RESUMEN
The new 2,3-diphenylquinoxalin-6-amine (dpqa) was designed and synthesized through an efficient and high yield condensation process. Data from FTIR and 1H-NMR spectroscopy have been adopted to ascertain the molecular structure of benzoxazine compounds. Furthermore, the quinoxaline amine based benzoxazine (BA-dpqa) was synthesized using bisphenol-A and paraformaldehyde followed by combining different weight percentages (1, 5 and 10 wt%) of (3-glycidyloxypropyl)trimethoxysilane functionalized CNT-PbS with benzoxazine to obtain nanocomposites. The thermal and morphological properties of the quinoxaline amine based neat polybenzoxazine matrix poly(BA-dpqa) and CNT-PbS/poly(BA-dpqa) composites were analysed by XRD, TGA and SEM analysis. The values of the degradation temperature (Td) obtained for neat poly(BA-dpqa) and 10 wt% CNT-PbS/poly(BA-dpqa) composites are 414 °C and 424 °C. Furthermore, the chair yield percentage was calculated as 33% and 35% respectively. The water contact angle of polybenzoxazine gradually increased from 89° to 127° proportional to the content of CNT-PbS. Among the composites, 10 wt% CNT-PbS reinforced poly(BA-dpqa) nanocomposites possess higher dielectric constant (k = 11.0) than other composites. The pseudocapacitor nature of the prepared electrodes is demonstrated by the good electrochemical performance according to the CV curve. Also, the prepared 10 wt% CNT-PbS/poly(BA-dpqa) (637 F g-1 at 5 A g-1 and 11.8 Ω) electrode shows better capacitance and lower charge transfer resistance values than 5 wt% CNT-PbS/poly(BA-dpqa) (613 F g-1 at 5 A g-1 and 13.2 Ω) and neat poly(BA-dpqa) (105 F g-1 at 5 A g-1 and 15.6 Ω) according to the charge/discharge curves and EIS spectra. 10 wt% CNT-PbS/poly(BA-dpqa) shows 99.2% cycling efficiency even at the 2000th cycle, which indicates the good electrochemical performance of the prepared electrode.
RESUMEN
Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.
Asunto(s)
Anoctaminas/ultraestructura , Proteínas de Arabidopsis/ultraestructura , Canales de Calcio/ultraestructura , Oryza/ultraestructura , Conformación Proteica , Secuencia de Aminoácidos/genética , Anoctaminas/química , Anoctaminas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Citoplasma/genética , Espectrometría de Masas , Potenciales de la Membrana/genética , Oryza/genética , Oryza/crecimiento & desarrollo , Presión Osmótica/fisiología , Agua/químicaRESUMEN
Antibiotics, a highly prevalent class of environmental organic pollutants, are becoming a matter of global concern. Clay minerals that are ubiquitous in subsurface environments may play an important role in the fate and transport of antibiotics. Taking ciprofloxacin (CIP) as a model antibiotic, this work explored the role of clay colloids (kaolinite and montmorillonite) on the adsorption and transport of CIP under different chemical solution conditions. The adsorption isotherms showed that montmorillonite colloids had a larger CIP sorption capacity than kaolinite colloids. The results of transport experiments indicated that montmorillonite colloids could promote CIP transport in saturated sand columns, but the addition of kaolinite colloids affected CIP mobility to a much smaller extent. The much stronger transport-enhancement effect of montmorillonite colloids was due to CIP adsorbed strongly to the colloids and desorption hysteresis of colloid-adsorbed CIP, likely stemming from the intercalation of this antibiotic in the interlayer of montmorillonite. Interestingly, transport of clay colloids increased with the increasing pH from 5.0 to 9.0; however, CIP transport decreased with the increasing pH in the presence of clay colloids. The observations were likely attributable to pH-dependent ciprofloxacin adsorption/desorption to clay minerals. Increasing the concentrations of NaCl and CaCl2 generally decreased the contaminant-mobilizing ability of montmorillonite colloids, mainly by increasing the aggregation of colloids and thus, decreasing the transport of colloid-adsorbed CIP. Moreover, under the test conditions (1 mM NaCl and pH 7.0), the presence of CIP inhibited the transport of clay colloids due to the increase in aggregate size of clay colloids with the addition of CIP. Overall, these findings suggest that clay colloids with high adsorption abilities for antibiotics in the subsurface environment may act as a carrier for certain antibiotic compounds.
