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1.
Health Psychol Behav Med ; 6(1): 162-179, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30009092

RESUMO

Introduction: Previous research demonstrating emotional influences on eating and weight suggest that emotionally expressive writing may have a significant impact on reducing risk of eating pathology. This study examined the effects of writing about Intensely Positive Experiences on weight and disordered eating during a naturalistic stressor. Method: Seventy-one female students completed an expressive or a control writing task before a period of exams. Both groups were compared on BMI (kg/m2) and the Eating Disorder Examination - Questionnaire (EDE-Q) before the writing task and at 8-week follow-up. A number of secondary analyses were also examined (to identify potential mediators) including measures of attachment, social rank, self-criticism and self-reassurance, stress and mood. Results: There was a significant effect of intervention on changes in the subscales of the EDE-Q (p = .03). Specifically, expressive writers significantly reduced their dietary restraint while those in the control group did not. There was no significant effect of the intervention on changes in BMI or the other subscales of the EDE-Q (Eating, Weight and Shape Concern). There was also no effect of writing on any of the potential mediators in the secondary analyses. Discussion: Emotionally expressive writing may reduce the risk of dietary restraint in women but these findings should be accepted with caution. It is a simple and light touch intervention that has the potential to be widely applied. However, it remains for future research to replicate these results and to identify the mechanisms of action.

2.
Bone Joint J ; 98-B(5): 647-53, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27143736

RESUMO

AIMS: Total wrist arthrodesis (TWA) produces a spectrum of outcomes. We investigated this by reviewing 77 consecutive TWA performed for inflammatory and post-traumatic arthropathies, wrist instability and as a salvage procedure. PATIENTS AND METHODS: All operations were performed by a single surgeon using a specifically designed pre-contoured dorsally applied non-locking wrist arthrodesis plate at a single centre. RESULTS: Median post-operative Buck-Gramcko Lohman (BGL), Disabilities of the Arm, Shoulder and Hand and Patient Rated Wrist Evaluation scores at six years (interquartile range (IQR) 3 to 11) were 9 (IQR = 6 to 10), 19 (IQR = 7 to 45) and 13 (IQR = 1 to 31) respectively. Polyarticular inflammatory arthritis and female gender were associated with poorer patient-reported outcomes, although the effect of gender was partly explained by higher rates of inflammatory disease among women. Return to work was negatively influenced by workers' compensation and non-inflammatory wrist pathology. There was no difference in complication rates for inflammatory and non-inflammatory indications. TAKE HOME MESSAGE: Polyarticular inflammatory arthritis is a risk factor for adverse patient-reported outcomes in TWA. Furthermore, when compared with patients without inflammatory arthritis, dorsally applied pre-contoured plates can be used for wrist arthrodesis in patients with inflammatory arthritis without an increased risk of complications. Cite this article: Bone Joint J 2016;98-B:647-53.


Assuntos
Artrodese , Instabilidade Articular/cirurgia , Articulação do Punho/cirurgia , Idoso , Artrite/cirurgia , Placas Ósseas , Avaliação da Deficiência , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Complicações Pós-Operatórias , Retorno ao Trabalho/estatística & dados numéricos , Fatores Sexuais , Sinovite/cirurgia , Indenização aos Trabalhadores/estatística & dados numéricos
3.
Protein Eng Des Sel ; 29(6): 231-243, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27160179

RESUMO

Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms.


Assuntos
Mutagênese Sítio-Dirigida , Agregados Proteicos , gama-Cristalinas/química , gama-Cristalinas/genética , Clonagem Molecular , Escherichia coli/genética , Humanos , Modelos Moleculares , Mutação Puntual , Mapas de Interação de Proteínas , Estabilidade Proteica , Eletricidade Estática , Temperatura , gama-Cristalinas/metabolismo
4.
J Microsc ; 249(3): 165-72, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23305211

RESUMO

In this study, nano- and microscale fibrillar and tubular structures formed by mixing two aromatic peptides known to self-assemble separately, (diphenylalanine and di-D-2-napthylalanine) have been investigated. The morphology, mechanical strength and thermal stability of the tubular structures formed have been studied. The tubes are shown to consist of both peptides with some degree of nanoscale phase separation. The ability of the mixed peptides to form structures, which display variable mechanical properties dependent on the percentage composition of the peptides is presented. Such materials with tuneable properties will be required for a range of applications in nanotechnology and biotechnology.


