RESUMEN
HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin ß (Impß), but how Rev associates with Impß is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impß/Rev complex. We show that Impß binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impß cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impß-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impß with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impß and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impß cargos.
Asunto(s)
VIH-1 , beta Carioferinas , VIH-1/metabolismo , Modelos Estructurales , Simulación del Acoplamiento Molecular , ARN Viral/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
INTRODUCTION: In arthroplasty surgery, positioning of the components must be accurate and reproducible to avoid complications. Conventional guides are often used to align a component, but they require surgical skill and experience, and are prone to error. To this end, a single-use, size-specific, nylon guide (single-use nylon guide) has been developed for the purpose of increasing the accuracy without adding extra cost to the operation. The effectiveness of this type of guide was evaluated in using a synthetic bone study. METHODS: A total of 66 synthetic femurs with the same osteoarthritic morphology were prepared. 3 surgeons participated in the experiments, and each surgeon created a drill hole for the femoral component by using the single-use nylon guide or a commercially-available, conventional, metal, neck-based guide (conventional guide). Anteversion, inclination, and insertion point acquired by the guide were compared between the guides, between surgeons, and to the computer-based plan. RESULTS: Anteversion acquired by the single-use nylon guide (6.7° [4.9-11.5°]) was significantly closer to the plan (14.6°) than that acquired by the conventional guide (4.3° [2.4-8.6°]) (p = 0.03). The insertion point was also significantly closer to the plan for the single-use nylon guide (3.8 mm ± 1.6 mm) than the conventional guide (5.7 mm ± 2.4 mm) (p < 0.001). No significant difference was found for the inclination (p = 0.76). CONCLUSION: A single-use, size-specific nylon guide was effective in acquiring a higher accuracy and precision in anteversion and insertion point than a conventional guide in this synthetic bone, hip resurfacing arthroplasty study. The use of single-use guides in other orthopaedic procedures should be explored.
Asunto(s)
Artroplastia de Reemplazo de Cadera/métodos , Fémur/cirugía , Prótesis de Cadera , Nylons , Cirugía Asistida por Computador/métodos , Adulto , Femenino , Humanos , Masculino , Diseño de PrótesisRESUMEN
Molecularly imprinted polymers (MIPs) templated with either the phytoalexin, (E)-resveratrol, or its structural analog, 3,5-dihydroxy-N-(4-hydroxyphenyl)benzamide, have been used in tandem for the sequential extraction of (E)-resveratrol from aqueous peanut meal extracts in high purity and in near quantitative yields. Re-processing of the (E)-resveratrol-depleted peanut meal extract with the 3,5-dihydroxy-N-(4-hydroxyphenyl)benzamide imprinted MIP yielded additional polyphenolic components, identified as A-type procyanidins. Tandem liquid chromatography-electrospray ionization mass spectrometry confirmed the identity and purity of the isolated products. This study documents the advantages of tandem approaches with MIPs for the solid phase extraction and analysis of multiple bioactive compounds present in complex biomass waste streams.