Asunto(s)
Antibacterianos/análisis , Ciprofloxacina/análisis , Arcilla/química , Contaminantes Ambientales/análisis , Cuarzo/química , Arena/química , Adsorción , Bentonita/química , Coloides , Caolín/química , Tamaño de la Partícula , Porosidad , Soluciones , Propiedades de SuperficieRESUMEN
An electrochemical sensor is described for the simultaneous voltammetric determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). An indium-tin oxide (ITO) electrode was modified with a hierarchical core-shell metal-organic framework and Ag-doped mesoporous metal-oxide based hybrid nanocomposites on g-C3N4 nanosheets. The morphology, structural and chemical composition of the hybrid nanocomposite was characterized using different analytical methods. The modified ITO showed superior electrocatalytic performance towards the oxidation of AA, DA and UA due to the enhanced surface area, synergistic effects and well-organized porous assembly. Figures of merit, include (a) linear responses from 0.1 to 200 µM, 2.5 to 100 µM and 2.5 to 625 µM; (b) detection limits (at S/N = 3) of 0.02, 0.01 and 0.06 µM, and (c) well separated oxidation peaks near -50, 186 and 390 mV (vs. Ag/AgCl) for simultaneous sensing AA, DA and UA, respectively. The sensor was evaluated by analysing spiked serum samples and gave data with precision, with recoveries of >98%. Graphical abstractSchematic Representation of a Mesoporous Silver-doped TiO2-SnO2 Nanocomposite (h-ATS) on g-C3N4 Nanosheets and Decorated with a Hierarchical Core-Shell Metal-Organic Framework (NC@GC) Based Electrochemical Sensor for Simultaneous Voltammetric Detection of Ascorbic acid, Dopamine and Uric acid.
Asunto(s)
Ácido Ascórbico/análisis , Dopamina/análisis , Electroquímica/métodos , Estructuras Metalorgánicas/química , Nanocompuestos/química , Nanoestructuras/química , Ácido Úrico/análisis , Ácido Ascórbico/sangre , Dopamina/sangre , Electroquímica/normas , Electrodos , Grafito/química , Compuestos de Nitrógeno/química , Plata , Compuestos de Estaño , Titanio , Ácido Úrico/sangreRESUMEN
In addition to gap junctional channels that mediate cell-to-cell communication, connexins form hemichannels that are present at the plasma membrane. Since hemichannels are permeable to small hydrophilic compounds, including metabolites and signaling molecules, their abnormal opening can cause or contribute to cell damage in disorders such as cardiac infarct, stroke, deafness, skin diseases, and cataracts. Therefore, hemichannels are potential pharmacological targets. A few aminoglycosides, well-known broad-spectrum antibiotics, have been shown to inhibit hemichannels. Here, we tested several commercially available aminoglycosides for inhibition of human connexin hemichannels using a cell-based bacterial growth complementation assay that we developed recently. We found that kanamycin A, kanamycin B, geneticin, neomycin, and paromomycin are effective inhibitors of hemichannels formed by connexins 26, 43, and 46 (Cx26, Cx43, and Cx46). Because of the >70 years of clinical experience with aminoglycosides and the fact that several of the aminoglycosides tested here have been used in humans, they are promising starting points for the development of effective connexin hemichannel inhibitors.
Asunto(s)
Aminoglicósidos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Conexinas/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Aminoglicósidos/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Isoformas de ProteínasRESUMEN
Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K+ uptake. These modified cells do not grow in low-K+ medium, but functional expression of connexin hemichannels allows K+ uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to high-throughput multi-well platforms.
