RESUMEN

In this contribution, ethanolic extracts of Cuminum cyminum (C. cyminum) seeds were evaluated in terms of phytochemical content, total phenol and flavonoid contents. As far as the analytical techniques are concerned, UV-Vis, FTIR, HPLC, NMR (1H and 13C) and ESI-MS were performed. The binding capacity of five different antidiabetic enzymes was tested by in silico molecular docking studies. The HPLC, UV-Vis, FTIR, NMR and ESI-MS data highlighted the presence of seven biologically active molecules e.g. α-pinene, ß-pinene, Δ3-carene, ρ-cymene, α-terpineol, cuminaldehyde and linalool. The results coming from the in silico molecular docking studies showed that such phytochemicals present in the cumin seed extracts play an important role in the activity of key enzymes involved in carbohydrate metabolism. Therefore, C. cyminum is proven to be useful for the treatment of diabetes mellitus and its major secondary complications.

Asunto(s)

Cuminum , Hipoglucemiantes , Hipoglucemiantes/farmacología , Hipoglucemiantes/análisis , Cuminum/química , Simulación del Acoplamiento Molecular , Fitoquímicos/farmacología , Fitoquímicos/análisis , Extractos Vegetales/química , Semillas/química

RESUMEN

Pharmaceuticals are a new developing pollutant that is threatening aquatic ecosystems and impacting numerous species in the ecosystem. The aim of this study is the green synthesis of TiO2-Fe2O3-Chitosan nanocomposites in conjunction with Moringa olifera leaves extract and its applicability for ibuprofen removal. Various characterization studies were performed for the synthesized nanocomposites. Box-Behnken design (BBD) is employed to optimize pH, agitation speed, and composite dosage. Equilibrium results show that adsorption process matches with Langmuir isotherm, demonstrating adsorption on the nanocomposite's homogenous surface and follows pseudo-first-order kinetics. Using the BBD, pH, adsorbent dose, and agitation speed were examined as adsorption parameters. Ibuprofen elimination was demonstrated to be most successful at a pH of 7.3, using 0.05 g of nanocomposites at a rotational speed of 200 rpm. Thermodynamic parameters for ibuprofen sorption were carried out and the ΔH and ΔS was found to be 76.23 & 0.233. Molecular Docking was performed to find the interaction between the pollutant and the nanocomposite. UV-vis spectra confirm the 243 nm absorption band corresponding to the nanocomposite's surface plasmon resonances. Fourier transform infrared spectroscopy spectra relate this band to a group of nanocomposites. The findings of this work emphasize the importance of TiO2-Fe2O3-Chitosan nanocomposites for removing ibuprofen from wastewater.

Asunto(s)

Quitosano , Contaminantes Ambientales , Nanocompuestos , Contaminantes Químicos del Agua , Aguas Residuales , Ibuprofeno , Ecosistema , Simulación del Acoplamiento Molecular , Quitosano/química , Porosidad , Concentración de Iones de Hidrógeno , Termodinámica , Adsorción , Espectroscopía Infrarroja por Transformada de Fourier , Nanocompuestos/química , Cinética , Contaminantes Químicos del Agua/análisis

RESUMEN

Non-steroidal anti-inflammatory medicines (NSAIDs) like paracetamol and other substances released into the water system pose serious environmental issues. The current work examines the synthesis of a nanocomposite combined with Moringa olifera aqueous leaf extract as a reducing and stabilizing agent for the green synthesis of nanocomposites. Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Thermogravimetric analysis (TGA), Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM) were used to investigate metal based functional nanocomposites. The absorption band centered at a wavelength of 243 nm, which corresponds to the surface plasmon resonances of the produced nanocomposite, is confirmed in UV-vis spectra. The distinctive band at this particular wavelength is attributed to a particular group of nanocomposites based on the result from the Fourier transform infrared spectroscopy spectra. The spherical with irregularly shaped aggregates was confirmed by transmission electron microscopy, and the average size of nanoparticles was found to be 1 nm. For the elimination of pharmaceutical contaminants such as paracetamol from aqueous solutions, the adsorptive characteristics of nanocomposites were examined. Temperature, pH, adsorbent dosage, and agitation speed were investigated as adsorption parameters using Box-Behnken Design (BBD). The best removal outcomes were found under the following circumstances: temperature at 303.15 K, pH = 7.5, 0.05 g of nanocomposites at 200 rpm. Based on the adsorption study, the kinetics was found to be pseudo first order (R2 > 0.9481) which was validated and fitted by Langmuir isotherm (R2 > 0.9973). The adsorption study confirms that it was adsorbed onto the synthesized nanocomposite and found to be present on the homogeneous surface.