Asunto(s)
Técnicas de Química Analítica/métodos , Polifenoles/análisis , Polifenoles/aislamiento & purificación , Extracción en Fase Sólida , Estilbenos/análisis , Estilbenos/aislamiento & purificación , Arachis/química , Cromatografía Líquida de Alta Presión , Residuos Industriales/análisis , Impresión Molecular , Resveratrol , Espectrometría de Masa por Ionización de Electrospray , Agua/químicaRESUMEN
The effects of different adenosine receptor antagonists and cyclic nucleotide phosphodiesterase (PDE) inhibitors on monoclonal antibody (mAb) titer and cell viability of murine hybridoma cells in culture were measured as part of our investigations to discover additives that enhance mAb production. Specific adenosine receptor antagonists and PDE inhibitors were found to enhance or decrease the titer of immunoglobulin G1 (IgG1) mAbs relative to negative controls, depending on the specific compound and cell line employed. The observed enhancements or decreases in IgG1 mAb titer appeared to be mainly due to an increase or decrease in specific productivity rates (ngmAb/cell), respectively. The different effects of the selective adenosine antagonists suggest that antagonism at the level of the adenosine A2A and A1 or the adenosine A3 receptors result in either enhancement or suppression of IgG1 mAb production by hybridoma cells. Overall, these studies have identified hitherto unknown activities of specific adenosine antagonists and PDE inhibitors which indicate they may have valuable roles as cell culture additives in industrial biomanufacturing processes designed to enhance the yields of mAbs or other recombinant proteins produced by mammalian cell culture procedures.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/biosíntesis , Inmunoglobulina G/biosíntesis , Inhibidores de Fosfodiesterasa/farmacología , Antagonistas de Receptores Purinérgicos P1/farmacología , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Cafeína/análogos & derivados , Cafeína/farmacología , Supervivencia Celular , Hibridomas , RatonesRESUMEN
Molecularly imprinted polymers are versatile materials with wide application scope for the detection, capture and separation of specific compounds present in complex feed stocks. A major challenge associated with their preparation has been the need to sacrifice one mole equivalent of the template molecule to generate the complementary polymer cavities that selectively bind the target molecule. Moreover, template molecules can often be difficult to synthesise, expensive or lack stability. In this study, we describe a new approach, directed at the use of synthetic selectophores, chosen as readily prepared and low cost structural analogues with recognition groups in similar three-dimensional arrangements as found in the target molecule. To validate the approach, a comparative study of selectophores related to the polyphenolic compound (E)-resveratrol has been undertaken using traditional and green chemical synthetic approaches. These molecular mimic compounds were employed as polymer templates and also as binding analytes to interrogate the recognition sites associated with the molecularly imprinted polymers. Importantly, the study confirms that the use of selectophores has the potential to confer practical advantages, including access to more efficient methods for selection and preparation of suitable template molecules with a broader range of molecular diversity, as well as delivering imprinted polymers capable of recognizing the target compound and structurally related products.
Asunto(s)
Impresión Molecular/métodos , Polímeros/síntesis química , Estilbenos/química , Adsorción , Cromatografía Líquida de Alta Presión , Tecnología Química Verde , Imitación Molecular , Estructura Molecular , Polímeros/química , ResveratrolRESUMEN
PURPOSE: The aim of the study was to investigate varus and normal knee morphologies to identify differences that may affect knee replacement alignment or design for varus knees. METHODS: Computed tomography scans of varus and normal knees were analyzed, and geometric shapes, points and axes were fit to the femur and tibia independently. These points were then projected in the three anatomical planes to measure the variations between the two groups. RESULTS: In the femur, varus knees had less femoral anteversion (p < 0.0001) and a larger medial extension facet (p < 0.05) compared with normal knees. In the tibia, the tubercle was found to be externally rotated in varus knees (12°), with a significant increase in the coronal slope (p = 0.001) and the extension facet angle (p = 0.002). CONCLUSIONS: The study highlighted the differences and similarities found between the two groups, which raises awareness on changes required during surgical intervention and component placement or design for a varus knee. This is particularly relevant for the design of patient-specific instrumentation and implants. LEVELS OF EVIDENCE: Diagnostic study, Level III.