Asunto(s)
Bioensayo/métodos , Animales , Conexinas/metabolismo , Humanos , Potasio/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
BACKGROUND: We assessed the treatment effect of panitumumab plus best supportive care (BSC) vs BSC on overall survival (OS) in patients with chemorefractory wild-type KRAS exon 2 metastatic colorectal cancer (mCRC) and report the first prospective extended RAS analysis in a phase 3 trial. METHODS: Patients with wild-type KRAS exon 2 mCRC were randomised 1 : 1 to panitumumab (6 mg kg-1 Q2W) plus BSC or BSC. On-study crossover was prohibited. RAS mutation status was determined by central laboratory testing. The primary endpoint was OS in wild-type KRAS exon 2 mCRC; OS in wild-type RAS mCRC (KRAS and NRAS exons 2, 3, and 4) was a secondary endpoint. RESULTS: Three hundred seventy seven patients with wild-type KRAS exon 2 mCRC were randomised. Median OS was 10.0 months with panitumumab plus BSC vs 7.4 months with BSC (HR=0.73; 95% CI=0.57-0.93; P=0.0096). RAS ascertainment was 86%. In wild-type RAS mCRC, median OS for panitumumab plus BSC was 10.0 vs 6.9 months for BSC (HR=0.70; 95% CI=0.53-0.93; P=0.0135). Patients with RAS mutations did not benefit from panitumumab (OS HR=0.99; 95% CI=0.49-2.00). No new safety signals were observed. CONCLUSIONS: Panitumumab significantly improved OS in wild-type KRAS exon 2 mCRC. The effect was more pronounced in wild-type RAS mCRC, validating previous retrospective analyses.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas ras/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales/genética , Terapia Combinada/métodos , Estudios Cruzados , Exones/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Panitumumab , Estudios Prospectivos , Adulto JovenRESUMEN
INTRODUCTION: In a world of fast moving vehicles, heavy machinery and industries crush injury to limbs with vascular compromise and soft tissue defect is common. The traditional treatment is a 2 step one dealing with vascular repair and soft tissue cover separately, in the same operation. We report a series of single step vascular repair and soft tissue cover with flow through anterolateral thigh flap (ALT) flap for limb salvage. MATERIALS AND METHODS: Ten patients with soft tissue defect and vascular injury were included in this study. A two team approach was used to minimise operative time, team one prepared the vessels and team 2 harvested the flap. OBSERVATIONS AND RESULTS: Of the ten patients operated (8 males), eight flaps were done for upper limb and two for lower limb salvage. Six anastomosis were done with ulnar vessels, two with radial and two with posterior tibial vessels. Nine extremities could be salvaged while one patient developed progressive thrombosis leading to amputation. CONCLUSION: The ALT flow-through flap is a versatile single step procedure that can be used to salvage an ischemic limb with soft tissue loss avoiding the need for interpositional vein graft.
RESUMEN
Vacuolar malic acid accumulation largely determines fruit acidity, a key trait for the taste and flavor of apple and other fleshy fruits. Aluminum-activated malate transporter 9 (ALMT9/Ma1) underlies a major genetic locus, Ma, for fruit acidity in apple, but how the protein transports malate across the tonoplast is unclear. Here, it is shown that overexpression of the coding sequence of Ma1 (Ma1α) drastically decreases fruit acidity in "Royal Gala" apple, leading to uncovering alternative splicing underpins Ma1's function. Alternative splicing generates two isoforms: Ma1ß is 68 amino acids shorter with much lower expression than the full-length protein Ma1α. Ma1ß does not transport malate itself but interacts with the functional Ma1α to form heterodimers, creating synergy with Ma1α for malate transport in a threshold manner (When Ma1ß/Ma1α ≥ 1/8). Overexpression of Ma1α triggers feedback inhibition on the native Ma1 expression via transcription factor MYB73, decreasing the Ma1ß level well below the threshold that leads to significant reductions in Ma1 function and malic acid accumulation in fruit. Overexpression of Ma1α and Ma1ß or genomic Ma1 increases both isoforms proportionally and enhances fruit malic acid accumulation. These findings reveal an essential role of alternative splicing in ALMT9-mediated malate transport underlying apple fruit acidity.
Asunto(s)
Empalme Alternativo , Malatos , Malus , Malatos/metabolismo , Empalme Alternativo/genética , Malus/genética , Malus/metabolismo , Frutas/metabolismo , Frutas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Vacuolas/metabolismo , Vacuolas/genética , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,â³ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.