Asunto(s)

Nanocompuestos , Contaminantes Químicos del Agua , Acetaminofén , Adsorción , Antiinflamatorios , Antiinflamatorios no Esteroideos , Excipientes , Concentración de Iones de Hidrógeno , Cinética , Nanocompuestos/química , Extractos Vegetales , Espectroscopía Infrarroja por Transformada de Fourier , Aguas Residuales , Agua/química , Contaminantes Químicos del Agua/química

RESUMEN

Green technology for the synthesis of nanoparticles has gained momentum due to its cost-effectiveness and eco-friendly nature. In this research study, silver nanoparticles (AgNps) were synthesized using an eco-friendly biological method involving the use of marine algae, Halimeda gracilis. The surface properties of the synthesized silver nanoparticles were studied using UV-visible spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy methods. During the synthesis of nano particles, the parameters namely temperature (30 °C to 90 °C), pH (6-10), silver nitrate (AgNO3) concentration (1-3 mg/ml) and quantity of algal extract (1-3 ml) were optimized to improve the production of AgNPs. The application of the synthesized silver nanoparticles for the adsorptive removal of copper from aqueous and industrial wastewater was investigated. Intra-particle diffusion mechanism was identified to be controlling step in metal removal. Regeneration of sorbent was carried out using 2.0 M HCl and the reusability was verified for 6 cycles. A removal efficiency of copper (64.8%) from electroplating wastewater demonstrated the industrial application potential of the synthesized silver nanoparticles.

Asunto(s)

Cobre , Nanopartículas del Metal , Antibacterianos , Extractos Vegetales , Plata , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

The diseases vectored by mosquitoes continue to be a main cause of illnesses and death throughout the world. The methanol extract of Juglans regia male flower was screened for larvicidal activity against three therapeutically important mosquito vectors viz., malarial vector, Anopheles stephensi; dengue vector, Aedes aegypti and the filarial vector, Culex quinquefasciatus. The larvicidal activity was assayed against the early fourth-instar larvae of tested mosquito species at a concentration ranging from 12.5 to 200 ppm under laboratory conditions. The methanol extract recorded significant mortality against the early fourth-instar larvae of the tested species. After 12 and 24 h of exposure period, the highest effect was recorded in An. stephensi with LC50 values of 139.87 and 59.80 ppm and LC90 values of 288.96 and 166.73 ppm, respectively, followed by Ae. aegypti and Cx. quinquefasciatus. The results could be useful in search for newer, safer and more effective natural larvicidal agents.

Asunto(s)

Aedes/efectos de los fármacos , Anopheles/efectos de los fármacos , Culex/efectos de los fármacos , Insecticidas/farmacología , Juglans/química , Animales , Flores/química , Larva/efectos de los fármacos , Dosificación Letal Mediana , Extractos Vegetales/farmacología , Hojas de la Planta

RESUMEN

Vector-borne diseases transmitted by mosquitoes cause globally important diseases such as malaria, dengue fever, and filariasis. The incidence of these diseases can be reduced through mosquito control programs but these control programs currently rely on synthetic insecticides that can impact the environment, and has selected widespread mosquito resistance. Environment friendly and biodegradable natural insecticides discovered in plants offer an alternative approach to mosquito control. Here, we investigated extracts from root or aerial parts of Chicory (Cichorium intybus) and wormwood (Artemisia absinthium) against the early 4th instar larvae of Anopheles stephensi (malaria vector), Aedes aegypti (dengue fever vector), and Culex quinquefasciatus (filariasis vector). The root and aerial parts extracts of A. absinthium and C. intybus at 200, 100, 50, 25 and 12.5 ppm caused significant mortality of the tested mosquito species. Root extracts exhibited higher larvicidal activity that aerial part extracts. The highest larvicidal activity was recorded in methanol extract of roots of C. intybus with LC50 = 66.16, 18.88 and LC¬90 = 197.56, 107.16 ppm for An. stephensi; LC50 = 78.51, 40.15 and LC90 = 277.31, 231.28 ppm for Ae. aegypti and LC50 = 103.99, 64.56 and LC¬90 = 314.04, 247.54 ppm for Cx. quinquefasciatus. These results reveal potent mosquito larvicidal activity against vectors of malaria, dengue fever, and filariasis is present in extracts of chicory and wormwood.