Asunto(s)
Desviación Ósea/diagnóstico por imagen , Articulación de la Rodilla/diagnóstico por imagen , Osteoartritis de la Rodilla/diagnóstico por imagen , Fenómenos Biomecánicos , Fémur/diagnóstico por imagen , Fémur/fisiopatología , Humanos , Articulación de la Rodilla/fisiología , Articulación de la Rodilla/fisiopatología , Masculino , Tomografía Computarizada Multidetector , Osteoartritis de la Rodilla/fisiopatología , Tibia/diagnóstico por imagen , Tibia/fisiopatologíaRESUMEN
OBJECTIVE: The conventional reference frame for the femur has limited relevance for the planning of hip surgery as the femoral neck axis, a crucial reference for surgeons, has to be independently derived. The purpose of this study is to develop and validate a reliable frame of reference for the proximal femur that can be applied in clinical settings. MATERIALS AND METHODS: Ten three-dimensional models of femurs were obtained. An iterative method was developed to find the femoral neck axis (X-axis). A second axis was also created from the lesser trochanter to the piriformis fossa (LTPF). The origin was defined as the femoral head centre. The cross product of the neck and LTPF axes provided the Z-axis and the third axis (Y-axis) was perpendicular to the other two. Intra-/inter-investigator reliability was assessed on the ten femur models; ten times by one investigator and twice by three investigators respectively. The results were then compared with the conventional reference frame using landmarks on the distal femur. RESULTS: The femoral neck and LTPF axes had mean intra-/inter-investigator angle differences of 0.5° (SD 0.4°) and 0.7° (SD 0.5°), and 0.8° (SD 0.5°) and 0.9° (SD 0.6°) respectively while the variations of the X-, Y- and Z- axes were SD 0.6°, 0.7° and 0.5°. CONCLUSIONS: A reliable method of obtaining the three-dimensional proximal femoral frame was developed, using the femoral neck axis, with greater relevance to clinical settings, preoperative planning and accurate assessment of procedures post-operatively.
Asunto(s)
Puntos Anatómicos de Referencia/diagnóstico por imagen , Cabeza Femoral/anatomía & histología , Cabeza Femoral/diagnóstico por imagen , Imagenología Tridimensional/métodos , Modelos Anatómicos , Interpretación de Imagen Radiográfica Asistida por Computador/métodos , Puntos Anatómicos de Referencia/anatomía & histología , Simulación por Computador , Articulación de la Cadera , Humanos , Imagenología Tridimensional/normas , Variaciones Dependientes del Observador , Interpretación de Imagen Radiográfica Asistida por Computador/normas , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tomografía Computarizada por Rayos X/métodosRESUMEN
We have investigated the binding characteristics of a potent member of the bis-ortho-substituted five-membered nitrogen heterocycle class of ALK-5 kinase inhibitors with lysates of cultured HEK-293 cells to identify protein binding partners of potential biological significance. An affinity chromatographic resin containing an immobilized ALK-5 kinase inhibitor, 2-phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine, was used to capture specific proteins from the cell lysate. The soluble inhibitor was then used to specifically elute the proteins which selectively bound to the pharmacophore ligand structure. Application of 2-D SDS-PAGE analysis with positive and negative controls demonstrated the inhibitor bound several different proteins via selective molecular recognition processes. The structural features of the specifically eluted proteins were identified by peptide mass fingerprinting (PMF) methods and included proteins with structural, metabolic and chaperone functions. Furthermore, these PMF results identified the therapeutic target in various cancer treatment studies, HSP-70, as a potential high-affinity binding partner. These observations warrant examination of bis-ortho-substituted five-membered nitrogen heterocycles as dual ALK-5/HSP-70 inhibitors for anti-cancer drug development.
Asunto(s)
Antineoplásicos/química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Células HEK293 , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/síntesis química , Piridinas/química , Piridinas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
Precise modelling of the proximal femur can be used for detecting and planning corrective surgery for subjects with deformed femurs using robotic technology or navigation systems. In this study, the proximal femoral geometry has been modelled mathematically. It is hypothesised that it is possible to fit a quadratic surface or combinations of them onto different bone surfaces with a relatively good fit. Forty-six computed tomography datasets of normal proximal femora were segmented. A least-squares fitting algorithm was used to fit a quadratic surface on the femoral head and neck such that the sum of distances between a set of points on the femoral neck and the quadratic surface was minimised. Furthermore, the position of the head-neck articular margin was also measured. The femoral neck was found to be represented as a good fit to a hyperboloid with an average root mean-squared error of 1.0 ± 0.13 mm while the shape of the femoral articular margin was a reproducible sinusoidal wave form with two peaks. The mathematical description in this study can be used for planning corrective surgery for subjects with cam-type femoroacetabular impingement.
