Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Toxicon X ; 7: 100040, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32875289

RESUMO

Etiology-specific onabotulinumtoxinA utilization to manage spasticity is largely unknown. In this 1-year interim analysis, we evaluated real-world onabotulinumtoxinA utilization and effectiveness across several etiologies from the Adult Spasticity International Registry (ASPIRE) study. ASPIRE is a multicenter, prospective, observational registry (NCT01930786) examining stroke, multiple sclerosis [MS], cerebral palsy [CP], traumatic brain injury [TBI], and spinal cord injury [SCI] patients with spasticity treated with onabotulinumtoxinA at the clinician's discretion. Assessments included onabotulinumtoxinA utilization (each session), clinician (subsequent session)/patient (5±1 weeks post-treatment) satisfaction, and the Disability Assessment Scale (DAS; subsequent session). 730 patients received ≥1 onabotulinumtoxinA treatment, with 37% naïve to botulinum toxin(s) for spasticity. The most common etiology was stroke (n=411, 56%), followed by MS (N=119, 16%), CP (N=77, 11%), TBI (N=45, 6%), and SCI (N=42, 6%). The total body mean cumulative dose (±SD) of onabotulinumtoxinA per session ranged from 296 U (±145) in CP to 406 U (±152) in TBI. The most commonly treated upper limb presentations were clenched fist (stroke, MS, and SCI), flexed wrist (CP), and flexed elbow (TBI). Equinovarus foot was the most commonly treated lower limb presentation in all etiologies. Stroke patients showed improved DAS scores for nearly all subscales in both limbs, indicative of improved global function. All etiologies showed improved lower limb mobility DAS scores. Across all sessions, clinicians (range: 87.4% [SCI]-94.2% [CP]) and patients (range: 67.6% [TBI]-89.7% [SCI]) reported extreme satisfaction/satisfaction that onabotulinumtoxinA helped manage spasticity, and clinicians (range: 94.6% [TBI]-98.8% [CP]) and patients (range: 88.4% [stroke]-91.2% [TBI]) would definitely/probably continue treatment. Treatment-related adverse events (TRAEs) and treatment-related serious adverse events (TRSAEs) were reported as follows: stroke: 10 TRAEs (2.2% patients), 3 TRSAEs (0.5%); MS: 5 TRAEs (4.2%), 0 TRSAEs; CP: 0 TRAEs, 0 TRSAEs; TBI: 1 TRAEs (2.2%), 0 TRSAEs; SCI: 0 TRAEs, 0 TRSAEs. No new safety signals were identified. High clinician- and patient-reported satisfaction were observed following individualized onabotulinumtoxinA treatment, as well as improved global function. Interim results from ASPIRE demonstrate etiology-specific similarities and differences in clinical approaches to manage spasticity.

2.
J Child Adolesc Psychopharmacol ; 30(6): 355-365, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32460523

RESUMO

Objective: To evaluate the efficacy and long-term safety of vilazodone in children and adolescent outpatients with major depressive disorder (MDD). Methods: Children and adolescents aged 7-17 years of age with MDD were randomized 2:2:1 to 8 weeks of double-blind placebo, vilazodone 15 or 30 mg/day or fluoxetine 20 mg/day, respectively. The primary and secondary efficacy outcomes, respectively, were change from baseline to week 8 in Children's Depression Rating Scale-Revised (CDRS-R) score total score and Clinical Global Impressions-Severity (CGI-S) score analyzed using a mixed model for repeated measurement approach. Patients who completed the 8-week randomized controlled trial (RCT), as well as new (de novo) patients, could participate in a 26-week, vilazodone-only, open-label extension (OLE) study. Results: The RCT enrolled 473 patients (60% female) with an average age of 13 years. Change in CDRS-R and CGI-S scores from baseline to week 8 did not differ between patients who received vilazodone and those randomized to placebo. The least-squares mean change from baseline in CDRS-R scores was similar for vilazodone and placebo (-20.7 vs. -20.3, p = 0.77; least-squares mean difference [LSMD] = -0.40). For fluoxetine, the LSMD versus placebo was -2.3 (p = 0.14). The OLE enrolled 330 patients (60% female) with an average age of 13-14 years. Overall, no new safety concerns were identified compared to what is known in adults. Conclusions: Similar improvements in depressive symptoms were observed in all arms. This study does not support the efficacy of vilazodone 15 or 30 mg/day for pediatric patients with MDD. No new or unexpected safety concerns were detected during the RCT or OLE studies.