Asunto(s)

Artemisia absinthium/química , Cichorium intybus/química , Insecticidas , Control de Mosquitos , Mosquitos Vectores , Extractos Vegetales , Aedes , Animales , Anopheles , Culex , Dengue/prevención & control , Filariasis/prevención & control , Larva/crecimiento & desarrollo , Malaria/prevención & control , Mosquitos Vectores/crecimiento & desarrollo

RESUMEN

Achillea millefoilum L. (Yarrow) is an important species of Asteraceae family with common utilization in traditional medicine of several cultures from Europe to Asia for the treatment of spasmodic gastrointestinal disorders, hepatobiliary, gynecological disorders, against inflammation and for wound healing. An extensive review of literature was made on A. millefoilum L. using ethno botanical text books, published articles in peer-reviewed journals, unpublished materials and scientific databases. The Plant List, International Plant Name Index and Kew Botanical Garden databases were used to authenticate the scientific names. Monoterpenes are the most representative metabolites constituting 90% of the essential oils in relation to the sesquiterpenes, and a wide range of chemical compounds have also been reported. Different pharmacological experiments in many in-vitro and in-vivo models have proved the potential of A. millefoilum with antiinflammatory, antiulcer, anticancer activities etc. lending support to the rationale behind numerous of its traditional uses. Due to the noteworthy pharmacological activities, A. millefoilum will be a better option for new drug discovery. The present review will comprehensively summarize the pharmacognosy, phytochemistry and ethnopharmacology of A. millefoilum reported to date, with emphasis on more in vitro, clinical and pathological studies needed to investigate the unexploited potential of this plant. Copyright © 2017 John Wiley & Sons, Ltd.

Asunto(s)

Achillea/química , Extractos Vegetales/farmacología , Animales , Etnofarmacología , Humanos , Medicina Tradicional , Farmacognosia , Fitoquímicos/farmacología , Fitoterapia , Ensayos Clínicos Controlados Aleatorios como Asunto

RESUMEN

Molecular dynamics (MD) simulations were carried out for inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) isoforms complexed with substrate (L-arginine) and the iNOS specific inhibitor GW 273629, 2 for a time period of 1.2ns. The simulations were compared both within and across the isoforms. iNOS specificity of inhibitor 2 is attributed to water mediated interactions and cooperative hydrogen bond networks. Juxtaposition of the carboxylic and ammonium groups in the substrate and inhibitor serve as a modulating key in binding to the isoforms. Based on these investigations, molecules 3 and 4 were rationally designed to attain specificity among the isoforms. The capability of the designed ligands was theoretically tested through MD simulations to envisage binding patterns with both isoforms. A detailed analysis of the molecular recognition pattern shows molecule 4 to be more selective to iNOS when compared to eNOS.

Asunto(s)

Ligandos , Modelos Moleculares , Óxido Nítrico Sintasa/química , Arginina/química , Arginina/metabolismo , Sitios de Unión , Simulación por Computador , Estructura Molecular , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismo , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-Actividad Cuantitativa , Sulfonas/química , Sulfonas/metabolismo , Termodinámica , Agua/química

RESUMEN

Three dimensional quantitative structure-activity relationship (3D-QSAR) studies were carried out on deoxythymidine monophosphate (dTMP) derivatives inhibiting thymidine monophosphate kinase (TMPK) in Mycobacterium tuberculosis. Molecular field analysis (MFA) models with three different alignment techniques, namely, least squares, pharmacophore based and receptor based methods were developed. Receptor based MFA model showed better results when compared with least squares and pharmacophore based models. The results help us to understand the nature of substituents required for activity and thereby provide guidelines to design novel and potent inhibitors as antitubercular agents.