Asunto(s)
Cabeza Femoral/patología , Fémur/patología , Modelos Anatómicos , Algoritmos , Fenómenos Biomecánicos , Biofisica , Fémur/anatomía & histología , Cuello Femoral/patología , Articulación de la Cadera/cirugía , Humanos , Análisis de los Mínimos Cuadrados , Modelos Teóricos , Robótica , Programas Informáticos , Estrés Mecánico , Propiedades de Superficie , Tomografía Computarizada por Rayos X/métodosRESUMEN
OBJECTIVE: Cam hips are commonly quantified using the two-dimensional α angle. The accuracy of this measurement may be affected by patient position and the technician's experience. In this paper, we describe a method of measurement that provides a quantitative definition of cam hips based upon three-dimensional computed tomography (CT) images. MATERIALS AND METHODS: CT scans of 47 (24 cam, 23 normal) femurs were segmented. A sphere was fitted to the articulating surface of the femoral head, the radius (r) recorded, and the femoral neck axis obtained. The cross sectional area at four locations spanning the head neck junction (r/4, r/2, 3r/4 and r), perpendicular to the neck axis, was measured. The ratios (Neck/Head) between the areas at each cut relative to the surface area at the head centre were calculated and aggregated. RESULTS: Normal and cam hips were significantly different: the sum of the head-neck ratios (HNRs) of the cam hips were always smaller than normal hips (p < 0.01). A cut off point of 2.55 with no overlap was found between the two groups, with HNRs larger than this being cam hips, and smaller being normal ones. CONCLUSION: Owing to its sensitivity and repeatability, the method could be used to confirm or refute the clinical diagnosis of a cam hip. Furthermore it can be used as a tool to measure the outcome of cam surgery.
Asunto(s)
Pinzamiento Femoroacetabular/diagnóstico por imagen , Cabeza Femoral/anomalías , Cabeza Femoral/diagnóstico por imagen , Cuello Femoral/anomalías , Cuello Femoral/diagnóstico por imagen , Imagenología Tridimensional/métodos , Tomografía Computarizada por Rayos X/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Interpretación de Imagen Radiográfica Asistida por Computador/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Femoroacetabular impingement is caused by abnormal morphology of either the femur or acetabulum or both. Diagnostic criteria currently include an alpha angle of over 50° on a lateral radiograph. In this study, CT scans of symptomatic hips (n = 37) were compared with normal hips (n = 34) obtained from CT colonoscopy procedures. The femoral head described in terms of a three dimensional (3D) alpha angle and a 3D head neck margin (epiphysis) angle '3Dµ' using a semi-automated algorithm. In normal hips 70% have a maximum 3Dα angle of more than 50° at some point around their femoral head (mean 53° ± 5°, range 42° - 64°), while in cam hips, it was significantly larger (mean 69° ± 10°, range 54° - 94°, p<0.001). The 3Dµ also varied significantly and had a reverse relationship to that of the alpha angle: cam hips have an articular extent that crossed over spherical limit of the hip joint (mean minimum 41° ± 7°) while the articular margin of normal hips always remained within the spherical limit (mean minimum 49° ± 6°). This semi-automated algorithm provides an objective measure of the femoral head in health and disease. It can reliably distinguish cam hips from normal, enabling cam hips to have their cam quantified and their surgery planned objectively.
Asunto(s)
Pinzamiento Femoroacetabular/diagnóstico por imagen , Imagenología Tridimensional , Tomografía Computarizada por Rayos X , Femenino , Humanos , Masculino , Índice de Severidad de la EnfermedadRESUMEN
Novel inhibitors of TGF-ß1 and activin A signalling based on a 2-aryl-4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridine pharmacophore have been synthesised. Compounds containing phenyl or aromatic nitrogen heterocycle substituents inhibited both types of signalling with HEK-293T cells in culture, with a selectivity preference for TGF-ß1. Synthetic compounds containing pyridin-3-yl, pyrazol-4-yl, pyrazol-1-yl or 1H-imidazoyl-1-yl substituents exhibited structural and functional attributes suitable for further investigation related to the development of more potent TGF-ß inhibitors.