3.
Biochim Biophys Acta ; 1848(2): 603-14, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25445670

RESUMO

G protein-coupled receptors (GPCRs) are integral membrane proteins involved in cellular signaling and constitute major drug targets. Despite their importance, the relationship between structure and function of these receptors is not well understood. In this study, the role of extracellular disulfide bonds on the trafficking and ligand-binding activity of the human A2A adenosine receptor was examined. To this end, cysteine-to-alanine mutations were conducted to replace individual and both cysteines in three disulfide bonds present in the first two extracellular loops. Although none of the disulfide bonds were essential for the formation of plasma membrane-localized active GPCR, loss of the disulfide bonds led to changes in the distribution of the receptor within the cell and changes in the ligand-binding affinity. These results indicate that in contrast to many class A GPCRs, the extracellular disulfide bonds of the A2A receptor are not essential, but can modulate the ligand-binding activity, by either changing the conformation of the extracellular loops or perturbing the interactions of the transmembrane domains.


Assuntos
Alanina/química , Membrana Celular/química , Cisteína/química , Dissulfetos/química , Receptor A2A de Adenosina/química , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Membrana Celular/metabolismo , Cisteína/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Receptor A2A de Adenosina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Relação Estrutura-Atividade
4.
J Gen Physiol ; 145(1): 5-16, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25512599

RESUMO

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels.


Assuntos
Proteínas de Bactérias/química , Ativação do Canal Iônico , Canais de Sódio Disparados por Voltagem/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Canais de Sódio Disparados por Voltagem/metabolismo
5.
PLoS One ; 8(4): e61216, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23579615

RESUMO

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative (22)Na(+) uptake into proteoliposomes containing a 3∶1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different "guest" glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains.


Assuntos
Bactérias/metabolismo , Metabolismo dos Lipídeos , Canais de Sódio Disparados por Voltagem/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Expressão Gênica , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Conformação Proteica , Alinhamento de Sequência , Sódio/metabolismo , Eletricidade Estática , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/isolamento & purificação
6.
Proc Natl Acad Sci U S A ; 110(16): 6364-9, 2013 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-23542377

RESUMO

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channel's full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate.


Assuntos
Alphaproteobacteria/metabolismo , Transporte de Íons/fisiologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo , Células HEK293 , Humanos , Ferro/metabolismo , Água/metabolismo
7.
Nat Commun ; 3: 1102, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23033078

RESUMO

Voltage-gated sodium channels are vital membrane proteins essential for electrical signalling; in humans, they are key targets for the development of pharmaceutical drugs. Here we report the crystal structure of an open-channel conformation of NavMs, the bacterial channel pore from the marine bacterium Magnetococcus sp. (strain MC-1). It differs from the recently published crystal structure of a closed form of a related bacterial sodium channel (NavAb) by having its internal cavity accessible to the cytoplasmic surface as a result of a bend/rotation about a central residue in the carboxy-terminal transmembrane segment. This produces an open activation gate of sufficient diameter to allow hydrated sodium ions to pass through. Comparison of the open and closed structures provides new insight into the features of the functional states present in the activation cycles of sodium channels and the mechanism of channel opening and closing.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
8.
J Biol Chem ; 286(18): 16386-91, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21454659