RESUMEN
Microbial plant pathogens deploy amphipathic cyclic lipopeptides to reduce surface tension in their environment. While plants can detect these molecules to activate cellular stress responses, the role of these lipopeptides or associated host responses in pathogenesis are not fully clear. The gramillin cyclic lipopeptide is produced by the Fusarium graminearum fungus and is a virulence factor and toxin in maize. Here, we show that gramillin promotes virulence and necrosis in both monocots and dicots by disrupting ion balance across membranes. Gramillin is a cation-conducting ionophore and causes plasma membrane depolarization. This disruption triggers cellular signaling, including a burst of reactive oxygen species (ROS), transcriptional reprogramming, and callose production. Gramillin-induced ROS depends on expression of host ILK1 and RBOHD genes, which promote fungal induction of virulence genes during infection and host susceptibility. We conclude that gramillin's ionophore activity targets plant membranes to coordinate attack by the F. graminearum fungus.
Asunto(s)
Membrana Celular , Fusarium , Lipopéptidos , Enfermedades de las Plantas , Fusarium/patogenicidad , Fusarium/metabolismo , Lipopéptidos/farmacología , Lipopéptidos/metabolismo , Virulencia , Membrana Celular/metabolismo , Enfermedades de las Plantas/microbiología , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Zea mays/microbiologíaRESUMEN
ATP-binding cassette (ABC) proteins have two nucleotide-binding domains (NBDs) that work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. In particular, it is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or opening of the dimers, with the NBDs remaining in contact during the hydrolysis cycle. We studied a prototypical ABC NBD, the Methanococcus jannaschii MJ0796, using spectroscopic techniques. We show that fluorescence from a tryptophan positioned at the dimer interface and luminescence resonance energy transfer between probes reacted with single-cysteine mutants can be used to follow NBD association/dissociation in real time. The intermonomer distances calculated from luminescence resonance energy transfer data indicate that the NBDs separate completely following ATP hydrolysis, instead of opening. The results support ABC protein NBD association/dissociation, as opposed to constant-contact models.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/química , Proteínas Bacterianas/química , Methanococcus/enzimología , Modelos Moleculares , Multimerización de Proteína , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/genética , Proteínas Bacterianas/genética , Sitios de Unión , Escherichia coli/enzimología , Escherichia coli/genética , Hidrólisis , Methanococcus/genéticaRESUMEN
Transporters contribute to renal elimination of drugs; therefore drug disposition can be impacted if transporters are inhibited by comedicant drugs. Regulatory agencies have provided guidelines to assess potential drug-drug interaction (DDI) risk for renal organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 and 2-K (MATE1/2-K) transporters. Despite this, there are challenges with translating in vitro data using currently available tools to obtain a quantitative assessment of DDI risk in the clinic. Given the high number of drugs and new molecular entities showing in vitro inhibition toward OCT2 and/or MATE1/2-K and the lack of translation to clinically significant effects, it is reasonable to question whether the current in vitro assay design and modeling practice has led to unnecessary clinical evaluation. The aim of this review is to assess and discuss available in vitro and clinical data along with prediction models intended to provide clinical context of risk, including static models proposed by regulatory agencies and physiologically-based pharmacokinetic models, in order to identify best practices and areas of future opportunity. This analysis highlights that different in vitro assay designs, including substrate and cell systems used, strongly influence the derived concentration of drug producing 50% inhibition values and contribute to high variability observed across laboratories. Furthermore, the lack of sensitive index substrates coupled with specific inhibitors for individual transporters necessitates the use of complex models to evaluate clinical DDI risk.
Asunto(s)
Riñón , Proteínas de Transporte de Catión Orgánico , Interacciones Farmacológicas , Células HEK293 , Humanos , Riñón/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 2 de Cátion Orgánico/metabolismo , Eliminación RenalRESUMEN
BACKGROUND: Growth and differentiation factor (GDF)-11 controls embryonic development and has been proposed as an antiaging factor. GDF-8 (myostatin) inhibits skeletal muscle growth. Difficulties in accurately measuring circulating GDF-11 and GDF-8 have generated controversy. METHODS: We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of circulating GDF-8 and GDF-11 that employs denaturation, reduction, and alkylation; cation-exchange solid-phase extraction; tryptic digestion; followed by separation and quantification using 2 signature peptides for multiple reaction monitoring and C-terminal [13C615N4]-Arg peptides as internal standards. We evaluated age trends in serum GDF-11 and GDF-8 concentrations in community-dwelling healthy men, 19 years or older, and determined the effects of graded testosterone doses on GDF-8 and GDF-11 concentrations in healthy men in a randomized trial. RESULTS: The assay demonstrated linearity over a wide range, lower limit of quantitation 0.5 ng/mL for both proteins, and excellent precision, accuracy, and specificity (no detectable cross-reactivity of GDF-8 in GDF-11 assay or of GDF-11 in GDF-8 assay). Mean ± SD (median ± 1QR) GDF-8 and GDF-11 levels in healthy community-dwelling men, 19 years and older, were 7.2â ±â 1.9 (6.8â ±â 1.4) ng/mL. Neither GDF-8 nor GDF-11 levels were related to age or body composition. Testosterone treatment significantly increased serum GDF-8 but not GDF-11 levels. CONCLUSIONS: The LC-MS/MS method for the simultaneous measurement of circulating total GDF-8 and GDF-11 demonstrates the characteristics of a valid assay. Testosterone treatment increased GDF-8 levels, but not GDF-11. Increase in GDF-8 levels by testosterone treatment, which increased muscle mass, suggests that GDF-8 acts as a chalone to restrain muscle growth.
Asunto(s)
Miostatina , Testosterona , Adulto , Cromatografía Liquida/métodos , Factores de Diferenciación de Crecimiento/sangre , Humanos , Masculino , Miostatina/sangre , Espectrometría de Masas en Tándem/métodos , Testosterona/administración & dosificaciónRESUMEN
Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.
Asunto(s)
Materiales Biocompatibles/química , Sistema Libre de Células/química , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Ensayo de Materiales , Tamaño de la PartículaRESUMEN
The human cannabinoid 1 receptor (hCB1), a ubiquitous G protein-coupled receptor (GPCR), transmits cannabinergic signals that participate in diverse (patho)physiological processes. Pharmacotherapeutic hCB1 targeting is considered a tractable approach for treating such prevalent diseases as obesity, mood disorders, and drug addiction. The hydrophobic nature of the transmembrane helices of hCB1 presents a formidable difficulty to its direct structural analysis. Comprehensive experimental characterization of functional hCB1 by mass spectrometry (MS) is essential to the targeting of affinity probes that can be used to define directly hCB1 binding domains using a ligand-assisted experimental approach. Such information would greatly facilitate the rational design of hCB1-selective agonists/antagonists with therapeutic potential. We report the first high-coverage MS analysis of the primary sequence of the functional hCB1 receptor, one of the few such comprehensive MS-based analyses of any GPCR. Recombinant C-terminal hexa-histidine-tagged hCB1 (His6-hCB1) was expressed in cultured insect (Spodoptera frugiperda) cells, solubilized by a procedure devised to enhance receptor purity following metal-affinity chromatography, desalted by buffer exchange, and digested in solution with (chymo)trypsin. "Bottom-up" nanoLC-MS/MS of the (chymo)tryptic digests afforded a degree of overall hCB1 coverage (>94%) thus far reported for only two other GPCRs. This MS-compatible procedure devised for His6-hCB1 sample preparation, incorporating in-solution (chymo)trypsin digestion in the presence of a low concentration of CYMAL-5 detergent, may be applicable to the MS-based proteomic characterization of other GPCRs. This work should help enable future ligand-assisted structural characterization of hCB1 binding motifs at the amino-acid level using rationally designed and targeted covalent cannabinergic probes.
Asunto(s)
Fragmentos de Péptidos/metabolismo , Proteómica/métodos , Receptor Cannabinoide CB1/química , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Western Blotting , Diseño de Fármacos , Histidina/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Oligopéptidos/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Transducción de SeñalRESUMEN
Carcinoembryonic antigen (CEA) is a well-known cancer biomarker for the detection of several malignancies. The development of ultrasensitive CEA diagnostic tools is crucial for early detection and progression observation of tumors. Herein, a dual signal amplified sandwich-type electrochemical immunoassay was developed based on dual-labeled mesoporous silica nanospheres as a signal amplifier, combined with NiO@Au decorated graphene as a conductive layer for ultrasensitive and rapid determination of CEA. The dual-labeled mesoporous silica (DLMS) nanosphere, which was synthesized by entrapping Au nanorod (Au NR) and horseradish peroxidase (HRP) in the channels of amine-functionalized SBA-15 followed by subordinate antibody (Ab2) conjugation which was denoted as Au NR@SBA-15/Ab2-HRP. The dual signal amplification from Au NR@SBA-15 and HRP enhanced the sensitivity of the proposed immunoassay. Consequently, the developed DLMS based immunosensor displayed ultra-low limits of detection of 5.25 fg/mL and a wide range of linearity (0.1-5 pg/mL), which was extended for CEA determination in real-time samples with improved recoveries of >98%. Therefore, this dual amplification prototype would cater to the clinical requirements for the ultrasensitive detection of CEA biomarkers.