Asunto(s)

Antituberculosos , Modelos Moleculares , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Relación Estructura-Actividad Cuantitativa , Timidina Monofosfato , Antituberculosos/química , Antituberculosos/farmacología , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Conformación Molecular , Mycobacterium tuberculosis/enzimología , Timidina Monofosfato/análogos & derivados , Timidina Monofosfato/química , Timidina Monofosfato/farmacología

RESUMEN

To evaluate the free radical scavenging activity of flavonoids containing extracts of Stevia leaf. Alcoholic. Successive butanolic and alcoholic extracts of leaves were examined for free radical scavenging activity using BHT (tert-butylhydroxytolune) as a positive control by in vitro models. Successive alcoholic extract showed remarkable free radical scavenging activity. The IC(50) was found to be 140 µg and 76 ig of successive alcoholic and BHT respectively in DPPH (2,2-diphenyl-1-picrylhydrazyl) assay method. The Dot-Blot test on silica layers also showed thescavenging activity of free radical of flavonoids containing extracts. The successive alcoholic extract shown significant antioxidant activity.

RESUMEN

Docking and pharmacophore screening tools were used to examine the binding of ligands in the active site of thymidine monophosphate kinase of Mycobacterium tuberculosis. Docking analysis of deoxythymidine monophosphate (dTMP) analogues suggests the role of hydrogen bonding and other weak interactions in enzyme selectivity. Water-mediated hydrogen-bond networks and a halogen-bond interaction seem to stabilize the molecular recognition. A pharmacophore model was developed using 20 dTMP analogues. The pharmacophoric features were complementary to the active site residues involved in the ligand recognition. On the basis of these studies, a composite screening model that combines the features from both the docking analysis and the pharmacophore model was developed. The composite model was validated by screening a database spiked with 47 known inhibitors. The model picked up 42 of these, giving an enrichment factor of 17. The validated model was used to successfully screen an in-house database of about 500,000 compounds. Subsequent screening with other filters gave 186 hit molecules.

Asunto(s)

Antituberculosos/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Modelos Químicos , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Antituberculosos/farmacología , Bases de Datos como Asunto , Inhibidores Enzimáticos/farmacología , Ligandos , Modelos Moleculares , Estructura Molecular , Nucleósido-Fosfato Quinasa/química , Relación Estructura-Actividad

RESUMEN

The 3D-QSAR studies of some indole derivatives as phosphodiesterase (PDE) type IV inhibitors was performed by Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) methods to determine the factors required for the activity of these compounds. The global minimum energy conformer of the template molecule, 3 the most active molecule of the series, was obtained by simulated annealing method and used to build the structures of the molecules in the dataset. The CoMFA model produced statistically significant results with cross-validated and conventional correlation coefficients of 0.494 and 0.986 respectively. The combination of steric, electrostatic and hydrophobic fields in CoMSIA gave the best results with cross-validated and conventional correlation coefficients of 0.541 and 0.967 respectively. The predictive ability of CoMFA and CoMSIA were determined using a test set of seven indole derivatives giving predictive correlation coefficients of 0.56 and 0.59 respectively indicating good predictive power. Further, the robustness of the models was verified by bootstrapping analysis. Based upon the information derived from CoMFA and CoMSIA, we have identified some key features that may be used to design new indole derivatives and predict their PDE IV affinities prior to synthesis.

Asunto(s)

3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Indoles/química , Inhibidores de Fosfodiesterasa/química , Relación Estructura-Actividad Cuantitativa , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Indoles/farmacología , Inhibidores de Fosfodiesterasa/farmacología

RESUMEN

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed for 100 anilinoquinazolines, inhibiting epidermal growth factor receptor (EGFR) kinase. The studies included molecular field analysis (MFA) and receptor surface analysis (RSA). The cross-validated r2 (r2cv) values are 0.81 and 0.79 for MFA and RSA, respectively. The predictive ability of these models was validated by 28 test set molecules. The results of the best QSAR model were further compared with structure-based investigations using docking studies with the crystal structure of EGFR kinase domain. The results helped to understand the nature of substituents at the 6- and 7-positions, thereby providing new guidelines for the design of novel inhibitors.