RESUMO

Eukaryotic sodium channels are important membrane proteins involved in ion permeation, homeostasis, and electrical signaling. They are long, multidomain proteins that do not express well in heterologous systems, and hence, structure/function and biochemical studies on purified sodium channel proteins have been limited. Bacteria produce smaller, homologous tetrameric single domain channels specific for the conductance of sodium ions. They consist of N-terminal voltage sensor and C-terminal pore subdomains. We designed a functional pore-only channel consisting of the final two transmembrane helices, the intervening P-region, and the C-terminal extramembranous region of the sodium channel from the marine bacterium Silicibacter pomeroyi. This sodium "pore" channel forms a tetrameric, folded structure that is capable of supporting sodium flux in phospholipid vesicles. The pore-only channel is more thermally stable than its full-length counterpart, suggesting that the voltage sensor subdomain may destabilize the full-length channel. The pore subdomains can assemble, fold, and function independently from the voltage sensor and exhibit similar ligand-blocking characteristics as the intact channel. The availability of this simple pore-only construct should enable high-level expression for the testing of potential new ligands and enhance our understanding of the structural features that govern sodium selectivity and permeability.


Assuntos
Proteínas de Bactérias/química , Membranas Artificiais , Dobramento de Proteína , Multimerização Proteica , Rhodobacteraceae/química , Canais de Sódio/química , Permeabilidade , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
9.
Protein Sci ; 18(11): 2356-70, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19760666

RESUMO

High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A(2)a (hA(2)aR) receptor is active for ligand-binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA(2)aR, as expected, and partially cleaved for hA(2)bR, hCCR5R, and hD(2L)R. Ligand-binding assays conducted on the adenosine family (hA(1)R, hA(2)aR, hA(2)bR, and hA(3)R) of receptors show that hA(2)aR and hA(2)bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae.


Assuntos
Transporte Proteico/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico/fisiologia , Sequência de Aminoácidos , Membrana Celular/metabolismo , Cromatografia de Afinidade , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Ensaio Radioligante , Receptor A2A de Adenosina/biossíntese , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Receptores Acoplados a Proteínas-G/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas
10.
Protein Expr Purif ; 58(2): 342-55, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18249008

RESUMO

Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The alpha subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the G(alpha) family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only approximately 10% of total G(alpha) protein expressed is active; the rest accumulates in inclusion bodies. Although G(ialpha) has been readily expressed in soluble form (to 10 mg/L), other alpha subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold G(alpha) from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of G(alpha) subunits (human G(ialpha(1)), human G(salpha(short)), human G(11alpha) and human G(talpha(cone))), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble G(ialpha) expressed from E. coli showed that although the refolded G(alpha) subunits were soluble and retained partial alpha-helicity characteristic of the native, folded G(alpha) subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded G(ialpha) protein has a native-like secondary structure, but is predominantly in a molten globular state.


Assuntos
Escherichia coli/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Corpos de Inclusão/metabolismo , Dicroísmo Circular , Subunidades alfa de Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Dobramento de Proteína , Estrutura Terciária de Proteína , Espectrofotometria Ultravioleta
11.
Biotechnol Prog ; 23(3): 540-7, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17397185

RESUMO

G protein-coupled receptors (GPCRs) are an important, medically relevant class of integral membrane proteins. Laboratories throughout all disciplines of science devote time and energy into developing practical methods for the discovery, isolation, and characterization of these proteins. Since the crystal structure of rhodopsin was solved 6 years ago, the race to determine high-resolution structures of more GPCRs has gained momentum. Since certain GPCRs are currently produced at sufficient levels for X-ray crystallography trials, it is speculated that heterologous expression of GPCRs may no longer be a bottleneck in obtaining crystal structures. This Review focuses on the current approaches in heterologous expression of GPCRs and explores the problems associated with obtaining crystal structures from GPCRs expressed in different systems. Although milligram amounts of certain GPCRs are attainable, the majority of GPCRs are still either produced at very low levels or not at all. Developing reliable expression techniques for GPCRs is still a major priority for the structural characterization of GPCRs.


Assuntos
Cristalografia por Raios X/métodos , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Escherichia coli/genética , Expressão Gênica , Receptores Acoplados a Proteínas-G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Leveduras/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA