences, and more than 400 three-dimensional structures of snake toxins deposited in public repositories, as well as of molecular modeling studies of the interaction between these toxins and their molecular targets. We also describe how modern bioinformatics have been used to study the snake venom protein phospholipase A2, the small basic myotoxin Crotamine, and the three-finger peptide Mambalgin.

19.
Mar Drugs ; 15(10)2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-29027927

RESUMO

Voltage-gated calcium (CaV) channels are widely expressed and are essential for the completion of multiple physiological processes. Close regulation of their activity by specific inhibitors and agonists become fundamental to understand their role in cellular homeostasis as well as in human tissues and organs. CaV channels are divided into two groups depending on the membrane potential required to activate them: High-voltage activated (HVA, CaV1.1-1.4; CaV2.1-2.3) and Low-voltage activated (LVA, CaV3.1-3.3). HVA channels are highly expressed in brain (neurons), heart, and adrenal medulla (chromaffin cells), among others, and are also classified into subtypes which can be distinguished using pharmacological approaches. Cone snails are marine gastropods that capture their prey by injecting venom, "conopeptides", which cause paralysis in a few seconds. A subset of conopeptides called conotoxins are relatively small polypeptides, rich in disulfide bonds, that target ion channels, transporters and receptors localized at the neuromuscular system of the animal target. In this review, we describe the structure and properties of conotoxins that selectively block HVA calcium channels. We compare their potency on several HVA channel subtypes, emphasizing neuronal calcium channels. Lastly, we analyze recent advances in the therapeutic use of conotoxins for medical treatments.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Conotoxinas/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio/metabolismo , Conotoxinas/química , Humanos , Potenciais da Membrana/efeitos dos fármacos , Caramujos
20.
Mol Pharm ; 14(7): 2197-2208, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28494157

RESUMO

A1899 is a potent and selective inhibitor of the two-pore domain potassium (K2P) channel TASK-1. It was previously reported that A1899 acts as an open-channel blocker and binds to residues of the P1 and P2 regions, the M2 and M4 segments, and the halothane response element. The recently described crystal structures of K2P channels together with the newly identified side fenestrations indicate that residues relevant for TASK-1 inhibition are not purely facing the central cavity as initially proposed. Accordingly, the TASK-1 binding site and the mechanism of inhibition might need a re-evaluation. We have used TASK-1 homology models based on recently crystallized K2P channels and molecular dynamics simulation to demonstrate that the highly potent TASK-1 blocker A1899 requires binding to residues located in the side fenestrations. Unexpectedly, most of the previously described residues that interfere with TASK-1 blockade by A1899 project their side chains toward the fenestration lumina, underlining the relevance of these structures for drug binding in K2P channels. Despite its hydrophobicity, A1899 does not seem to use the fenestrations to gain access to the central cavity from the lipid bilayer. In contrast, binding of A1899 to residues of the side fenestrations might provide a physical "anchor", reflecting an energetically favorable binding mode that after pore occlusion stabilizes the closed state of the channels.


Assuntos
Benzamidas/farmacologia , Benzenoacetamidas/farmacologia , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Animais , Benzamidas/química , Benzenoacetamidas/química , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/metabolismo
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