Asunto(s)
Activinas/antagonistas & inhibidores , Compuestos Heterocíclicos/farmacología , Piridinas/farmacología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Activinas/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/química , Humanos , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Transducción de Señal/efectos de los fármacos , Estereoisomerismo , Relación Estructura-Actividad , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
(E)-Resveratrol imprinted polymers have been rationally designed with the aid of molecular modelling and NMR spectroscopic titration techniques to determine the optimal ratio of the template to functional monomer for polymer formation. Based on this approach, (E)-resveratrol imprinted polymers were prepared via non-covalent self-assembly with the functional monomer 4-vinylpyridine (4VP) in a 1:3 molar ratio. Polymerisation in the presence of a cross-linker resulted in rigid block copolymers that had selective capacities towards (E)-resveratrol (e.g. 14 µmol/g) when compared to the non-imprinted reference polymer. The selectivity of these MIPs was also examined using several structurally related polyphenolic compounds to determine the influence of polyphenolic hydroxyl number and position on binding and molecular recognition.
Asunto(s)
Impresión Molecular , Polímeros/química , Estilbenos/química , Cromatografía Líquida de Alta Presión , Modelos Moleculares , Fenoles/química , Piridinas , Resveratrol , Extracción en Fase SólidaRESUMEN
Molecularly imprinted solid phase extraction (MISPE) has been employed to isolate and concentrate bioactive polyphenols from peanut press waste. To this end, a molecularly imprinted polymer (MIP) templated with the phytoalexin (E)-resveratrol has been prepared via self-assembly with the functional monomer 4-vinylpyridine (4VP) in a 1:3 molar ratio. Subsequent molecular interrogation of the MIP binding sites demonstrated preferential structural selectivity for (E)-resveratrol with respect to other structurally related naturally occurring compounds. This selectivity was subsequently exploited to achieve substantial sample cleanup of peanut press waste under aqueous conditions with significant enrichment of (E)-resveratrol (>60 fold) requiring minimal sample preparation.
Asunto(s)
Arachis/química , Polímeros/química , Estilbenos/análisis , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Resveratrol , Microextracción en Fase Sólida , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium formate, and an increasing content of acetonitrile or methanol, were employed. Compared to the analysis of the same tryptic peptides under low-pH conditions with an ion-pairing reagent, the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity. Thus, improved selectivity for the peptide separation and favourable tandem mass spectrometry analysis could be obtained with eluents in this intermediate pH range. The number of tryptic peptides identified by the new approach for the proteins investigated were significantly higher than that obtained by the conventional low-pH methods. Moreover, analysis of protein digests at very low concentrations was also performed under both acidic and intermediate pH conditions and similar improvements in selectivity and MS/MS detection limits were observed, i.e. identification of more distinct peptides and higher sequence coverage of the protein was obtained when eluents of intermediate pH were employed. This study therefore highlights the potential of conducting peptide mapping in the intermediate pH range to achieve more reliable and sensitive protein identifications with capillary RP-HPLC-ESI-MS/MS.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Mapeo Peptídico , Péptidos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Concentración de Iones de Hidrógeno , Péptidos/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
Angiogenesis is critical for cancer development and metastasis. Here we have employed a functional antibody library-based proteomic screen to identify proteins that participate in and might be used as therapeutic targets for tumor-related angiogenesis. Mice were immunized with human esophageal cancer endothelial cells (HECEC). The antibody library was established with the mouse spleen cells the serum of which had most anti-angiogenic effect. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with cell surface antigens of HECECs and influenced their behavior were selected. Antigens that recognized by the antibodies were obtained by immunoprecipitation and then identified by mass spectrometry analysis. Migration-stimulating factor (MSF), the antigen of 1D2 antibody was identified using this approach. Further studies demonstrated that the 1D2 antibody suppressed MSF-effected migration and adhesion of HECECs on fibronectin matrix. Biodistribution assay showed that MSF targeting antibody 1D2 could specifically home to the xenograft with humanized blood vessel. Targeting treatment with 1D2 antibody significantly suppressed tumor growth through inhibition of human tumor-related angiogenesis. These results indicate that the functional antibody library-based proteomic screen can successfully identify proteins that involved in tumor-related angiogenesis and MSF may be a target for the anti-angiogenic treatment of the esophageal cancer.