Assuntos
Nanotubos/ultraestrutura , Peptídeos/metabolismo , Multimerização Proteica , Nanotubos/química , Peptídeos/química , Estabilidade Proteica , Temperatura
5.
J Control Release ; 162(2): 321-9, 2012 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-22580112

RESUMO

Polymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5 nm thick) at the surface which was readily removed under sputtering with C(60). Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2 µm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations.


Assuntos
Portadores de Fármacos/química , Ácido Láctico/química , Muramidase/química , Ácido Poliglicólico/química , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Álcool de Polivinil/química , Porosidade , Análise Espacial , Espectrometria de Massa de Íon Secundário , Propriedades de Superfície , Tensoativos/química
6.
Neuroscience ; 204: 74-82, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22192839

RESUMO

UNLABELLED: Restraint stress exposures evoke progressively larger increases in 2-arachidonoylglycerol (2-AG) in limbic brain regions as the number of repetitions increases. The Porsolt swim test usually involves two swim exposures separated by 24 h, and we asked whether the 2-AG response differed between the first and second exposures. METHODS: Four groups of male C57/Bl6N mice were studied: control; exposed to a single 6 min swim and killed immediately; exposed to a single 6 min swim and killed 24 h later; and exposed to two swims, separated by 24 h, and killed after the second swim. Outcomes were swim behavior, serum corticosterone, and 2-AG and 2-oleoylglycerol (2-OG) contents in amygdala, hippocampus, and prefrontal cortex. RESULTS: Mean 2-AG contents were not significantly different among the four treatment groups in any brain region and did not correlate with immobility in either forced swim exposure. However, 2-AG contents in all three brain regions only of the mice exposed to two swims were significantly, positively correlated with serum corticosterone concentrations measured at the same time. 2-OG is present in brain and exhibits a striking regional heterogeneity in control mice. 2-OG concentrations in prefrontal cortex were significantly reduced in the mice killed on the second day compared with the mice killed on the first day. As the target of 2-OG in brain is not known, the significance of these observations await further studies. CONCLUSIONS: Although prior exposure to swim stress does not alter brain 2-AG contents upon re-exposure, 2-AG concentrations in brain become significantly correlated with the hypothalamic-pituitary-adrenal (HPA) axis response to stress when prior exposure to the stress has occurred. These data suggest that even a single exposure to a short period of intense stress sensitizes the 2-AG response to re-exposure to that situation and are consistent with a role for endocannabinoid signaling in modulating stress responses.


Assuntos
Ácidos Araquidônicos/metabolismo , Glicerídeos/metabolismo , Sistema Límbico/metabolismo , Estresse Fisiológico/fisiologia , Estresse Psicológico/metabolismo , Animais , Corticosterona/sangue , Endocanabinoides , Sistema Hipotálamo-Hipofisário/metabolismo , Masculino , Camundongos , Sistema Hipófise-Suprarrenal/metabolismo , Natação
7.
Br J Pharmacol ; 160(3): 736-46, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20590576

RESUMO

BACKGROUND AND PURPOSE: In vitro studies demonstrate that cannabinoid CB(1) receptors subserve activity-dependent suppression of inhibition in the neocortex. To examine this mechanism in vivo, we assessed the effects of local changes in CB(1) receptor activity on somatosensory cortex neuronal activation by whisker movement in rats. EXPERIMENTAL APPROACH: Laser Doppler flowmetry and c-Fos immunohistochemistry were used to measure changes in local blood flow and neuronal activation, respectively. All drugs were applied directly to the cranium above the whisker barrel fields of the primary somatosensory cortex. KEY RESULTS: The CB(1) receptor agonist WIN55212-2 potentiated the hyperaemia induced by whisker movement and this potentiation was occluded by bicuculline. The CB(1) receptor antagonists, rimonabant and AM251, inhibited hyperaemic responses to whisker movement; indicating that activation of endogenous CB(1) receptors increased during whisker movement. Whisker movement-induced expression of c-Fos protein in neurons of the whisker barrel cortex was inhibited by rimonabant. Movement of the whiskers increased the 2-arachidonoylglycerol content in the contralateral, compared to the ipsilateral, sensory cortex. CONCLUSIONS AND IMPLICATIONS: These results support the hypothesis that endocannabinoid signalling is recruited during physiologically relevant activation of the sensory cortex. These data support the hypothesis that the primary effect of CB(1) receptor activation within the activated whisker barrel cortex is to inhibit GABA release, resulting in disinhibition of neuronal activation. These studies provide physiological data involving endocannabinoid signalling in activity-dependent regulation of neuronal activation and provide a mechanistic basis for the effects of cannabis use on sensory processing in humans.


