RESUMO
The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 microM EUAC with 50 microM, 100 microM, or 500 microM carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 microM CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 microM) obtained with 100 microM and 500 microM CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations.
Assuntos
Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Receptores Nicotínicos/fisiologia , Animais , Linhagem Celular Transformada , Diterpenos/química , Diterpenos/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Interações Medicamentosas , Estimulação Elétrica , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Mioblastos/fisiologia , Mioblastos/efeitos da radiação , Técnicas de Patch-Clamp/métodosRESUMO
A recent investigation of the constituents of Eunicea sp. from Colombia and Puerto Rico has provided two novel cembrane glycosides, calyculaglycosides D (6) and E (7), and a new cembranoid diterpene in free form, (+)-nephthenol (4). Metabolites 6 and 7 possess a glycosyl-fused cembrane skeleton with a 1S configuration. Biogenetic considerations have led to a revision of the previously assigned dilophol skeleton of calyculaglycosides A-C, which was confirmed by extensive 2D NMR investigation and a chemical degradation study. Consequently, the true structures for calyculaglycosides A-C are 8-10, respectively, not 1-3. This is the first report of the occurrence of marine diterpene glycosides having a cembrane aglycon.
Assuntos
Cnidários/química , Diterpenos/isolamento & purificação , Glicosídeos/isolamento & purificação , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Colômbia , Diterpenos/química , Diterpenos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Glicosídeos/química , Glicosídeos/farmacologia , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Porto Rico , Espectrofotometria Infravermelho , EstereoisomerismoRESUMO
Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)-2,7,11-cembratriene-4-6-diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine-induced (86)Rb(+) flux with IC(50) in the low micromolar range. The cell lines used were the SH-EP1-halpha4beta2 cell line heterologously expressing human alpha4beta2-AChR, the SH-SY5Y neuroblastoma line naturally expressing human ganglionic alpha3beta4-AChR, and the TE671/RD cell line naturally expressing embryonic muscle alpha1beta1gammadelta-AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13-bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [(3)H]tenocyclidine to AChR from Torpedo californica electric organ. IC(50) values were in the submicromolar to low-micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S.
Assuntos
Diterpenos/farmacologia , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Nicotiana/química , Nicotina/farmacologia , Plantas Tóxicas , Receptores Colinérgicos/efeitos dos fármacos , Animais , Ligação Competitiva , Células Cultivadas , Diterpenos/metabolismo , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Técnicas In Vitro , Nicotina/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptores Colinérgicos/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
Four diterpenes and a nor-diterpenoid, all of which possess unusual carbocyclic skeletons, were isolated from the hexane solubles of the West Indian gorgonian Pseudopterogorgia elisabethae. The structures and relative configurations of novel metabolites elisabethin D (2), elisabethin D acetate (3), 3-epi-elisabanolide (5), elisapterosin A (6), and elisapterosin B (7) were elucidated by interpretation of overall spectral data, which included 2D NMR correlation methods, IR, UV, and accurate mass measurements (HREI-MS and HRFAB-MS), chemical reactions, and X-ray diffraction analyses. The tetracyclic carbon skeleton of the elisapterosins is undescribed and constitutes a new class of C(20) rearranged diterpenes. Elisapterosin B displays strong in vitro anti-tuberculosis activity.
Assuntos
Cnidários/química , Diterpenos/química , Animais , Antituberculosos/química , Antituberculosos/isolamento & purificação , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/química , Cristalografia por Raios X , Diterpenos/isolamento & purificação , Diterpenos/metabolismo , Diterpenos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Compostos Policíclicos/química , Células Tumorais Cultivadas , Índias OcidentaisRESUMO
A chemical study of the hexane extracts of the gorgonian octocoral Pseudopterogorgia elisabethae collected in San Andrés Island, Colombia, led to the isolation of two highly conjugated amphilectane-type diterpenes, compounds 1 and 2. Their structures were established by spectroscopic studies, which included 2D NMR correlation methods, IR, UV, and accurate mass measurements (HREIMS).
Assuntos
Cnidários/química , Diterpenos/química , Animais , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Índias OcidentaisRESUMO
The class of diterpenoids with a 14-carbon cembrane ring, the cembranoids, includes both competitive and noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR). All 20 coelenterate-derived cembranoids studied in this report inhibited [piperidyl-3,4-3H]-phencyclidine ([3H]-PCP) binding to its high-affinity site on the electric organ AChR, with IC50s ranging from 0.9 microM for methylpseudoplexaurate to 372 microM for lophotoxin. Inhibition was complete with all cembranoids but lophotoxin and most Hill coefficients were close to 1. Methylpseudoplexaurate and [3H]-PCP binding was competitive. Methylpseudoplexaurate and the fourth most potent cembranoid, eunicin, competed with each other for [3H]-PCP displacement, indicating that there exist one or more cembranoid sites on the AChR. Cembranoid affinity for the AChR correlated with hydrophobicity, but was also dependent on other features. Methylpseudoplexaurate and n-octanol also competed with each other for [3H]-PCP displacement, indicating that the cembranoid site is linked to the n-octanol site on the AChR. Unlike lophotoxin, the five cembranoids tested did not inhibit [125I]Tyr54-alpha-bungarotoxin binding to the AChR agonist sites. All seven cembranoids tested on oocyte-expressed electric organ AChR reversibly blocked acetylcholine-induced currents, although the inhibitor concentration curves were shallow and the inhibition was incomplete.