Asunto(s)
Anticuerpos Antineoplásicos/inmunología , Citocinas/análisis , Células Endoteliales/patología , Neoplasias Esofágicas/irrigación sanguínea , Neovascularización Patológica/inmunología , Biblioteca de Péptidos , Proteómica/métodos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígenos de Neoplasias/inmunología , Adhesión Celular , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/patología , Movimiento Celular , Proliferación Celular , Técnicas de Cocultivo , Células Endoteliales/inmunología , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/patología , Matriz Extracelular/metabolismo , Femenino , Fibronectinas , Humanos , Inmunización , Ratones , Pruebas de Neutralización , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The transforming growth factor-beta (TGF-beta) superfamily member, activin A, plays a central role in the regulation of multiple physiological processes including cell differentiation, mitogenesis, embryogenesis, apoptosis and inflammation. In normal cells, activin A signalling is regulated to maintain cellular and tissue health and suppress tumour growth. Disruption of activin A signalling has been implicated in tumour formation and progression. Hence, the availability of activin A is an important target for the development of diagnostics and drugs for therapeutic intervention. To this end, we have expressed human activin A in Pichia pastoris, permitting its secretion into culture medium and purification as the mature homodimer. A construct was engineered encoding the monomeric precursor protein with a N-terminal FLAG affinity tag (DYKDDDDK) and a cleavage site (EKR) for Kex2p protease. Procedures for the two-step purification of human activin A by ion-exchange and anti-FLAG antibody affinity chromatography, and for the removal of the FLAG affinity tag from purified recombinant human activin A by enteropeptidase, are described. The molecular weights of the FLAG-tagged and de-tagged human activin A were confirmed by MALDI-TOF mass spectroscopy. The biological activity of these recombinant activins was assessed for their effects on modulating the secretion of Endothelin-1 (ET-1) by human umbilical vein endothelial cells (HUVECs). The recombinant human activin A containing the intact FLAG tag resulted in a reduced ET-1 secretion from HUVECs, whereas upon removal of this affinity purification tag the purified recombinant human activin A restored ET-1 secretion to levels comparable to the positive control. These results document an approach of considerable potential for the simple, large-scale expression and purification of this important human growth factor for use in diagnostic and therapeutic purposes.
Asunto(s)
Subunidades beta de Inhibinas/biosíntesis , Pichia/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Cromatografía de Afinidad , Humanos , Subunidades beta de Inhibinas/genética , Subunidades beta de Inhibinas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Pichia/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We have previously shown that exposing rats to a relatively high dose of ethanol during early postnatal life can result in an alteration in spatial learning ability. The hippocampal formation is known to be involved in the control of this ability. The purpose of the present study was to determine whether exposure of rats to ethanol during early postnatal life had either immediate or delayed effects on the numbers of pyramidal cells in the CA1-CA3 subregion of the hippocampus. Wistar rats were exposed to a relatively high daily dose of ethanol at postnatal day 10-15 by placing them for 3 h/day in a chamber containing ethanol vapor. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anesthetized and killed at 16 and 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle was used to determine the volumes of the CA1 and CA2+CA3 regions. The physical disector method was used to estimate the numerical density of neurons in each of the subdivisions. The total number of pyramidal cells was calculated by multiplying the appropriate estimates of the numerical density by the volume. There were significant age-related reductions in the total numbers of pyramidal cells at 16-30 days of age irrespective of the groups examined. Ethanol treated rats were found to have slightly but significantly fewer pyramidal cell neurons than either the MRC or SC groups. These observations indicate that pyramidal cells in the hippocampus may be vulnerable to a relatively high dose of ethanol exposure during this short period of early postnatal life.