Asunto(s)
Técnicas Biosensibles , Antígeno Carcinoembrionario , Grafito , Nanopartículas del Metal , Nanosferas , Antígeno Carcinoembrionario/análisis , Técnicas Electroquímicas , Oro , Inmunoensayo , Límite de Detección , Dióxido de SilicioRESUMEN
The active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser(129) by AM6701 in the putative hMGL catalytic triad demonstrates this residue's essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve approximately 80% enzyme inhibition. Although Cys(215) and/or Cys(249) mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys(249) is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs.
Asunto(s)
Dominio Catalítico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/química , Mutación , Amidas/química , Amidas/farmacología , Secuencia de Aminoácidos , Diseño de Fármacos , Humanos , Isomerismo , Ligandos , Maleimidas/química , Maleimidas/farmacología , Espectrometría de Masas , Datos de Secuencia Molecular , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Mutagénesis Sitio-DirigidaRESUMEN
Connexins hemichannels (HCs) from adjacent cells form gap junctional channels that mediate cell-to-cell communication. Abnormal opening of "free" undocked HCs can produce cell damage and participate in the mechanism of disorders such as cardiac infarct, stroke, deafness, skin diseases, and cataracts. Therefore, inhibitors of connexin HCs have great pharmacological potential. Antibiotic aminoglycosides (AGs) have been recently identified as connexin HC inhibitors, but their antibiotic effect is an issue for the treatment of disorders where infections do not play a role. Herein, we synthesized and tested several amphiphilic AGs without antibiotic effect for their inhibition against connexin HCs, using a newly developed cell-based bacterial growth complementation assay. Several leads with superior potency than the parent compound, kanamycin A, were identified. Unlike traditional AGs, these amphiphilic AGs are not bactericidal and are not toxic to mammalian cells, making them better than traditional AGs as HC inhibitors for clinical use and other applications.
RESUMEN
INTRODUCTION: Tumor rat sarcoma gene (RAS) status is a negative predictive biomarker for anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC). We analyzed outcomes according to RAS and v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutational status, and evaluated early tumor shrinkage (ETS) and depth of response (DpR) for patients with wild type RAS. PATIENTS AND METHODS: Patients with confirmed metastatic colon or rectum adenocarcinoma, wild type Kristen rat sarcoma gene tumor exon 2 status, clinical/radiologic disease progression or toxicity during irinotecan or oxaliplatin treatment, and no previous anti-EGFR therapy were randomized 1:1 to receive best supportive care (BSC) with or without panitumumab (6.0 mg/kg, intravenously, on day 1 of each 14-day cycle) in this open-label, multicenter, phase III study (20100007). RAS and BRAF mutation status were determined using Sanger sequencing. ETS was evaluated as maximum percentage change from baseline to week 8; DpR was calculated as the percentage change for tumor shrinkage at nadir versus baseline. RESULTS: Overall, 270 patients had RAS wild type mCRC (panitumumab with BSC, n = 142; BSC, n = 128). For patients with wild type RAS tumors, median overall survival (OS; hazard ratio [HR], 0.72; P = .015) and progression-free survival (PFS; HR, 0.45; P < .0001) were improved with panitumumab with BSC versus BSC. Similar improvements were seen for patients with wild type RAS, and wild type BRAF tumors (OS: HR, 0.75; P = .04; PFS: HR, 0.45; P < .0001). Median DpR was 16.9% for the evaluable panitumumab with BSC wild type RAS population. Overall, 69.5% experienced any type of tumor shrinkage at week 8; 38.2% experienced ≥ 20% shrinkage. Similar improvements in OS and PFS were seen with stratification according to ETS. CONCLUSION: This analysis showed that panitumumab improved outcomes in wild type RAS mCRC and indicated that ETS and DpR could be used as additional efficacy markers.