Assuntos
Diterpenos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Feminino , Fenciclidina/metabolismo , Receptores Nicotínicos/metabolismo , Torpedo , Xenopus laevisRESUMO
A high level of DDT resistance and low levels of resistance to organophosphorus, carbamate and pyrethroid insecticides were detected by discriminating dose assays in field populations of Anopheles albimanus in Chiapas, southern Mexico, prior to a large-scale resistance management project described by Hemingway et al. (1997). Biochemical assays showed that the DDT resistance was caused by elevated levels of glutathione S-transferase (GST) activity leading to increased rates of metabolism of DDT to DDE. The numbers of individuals with elevated GST and DDT resistance were well correlated, suggesting that this is the only major DDT resistance mechanism in this population. The carbamate resistance in this population is conferred by an altered acetylcholinesterase (AChE)-based resistance mechanism. The level of resistance observed in the bioassays correlates with the frequency of individuals homozygous for the altered AChE allele. This suggests that the level of resistance conferred by this mechanism in its heterozygous state is below the level of detection by the WHO carbamate discriminating dosage bioassay. The low levels of organophosphate (OP) and pyrethroid resistance could be conferred by either the elevated esterase or monooxygenase enzymes. The esterases were elevated only with the substrate pNPA, and are unlikely to be causing broad spectrum OP resistance. The altered AChE mechanism may also be contributing to the OP but not the pyrethroid resistance. Significant differences in resistance gene frequencies were obtained from the F1 mosquitoes resulting from adults obtained by different collection methods. This may be caused by different insecticide selection pressures on the insects immediately prior to collection, or may be an indication that the indoor- and outdoor-resting A. albimanus collections are not from a randomly mating single population. The underlying genetic variability of the populations is currently being investigated by molecular methods.
Assuntos
Anopheles , Insetos Vetores , Controle de Mosquitos/métodos , Acetilcolinesterase/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , DDT/metabolismo , Feminino , Glutationa Transferase , Resistência a Inseticidas , Malária , MéxicoRESUMO
A new chemical study of the Caribbean gorgonian Eunicea succinea collected in Puerto Rico afforded seven new cembranolides (1-5, 7, and 8). Excepting 1, the new compounds described here possess an unusual unsaturation at either position C-1, C-6, or C-8, and all but 8 possess the C-12R, C-13S stereochemistry usually ascribed to cembranolides from E. succinea. Their chemical structures were carefully established by a combination of chemical and spectroscopic methods in addition to detailed NMR spectral comparisons with known cembranolide models.
Assuntos
Antineoplásicos/isolamento & purificação , Cnidários/química , Diterpenos/isolamento & purificação , Animais , Antineoplásicos/farmacologia , Diterpenos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Porto Rico , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Células Tumorais CultivadasRESUMO
We propose the use of generalized tree models (GTMs) to analyze data from entomological field studies. Generalized tree models can be used to characterize environments with different mosquito breeding capacity. A GTM simultaneously analyzes a set of predictor variables (e.g., vegetation coverage) in relation to a response variable (e.g., counts of Anopheles albimanus larvae), and how it varies with respect to a set of criterion variables (e.g., presence of predators). The algorithm produces a treelike graphical display with its root at the top and 2 branches stemming down from each node. At each node, conditions on the value of predictors partition the observations into subgroups (environments) in which the relation between response and criterion variables is most homogeneous.
Assuntos
Anopheles/fisiologia , Animais , Anopheles/crescimento & desenvolvimento , México , Plantas , ReproduçãoRESUMO
A blind test of two remote sensing-based models for predicting adult populations of Anopheles albimanus in villages, an indicator of malaria transmission risk, was conducted in southern Chiapas, Mexico. One model was developed using a discriminant analysis approach, while the other was based on regression analysis. The models were developed in 1992 for an area around Tapachula, Chiapas, using Landsat Thematic Mapper (TM) satellite data and geographic information system functions. Using two remotely sensed landscape elements, the discriminant model was able to successfully distinguish between villages with high and low An. albimanus abundance with an overall accuracy of 90%. To test the predictive capability of the models, multitemporal TM data were used to generate a landscape map of the Huixtla area, northwest of Tapachula, where the models were used to predict risk for 40 villages. The resulting predictions were not disclosed until the end of the test. Independently, An. albimanus abundance data were collected in the 40 randomly selected villages for which the predictions had been made. These data were subsequently used to assess the models' accuracies. The discriminant model accurately predicted 79% of the high-abundance villages and 50% of the low-abundance villages, for an overall accuracy of 70%. The regression model correctly identified seven of the 10 villages with the highest mosquito abundance. This test demonstrated that remote sensing-based models generated for one area can be used successfully in another, comparable area.