Asunto(s)

Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Quinazolinas/química , Quinazolinas/farmacología , Compuestos de Anilina/metabolismo , Animales , Sitios de Unión , Diseño de Fármacos , Activación Enzimática , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Receptores ErbB/antagonistas & inhibidores , Enlace de Hidrógeno , Ligandos , Modelos Químicos , Relación Estructura-Actividad Cuantitativa , Quinazolinas/metabolismo

RESUMEN

A series of 1,5-diarylpyrazoles having a substituted benzenesulfonamide moiety as pharmacophore was synthesized and evaluated for cyclooxygenase (COX-1/COX-2) inhibitory activities. Through SAR and molecular modeling, it was found that fluorine substitution on the benzenesulfonamide moiety along with an electron-donating group at the 4-position of the 5-aryl ring yielded selectivity as well as potency for COX-2 inhibition in vitro. Among such compounds 3-fluoro-4-[5-(4-methoxyphenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-1-benzenesulfonamide 3 displayed interesting pharmacokinetic properties along with antiinflammatory activity in vivo. Among the sodium salts tested in vivo, 10, the propionyl analogue of 3, showed excellent antiinflammatory activity and therefore represents a new lead structure for the development of injectable COX-2 specific inhibitors.

Asunto(s)

Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Ciclooxigenasa/farmacología , Isoenzimas/antagonistas & inhibidores , Pirazoles/síntesis química , Pirazoles/farmacología , Sodio/química , Sulfonamidas/química , Sulfonamidas/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacocinética , Antiinflamatorios no Esteroideos/farmacología , Sitios de Unión , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacocinética , Edema/inducido químicamente , Edema/tratamiento farmacológico , Humanos , Concentración 50 Inhibidora , Masculino , Proteínas de la Membrana , Modelos Moleculares , Prostaglandina-Endoperóxido Sintasas , Pirazoles/química , Pirazoles/farmacocinética , Ratas , Ratas Wistar , Ovinos , Spodoptera , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Bencenosulfonamidas

RESUMEN

A comparative molecular field analysis (CoMFA) of phthalazine class of phosphodiesterase IV (PDE IV) inhibitors has been performed to correlate their chemical structures with their observed biological activity. A statistically valid model with good correlative and predictive power is reported. The leave one out cross-validation study gave cross-validation r(2)(cv) of value 0.507 at six optimum components and conventional r(2) of value 0.98. The predictive ability of the model was tested by predicting the seven molecules belonging to the test set giving predictive correlation coefficient of 0.59. This model is potentially helpful in the design of novel and more potent PDE IV inhibitors.

Asunto(s)

3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacología , Ftalazinas/química , Ftalazinas/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Bases de Datos Factuales , Análisis de los Mínimos Cuadrados , Modelos Moleculares , Modelos Estadísticos , Conformación Molecular , Relación Estructura-Actividad Cuantitativa

RESUMEN

Cyclic nucleotide phosphodiesterase IV (PDE IV) inhibitors find utility in asthma and Chronic Obstructive Pulmonary Disease (COPD) therapy. A series of 29 thieno[3,2-d]pyrimidines with affinity for PDE IV was subjected to three dimensional quantitative structure activity relationship (3D-QSAR) studies using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Both CoMFA and CoMSIA provided statistically valid models with good correlative and predictive power. The incorporation of hydrophobic, hydrogen bond donor and hydrogen bond acceptor fields showed insignificant improvement in CoMSIA model. The 3D-QSAR models provide information for predicting the affinity of related compounds and designing more potent inhibitors.