Assuntos
Hiperemia/fisiopatologia , Receptor CB1 de Canabinoide/fisiologia , Córtex Somatossensorial/irrigação sanguínea , Córtex Somatossensorial/fisiologia , Animais , Ácidos Araquidônicos/metabolismo , Benzoxazinas/farmacologia , Bicuculina/farmacologia , Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Lateralidade Funcional , Glicerídeos/metabolismo , Hiperemia/metabolismo , Masculino , Morfolinas/farmacologia , Naftalenos/farmacologia , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Rimonabanto , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/metabolismo , Vibrissas/fisiologia
8.
Eur J Pharm Sci ; 38(1): 1-8, 2009 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-19447176

RESUMO

In this study we aim to explore the potential links between the mechanical properties, micronisation behaviour and surface energy of carbamazepine polymorphs using atomic force microscopy (AFM) measurements of material properties at the nanoscale. Carbamazepine Forms I, II and III were prepared and confirmed using X-ray powder diffraction (XRPD). AFM measurements of indentation hardness, Young's modulus and surface energy were made on the starting material. In addition, the surface energy was measured immediately after micronisation and after storage for four weeks. Carbamazepine polymorphs could be ranked by Young's modulus and hardness. Surface energy measurements showed an increase after micronisation in all cases, and a varying relaxation after storage for four weeks. Form I showed a smaller particle size distribution, indicating more complete micronisation. A promising correlation was observed between the hardness/Young's modulus ratio and the micronisation behaviour, in terms of particle size reduction and surface energy change. The results show potential for the predictive capacity of such an approach, and help to provide a greater understanding of material behaviour and properties during micronisation.


Assuntos
Carbamazepina/química , Microscopia de Força Atômica , Tamanho da Partícula , Fenômenos Químicos , Química Farmacêutica , Cristalização , Módulo de Elasticidade , Dureza , Microscopia Eletrônica de Varredura , Propriedades de Superfície , Difração de Raios X
9.
Biophys Chem ; 141(2-3): 214-21, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19258192

RESUMO

The beta-helix is an important protein fold in many pathogens, and is a challenging system for folding pathway prediction because it primarily is stabilized by non-local interactions along the primary sequence. A useful experimental model of this fold is a monomeric truncation of P22 tailspike protein, the beta-helix domain (bhx). This report describes a systematic in vitro study of the chemical denaturation and refolding of bhx. Results from equilibrium chemical denaturation experiments were consistent with a two-state folding mechanism, but showed only partial reversibility. Stopped-flow fluorescence studies showed a single unfolding step, but two refolding steps. The slow refolding step could be partly attributed to proline isomerization, based on an increased rate during refolding in the presence of PPIase and an increased relative amplitude of this step with increasing delay time in double-jump refolding experiments observed over delays of 5-100 s. However, double-jump refolding experiments with delay times longer than 100 s along with size exclusion chromatography and dynamic light scattering of refolding samples showed that the overall refolding yield decreased as bhx was unfolded for longer periods of time. Furthermore, the losses resulted from aggregate formation during refolding. This suggests that a change occurs over time in the unfolded or denatured state ensemble that increases the propensity for aggregation upon the shift to more native-favoring conditions. Alternatively aggregate nuclei may be able to form even under high denaturant conditions, and these subsequently grow and consume monomer when placed under native-favoring conditions.


Assuntos
Bacteriófago P22/química , Dobramento de Proteína , Proteínas da Cauda Viral/química , Cromatografia em Gel , Fluorescência , Glicosídeo Hidrolases , Isomerismo , Cinética , Luz , Modelos Moleculares , Peptidilprolil Isomerase/química , Prolina/química , Estrutura Terciária de Proteína , Espalhamento de Radiação , Tempo
10.
Colloids Surf B Biointerfaces ; 67(1): 1-13, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18775653

RESUMO

Understanding the movement of the active ingredient in relation to the other formulation components following application is crucial to an overall understanding of herbicide performance. We describe the novel use of time-of-flight secondary ion mass spectrometry (ToF-SIMS) as a tool for following the movement of herbicide formulation components into and across plant cuticles. This technique provides new insights since it provides both high (sub-micron) spatial resolution combined with the chemical specificity associated with organic mass spectrometry. The components studied include the oligomeric ethoxylate surfactants Synperonic A7 and A20 and active ingredient Sulfosate (trimesium glyphosate). The movement of these molecules, both separately and when combined in a simple formulation, into the surface of Prunus laurocerasus leaves and across the isolated plant cuticle was investigated and clear differences in penetration/diffusion behaviour were identified. ToF-SIMS was uniquely able to (simultaneously) spatially resolve all the species involved, including the anion and cation components of the active ingredient. Also, using spectral reconstructions from the imaging raw data streams, the behaviour of individual oligomers within the surfactant distributions, could be assessed. The observations are discussed with reference to the action of surfactants identified in parallel micro-structural studies and the current understanding of herbicide uptake.


Assuntos
Herbicidas/farmacocinética , Folhas de Planta/metabolismo , Espectrometria de Massa de Íon Secundário , Cromatografia Líquida de Alta Pressão , Herbicidas/química , Microscopia Confocal , Prunus/metabolismo
11.
Biophys J ; 95(7): 3400-6, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18599629

RESUMO

Dynamic light scattering and Fourier transform infrared spectroscopy were used to study the formation of prefibrillar aggregates and fibrils of bovine pancreatic insulin at 60 degrees C and at pH 1. The kinetics of disintegration of the prefibrillar aggregates were also studied using these techniques after a quench to 25 degrees C. These experiments reveal that formation of prefibrillar aggregates is reversible under the solution conditions studied and show that it is possible to significantly reduce the nucleation (lag) times associated with the onset of fibril growth in bovine pancreatic insulin solutions by increasing the concentration of prefibrillar aggregates in solution. These results provide convincing evidence that less structured prefibrillar aggregates can act as fibril-forming intermediates.


Assuntos
Insulina/química , Insulina/metabolismo , Animais , Bovinos , Concentração de Íons de Hidrogênio , Luz , Pâncreas/metabolismo , Tamanho da Partícula , Ligação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Fatores de Tempo
12.
Biophys J ; 93(6): 2143-51, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17496011

RESUMO

A technique was developed for studying the nucleation and growth of fibrillar protein aggregates. Fourier transform infrared and attenuated total reflection spectroscopy were used to measure changes in the intermolecular beta-sheet content of bovine pancreatic insulin in bulk solution and on model polystyrene (PS) surfaces at pH 1. The kinetics of beta-sheet formation were shown to evolve in two stages. Combined Fourier transform infrared, dynamic light scattering, atomic force microscopy, and thioflavin-T fluorescence measurements confirmed that the first stage in the kinetics was related to the formation of nonfibrillar aggregates that have a radius of 13 +/- 1 nm. The second stage was found to be associated with the growth of insulin fibrils. The beta-sheet kinetics in this second stage were used to determine the nucleation and growth rates of fibrils over a range of temperatures between 60 degrees C and 80 degrees C. The nucleation and growth rates were shown to display Arrhenius kinetics, and the associated energy barriers were extracted for fibrils formed in bulk solution and at PS surfaces. These experiments showed that fibrils are nucleated more quickly in the presence of hydrophobic PS surfaces but that the corresponding fibril growth rates decrease. These observations are interpreted in terms of the differences in the attempt frequencies and energy barriers associated with the nucleation and growth of fibrils. They are also discussed in the context of differences in protein concentration, mobility, and conformational and colloidal stability that exist between insulin molecules in bulk solution and those that are localized at hydrophobic PS interfaces.


Assuntos
Insulina/química , Animais , Fenômenos Biofísicos , Biofísica , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Luz , Microscopia de Força Atômica , Poliestirenos , Estrutura Secundária de Proteína , Espalhamento de Radiação , Soluções , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Termodinâmica
13.
J Anim Sci ; 85(1): 163-71, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17179552

RESUMO

Insulin-like growth factor-1 could be a useful marker in the horse for diagnostic, selection, or forensic purposes, provided its physiological regulation is well understood. The objective of this study was to investigate factors, such as acute exercise, fitness training, time of day, sex, and age, that may influence serum IGF-1 in normal, healthy horses. Throughout a 9-wk training program, 6 geldings maintained a mean (+/- SEM) IGF-1 concentration of 302 +/- 29 ng/mL. Moderate or high intensity exercise had no effect on IGF-1 concentrations, when pre- and postexercise values were compared. Over a 24-h period, there was some variation in IGF-1 concentrations but no clear diurnal rhythm. Concentrations of IGF-1 were measured in a large population of thoroughbred horses (1,880) on 3 continents. The population deviated slightly from a normal distribution (P < 0.001) because of large IGF-1 concentrations in 10 horses. The global mean IGF-1 concentration was 310 +/- 2.2 ng/mL, with a greater mean value (P < 0.001) in gonad-intact males (336 +/- 5.6 ng/mL) than in females (303 +/- 3.2 ng/mL) or geldings (302 +/- 3.2 ng/mL). However, the greatest IGF-1 concentrations observed for all stallions, mares, and geldings were 627, 676, and 709 ng/mL, respectively. In mares and geldings, IGF-1 concentrations showed a gradual decrease with advancing age (P < 0.001), but the effect was much less marked in stallions. This study confirms that IGF-1 concentrations are stable, compared with GH concentrations, in the horse and that a meaningful measure of IGF-1 status can be obtained from a daily serum sample.


Assuntos
Envelhecimento/fisiologia , Ritmo Circadiano/fisiologia , Cavalos/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Condicionamento Físico Animal/fisiologia , Caracteres Sexuais , Animais , Peso Corporal , Feminino , Frequência Cardíaca , Masculino , Fatores de Tempo
14.
Xenobiotica ; 36(10-11): 902-37, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17118914

RESUMO

To examine species-specific aspects of the induction of absorption, distribution, metabolism and excretion (ADME)-related genes, we used 25 000 gene oligonucleotide microarrays to construct a rodent gene-response compendium that compared hepatic gene expression profiles and developed consensus aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) ligand signatures relevant to drug clearance. Twenty-six inducer compounds were chosen from the literature. Rats and mice received one of six dose levels (log2 dose escalation, 32-fold dose range) of each compound daily for 3 days. Animals were necropsied 6-9 h after the last dose, and tissues were collected for RNA analysis. Hepatic gene expression profiles were obtained using Rosetta Resolver expression analysis system, and ADME-related genes were extracted. Cross-talk among nuclear receptors or hepatoxicity at high dose levels resulted in large signatures (usually >1000 genes at p < 0.01) for most compounds. After ADME gene transcript enrichment, agglomerative clustering separated AhR ligands from CAR/PXR ligands, but it was difficult to distinguish CAR from PXR ligands. Consensus signatures were derived from groups of AhR, CAR and PXR ligands; and cross-talk among responding genes was determined. Many compounds had distinct log dose-response profiles, and relative potencies for ligands were established. Robust responses by CYP1A1, CYP2B10 (CAR responsive in mice) and CYP2B15 (CAR responsive in rats) and CYP3A1 (PXR responsive in rats) were used to benchmark the relative potency of different ligands and to determine the relative selectivity for AhR, CAR or PXR. By using a compendium of gene expression profiles, we defined species-specific induction patterns across the ADME transcriptome.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fígado/metabolismo , Análise em Microsséries/métodos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Análise por Conglomerados , Receptor Constitutivo de Androstano , Relação Dose-Resposta a Droga , Feminino , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Receptor de Pregnano X , Ratos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Fatores de Transcrição/genética
15.
Xenobiotica ; 36(10-11): 963-88, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17118916