Asunto(s)
Envejecimiento/patología , Trastornos del Sistema Nervioso Inducidos por Alcohol/patología , Hipocampo/efectos de los fármacos , Hipocampo/patología , Células Piramidales/efectos de los fármacos , Células Piramidales/patología , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Trastornos del Sistema Nervioso Inducidos por Alcohol/fisiopatología , Animales , Animales Recién Nacidos , Cámaras de Exposición Atmosférica , Recuento de Células , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Esquema de Medicación , Etanol/sangre , Etanol/toxicidad , Femenino , Hipocampo/crecimiento & desarrollo , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Embarazo , Ratas , Ratas WistarRESUMEN
BACKGROUND: Buprenorphine is used in the treatment of opioid dependence. Due to its pharmacology, the transfer from methadone to buprenorphine may precipitate withdrawal symptoms. METHODS: Methadone maintained patients with clinical indicators of stability who were seeking withdrawal from methadone were recruited from three Australian states. Patients on methadone doses between 30 and 40 mg were randomised to transfer to buprenorphine by a fixed dose (transfer at 30 mg methadone) or by a variable dose induction (transfer when 'uncomfortable'). A third group of patients with methadone doses less than 30 mg were transferred to buprenorphine at their entry methadone dose. Fifty-one patients were inducted onto buprenorphine using the same dosing protocol with the first dose of 4 mg buprenorphine. Following stabilisation on buprenorphine, patients gradually reduced the buprenorphine dose to 0 mg. Withdrawal severity and drug use was monitored. RESULTS: There were no significant difference between the transfer at 30 mg and transfer when 'uncomfortable' dosing protocols in severity of withdrawal on transfer from methadone to buprenorphine. Those on doses less than 30 mg reported significantly less withdrawal discomfort at transfer. All but one patient stabilised on buprenorphine. Thirty-eight of the 51 patients inducted onto buprenorphine reached 0 mg. CONCLUSIONS: Transfer from methadone to buprenorphine can safely occur from doses of around 30 mg of methadone. Buprenorphine dose reductions were well tolerated. Thirty-one percent of patients were not using heroin or methadone at 1-month follow-up.
Asunto(s)
Buprenorfina/administración & dosificación , Metadona/administración & dosificación , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Adulto , Esquema de Medicación , Femenino , Estudios de Seguimiento , Humanos , Masculino , Proyectos Piloto , Síndrome de Abstinencia a Sustancias/psicología , Resultado del TratamientoRESUMEN
We previously showed that 16-day-old rats exposed to a relatively high dose of ethanol at 10-15 postnatal days of age have fewer neurons in the hilus region of the hippocampus compared with controls. Dentate gyrus granule cell numbers, however, showed no statistically significant changes attributable to the ethanol treatment. It is possible that some of the changes in brain morphology, brought about as a result of the exposure to ethanol during early life, may not be manifested until later in life. This question has been further addressed in an extension to our previous study. Wistar rats were exposed to a relatively high daily dose of ethanol on postnatal days 10-15 by placement in a chamber containing ethanol vapour, for 3 h/day. The blood ethanol concentration was found to be approximately 430 mg/dl at the end of the period of exposure. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anaesthetised and killed either at 16 or 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle and the physical disector methods were used to estimate, respectively, the regional volumes and neuron cell numerical densities in the hilus and granule cell regions of the dentate gyrus. The total numbers of neurons in the hilus region and granule cell layer were computed from these estimates. It was found that 16-day-old animals had 398,000-441,000 granule cells, irrespective of group. The numbers of granule cells increased such that by 30 days of age, rats had 487,000-525,500 granule cells. However, there were no significant differences between ethanol-treated rats and their age-matched controls in granule cell numbers. In contrast, ethanol-treated rats had slightly but significantly fewer neurons in the hilus region than did control animals at 16 days of age, but not at 30 days of age. Therefore, it appears that a short period of ethanol exposure during early life can have effects on neuron numbers of some hippocampal neurons, but not others. The effects on hilar neuron numbers, observed as a result of such short periods of ethanol treatment, appeared to be transitory.