Asunto(s)

3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Hidrocarburos Cíclicos/química , Hidrocarburos Cíclicos/farmacología , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Electricidad Estática , Termodinámica

RESUMEN

Comparative molecular field analysis and comparative molecular similarity indices analysis were performed on 114 analogues of 1,2-diarylimidazole to optimize their cyclooxygenase-2 (COX-2) selective antiinflammatory activities. These studies produced models with high correlation coefficients and good predictive abilities. Docking studies were also carried out wherein these analogues were docked into the active sites of both COX-1 and COX-2 to analyze the receptor ligand interactions that confer selectivity for COX-2. The most active molecule in the series (53) adopts an orientation similar to that of SC-558 (4-[5-(4-bromophenyl)-3-trifluoromethyl-1H-1-pyrozolyl]-1-benzenesulfonamide) inside the COX-2 active site while the least active molecule (101) optimizes in a different orientation. In the active site, there are some strong hydrogen-bonding interactions observed between residues His90, Arg513, and Phe518 and the ligands. Additionally, a correlation of the quantitative structure-activity relationship data and the docking results is found to validate each other and suggests the importance of the binding step in overall drug action.

Asunto(s)

Inhibidores de la Ciclooxigenasa/química , Imidazoles/química , Isoenzimas/química , Prostaglandina-Endoperóxido Sintasas/química , Sitios de Unión , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Diseño de Fármacos , Conformación Molecular , Relación Estructura-Actividad Cuantitativa

RESUMEN

In Drosophila, a BMP-related ligand Decapentaplegic (Dpp) is essential for cell fate specification during embryogenesis and in imaginal disc development. Dpp signaling culminates in the phosphorylation and nuclear translocation of Mothers against dpp (Mad), a receptor-specific Smad that can bind DNA and regulate the transcription of Dpp-responsive genes. Genetic analysis has implicated Schnurri (Shn), a zinc finger protein that shares homology with mammalian transcription factors, in the Dpp signal transduction pathway. However, a direct role for Shn in regulating the transcriptional response to Dpp has not been demonstrated. In this study we show that Shn acts as a DNA-binding Mad cofactor in the nuclear response to Dpp. Shn can bind DNA in a sequence-specific manner and recognizes sites within a well-characterized Dpp-responsive promoter element, the B enhancer of the Ultrabithorax (Ubx) gene. The Shn-binding sites are relevant for in vivo expression, since mutations in these sites affect the ability of the enhancer to respond to Dpp. Furthermore we find that Shn and Mad can interact directly through discrete domains. To examine the relative contribution of the two proteins in the regulation of endogenous Dpp target genes we developed a cell culture assay and show that Shn and Mad act synergistically to induce transcription. Our results suggest that cooperative interactions between these two transcription factors could play an important role in the regulation of Dpp target genes. This is the first evidence that Dpp/BMP signaling in flies requires the direct interaction of Mad with a partner transcription factor.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Insectos/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión/genética , Células COS , Células Cultivadas , ADN/genética , ADN/metabolismo , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Sistema Digestivo/embriología , Sistema Digestivo/metabolismo , Drosophila/embriología , Drosophila/genética , Elementos de Facilitación Genéticos , Genes de Insecto , Proteínas de Homeodominio/genética , Proteínas de Insectos/genética , Factores de Transcripción/genética , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Dedos de Zinc

RESUMEN

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of 'Sertoli cell only' animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of <9.8x10(-7) M for testosterone and 9x10(-6) M for oestradiol respectively.

Asunto(s)

Glutatión Transferasa/metabolismo , Túbulos Seminíferos/metabolismo , Esteroides/metabolismo , Animales , Busulfano/farmacología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Medios de Cultivo Condicionados , Femenino , Glutatión Transferasa/biosíntesis , Inmunohistoquímica , Isoenzimas/análisis , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Hígado/enzimología , Masculino , Peso Molecular , Mapeo Peptídico , Unión Proteica , Ratas , Ratas Wistar , Túbulos Seminíferos/citología , Túbulos Seminíferos/enzimología , Células de Sertoli/citología , Células de Sertoli/efectos de los fármacos , Células de Sertoli/enzimología , Células de Sertoli/metabolismo , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología

RESUMEN

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control.

Asunto(s)

Toxinas Bacterianas , Quitinasas/genética , Mariposas Nocturnas/crecimiento & desarrollo , Nicotiana/genética , Control Biológico de Vectores , Plantas Modificadas Genéticamente/genética , Plantas Tóxicas , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas , Quitinasas/metabolismo , Endotoxinas , Proteínas Hemolisinas , Manduca/enzimología , Manduca/genética , Manduca/crecimiento & desarrollo , Nicotiana/enzimología , Transformación Genética , Transgenes