RESUMO

Carrier-mediated transporters play a critical role in xenobiotic disposition and transporter research is complicated by species differences and their selective tissue expression. The purpose of this study was to generate a comprehensive data set of xenobiotic transporter gene expression profiles in humans and the pre-clinical species mouse, rat, beagle dog and cynomolgus monkey. mRNA expression profiles of 50 genes from the ABC, SLC and SLCO transporter superfamilies were examined in 40 human tissues by microarray analyses. Transporter genes that were identified as enriched in the liver or kidney, or that were selected for their known roles in xenobiotic disposition, were then compared in 22 tissues across the five species. Finally, as clinical variability in drug response and adverse reactions may be the result of variability in transporter gene expression, variability in the expression of selected transporter genes in 75 human liver donors were examined and compared with the highly variable drug metabolizing enzyme CYP3A4.


Assuntos
Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Xenobióticos/metabolismo , Animais , Feminino , Expressão Gênica , Humanos , Inativação Metabólica , Rim/metabolismo , Fígado/metabolismo , Masculino , Especificidade da Espécie , Doadores de Tecidos
16.
Langmuir ; 21(8): 3591-8, 2005 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-15807606

RESUMO

Poly[2-(dimethylamino)ethyl methacrylate-b-2-methacryloyloxyethyl phosphorylcholine] (DMA-MPC) is currently under investigation as a new vector candidate for gene therapy. The DMA block has been previously demonstrated to condense DNA effectively. The MPC block contains a phosphorylcholine (PC) headgroup, which can be found naturally in the outside of the cell membrane. This PC-based polymer is extremely hydrophilic and acts as a biocompatible steric stabilizer. In this study, we assess in detail the morphologies of DNA complexes obtained using the diblock copolymer series DMA(x)MPC30 (where the mean degree of polymerization of the MPC block was fixed at 30 and the DMA block length was systematically varied) using transmission electron microscopy (TEM) and liquid atomic force microscopy (AFM). Both techniques indicate more compact complex morphologies (more efficient condensation) as the length of the cationic DMA block increases. However, the detailed morphologies of the DMA(x)MPC30-DNA complexes observed by TEM in vacuo and by AFM in aqueous medium are different. This phenomena is believed to be related to the highly hydrophilic nature of the MPC block. TEM studies revealed that the morphology of the complexes changes from loosely condensed structures to highly condensed rods, toroids, and oval-shaped particles as the DMA moiety increases. In contrast, morphological changes from plectonemic loops to flower-like and rectangular block-like structures, with an increase in highly condensed central regions, are observed by in situ AFM studies. The relative population of each structure is clearly dependent on the polymer molecular composition. Enzymatic degradation assays revealed that only the DMA homopolymer provided effective DNA protection against DNase I degradation, while other highly condensed copolymer complexes, as judged from TEM and gel electrophoresis, only partially protected the DNA. However, AFM images indicated that the same highly condensed complexes have less condensed regions, which we believe to be the initiation sites for enzymatic attack. This indicates that the open structures observed by AFM of the DNA complexation by the DMA(x)MPC30 copolymer series are closer to in vivo morphology when compared to TEM.


Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Metacrilatos/química , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Biopolímeros/química , Biopolímeros/farmacologia , Cátions , Membrana Celular/metabolismo , DNA/química , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Eletroforese em Gel de Ágar , Terapia Genética , Metacrilatos/farmacologia , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Conformação de Ácido Nucleico , Fosforilcolina/química
17.
J Control Release ; 100(3): 437-50, 2004 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-15567508

RESUMO

Polyelectrolyte complexes between DNA and polyethylenimine (PEI) are promising non-viral delivery systems for pulmonary inhalation gene therapy and thus require sufficient stability during nebulization. The structure and stability of four different PEI-DNA polyplexes, namely branched (bPEI), linear (linPEI), poly(ethylene glycol)-grafted PEI (PEGPEI), biodegradable (bioPEI) PEI with DNA, were investigated. Using atomic force microscopy, the morphology of DNA and polyplexes before and after both air-jet and ultrasonic nebulization was characterized. The influence of nebulization on physico-chemical properties, particle size and zeta potential, was studied. Efficient DNA condensation to spherical particles was achieved with bPEI (90 nm) and PEGPEI (110 nm). By contrast, incomplete DNA condensations, seen as flower structures, were observed with linPEI (110 nm) and bioPEI (105 nm). Air-jet nebulization altered the polyplex structure to a greater extent than ultrasonic nebulization and resulted mainly in smaller and non-spherical particles (30-200 nm). Ultrasonic nebulization did not change the spherical structure or particle size of the polyplexes. In particular, the shape and size of the PEGPEI polyplexes did not change. We conclude that ultrasonic nebulization is a milder aerosolization method for gene delivery systems based on PEI. Additionally, PEGPEI-DNA polyplexes seem to be more stable than their counterparts, which may be advantageous in pulmonary inhalation gene therapy.


Assuntos
Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Polietilenoimina/química , Aerossóis , DNA/genética , Desoxirribonucleases/química , Eletroforese em Gel de Ágar , Fluxometria por Laser-Doppler , Luz , Microscopia de Força Atômica , Nebulizadores e Vaporizadores , Tamanho da Partícula , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Espalhamento de Radiação , Ultrassom
18.
J Microsc ; 215(Pt 3): 297-301, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15312194

RESUMO

Multivalent ions in solution are known to mediate attraction between two like-charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either 'salt bridging' or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re-explore current theories of like-charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.


Assuntos
Silicatos de Alumínio/metabolismo , DNA Bacteriano/metabolismo , Microscopia de Força Atômica/métodos , Sódio/farmacologia , Escherichia coli/genética , Plasmídeos/genética
19.
Int J Pharm ; 280(1-2): 185-98, 2004 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-15265558

RESUMO

Atomic force microscopy (AFM) has been used to investigate the (1 0 0) face of crystalline adipic acid, both in air and liquid environments. In air, surface reorganization occurred during scanning of the AFM probe, which has been investigated using single point force-distance analysis under a controlled relative humidity (RH) environment. We suggest such reorganization can be attributed to the influence of a network of water molecules bound to the hydrophilic (1 0 0) surface permitting local AFM tip-enhanced dissolution and reorganization of the solute. In situ imaging was also carried out on the crystals, revealing etch-pit formation during dissolution, and rapid growth at higher levels of supersaturation (sigma), both of which are direct consequences of the hydrophilic nature of the (1 0 0) face. Also presented here are nanoscale observations of the effect of octanoic acid, a structurally-related habit modifier, on crystalline adipic acid. Using AFM, we have been able to show that the presence of octanoic acid at low concentration has little observable affect on the development of the (1 0 0) face; however, as this concentration is increased, there are clear changes in step morphology and growth mode on the (1 0 0) face of the crystal. At a concentration of 1.26 mmol dm(-3) (a concentration corresponding to a molar ratio of approximately 1:175 octanoic acid:adipic acid), growth on the (1 0 0) face is inhibited, with in situ AFM imaging indicating this is a direct consequence of octanoic acid binding to the surface, and pinning the monomolecular growth steps.


Assuntos
Adipatos/química , Excipientes/química , Microscopia de Força Atômica/métodos , Adipatos/análise , Cristalização , Excipientes/análise
20.
J Microsc ; 214(Pt 3): 261-71, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15157194

RESUMO

Co-ordination polymers are currently attracting extensive interest due to their potential applications as supramolecular hosts, vessels, and frameworks for storage and separations. Many applications rely on the ion exchange capabilities of these compounds, and considerable debate surrounds the mechanism by which ion exchange occurs in co-ordination polymers. Here AFM and SEM were applied, for the first time, to investigate this class of materials. In situ AFM studies revealed the mechanism by which anion exchange and the subsequent structural transformations of the crystalline co-ordination polymers [[Ag(4,4'-bipy)]BF(4)](infinity) and [[Ag(4,4'-bipy)]NO(3)](infinity) occur. The process is initiated by the dissolution of the metastable crystalline polymer, followed by the subsequent crystallization of the new stable phase on the surface of the original crystal. The formation of deep clefts in the metastable polymer crystal during the transformation allows the solution to access the successive crystalline layers. Thus, the entire process can be viewed as a self-perpetuating cascade of dissolution and recrystallization throughout the macroscopic crystal. SEM data consolidate the findings of AFM. These techniques collectively illustrate that the anion exchange, and subsequent structural transformation, proceeds via a solvent-mediated mechanism, rather than a purely solid-state one.

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