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1.
Zhongguo Zhong Yao Za Zhi ; 44(20): 4476-4480, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31872635

RESUMO

The 70% ethanol extract of the whole plant of Souliea vaginata was purified by multi-chromatographic methods including macroporous resin,silica gel,Sephadex LH-20,and C18-reversed-phase column chromatography. A new spirocyclic cycloartane triterpenoid was isolated and identified as( 16 R*,20 R*,23 S*,24 R*,25 S*)-16,23: 23,26-diepoxy-15α,24,25-trihydroxy-9,19-cycloart-3ß-O-ß-D-xylopyranoside( 1),and named as soulieoside S. Its planar structure and relative configuration were determined by spectroscopic techniques including 2 D NMR and HRESI-MS. As one of the main components of S. vaginata,compound 1 was evaluated for its anti-inflammatory activity by a lipopolysaccharide( LPS)-stimulated NO production model in RAW264. 7 macrophages,but it didn't show NO production inhibitory effect.


Assuntos
Actaea/metabolismo , Triterpenos/metabolismo , Actaea/química , Glicosídeos , Lipopolissacarídeos , Estrutura Molecular , Triterpenos/análise
2.
Ann Transl Med ; 7(7): 145, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31157266

RESUMO

Background: To measure the direct effects of pre-operative magnetic resonance neurography (MRN), and analyze the procedure and clinical outcomes of the percutaneous endoscopic interlaminar discectomy, in order to demonstrate the feasibility and safety of the interlaminar approach. Methods: In this study, 127 patients who underwent percutaneous endoscopic lumbar discectomy (PELD) and were followed up by more than 12 months, were retrospectively evaluated. The pre-operative demographic data were collected. In addition, the coronal scan and the reconstruction of the lumbosacral plexus were examined to measure the distance between the nerve root and the dural sac at the coronal plane. Furthermore, the post-operative and pre-operative visual analog scale (VAS) scores and Oswestry Disability Index (ODI) were compared. Results: The mean and minimum values of distance T between the nerve root and dural sac of L5/S1 to L2/3 on the operation side of the MRN images were all larger than 7.3 mm which is the diameter of the working canal. During the follow-up, VAS and ODI data improved significantly compared with their corresponding pre-operative scores (P<0.01). Regarding the post-operative complications, there were 2 (1.57%) cases of hypesthesia and 3 (2.36%) cases of neuropathic pain, which were transient and alleviated in 3 months. In addition, there was 1 (0.79%) case of intervertebral space infection and 1 (0.79%) dural tear. No relapse of disc herniation and iatrogenic instability occurred by the end of the last follow-up. Conclusions: The MRN indicates that the incidence of herniated disc impingement increases over the distance between the nerve root and the dural sac, thus making the interlaminar approach more suitable for the treatment of herniation. The procedures and clinical outcomes of the IL-PELD demonstrate the safety and advantages of the interlaminar approach.

3.
Biomed Res Int ; 2016: 8356435, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27088091

RESUMO

Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined.


Assuntos
Anticorpos Monoclonais/genética , Formação de Anticorpos/genética , RNA/genética , Animais , Anticorpos Monoclonais/biossíntese , Células CHO , Cricetinae , Cricetulus , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Análise de Sequência de RNA
4.
Medicine (Baltimore) ; 95(8): e2880, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26937924

RESUMO

Dexmedetomidine (DEX) has been reported to have synergistic action with local anesthetics. This prospective, randomized, double-blind clinical study was designed to observe the efficacy of intravenous DEX without loading dose on spinal blockade duration, postoperative sedation, patient-controlled analgesia and its morphine-sparing effect in lower limb surgeries.Seventy-five patients, scheduled for lower limb surgery under spinal anesthesia, were randomly allocated into 2 groups: group BS (received 15 mg of 0.5% of bupivacaine for subarachnoid anesthesia and continuous intravenous infusion of saline in Ringer solution) and BD group (received 15 mg of 0.5% of bupivacaine for subarachnoid anesthesia and continuous intravenous infusion of DEX in Ringer solution at a rate of 0.25 µg/kg/h). Intravenous infusion started 15 minutes before spinal anesthesia.The onset time of sensory and motor blockade was shorten, the duration of sensory and motor blockade was significantly prolonged in BD patients when compared to BS patients. The Ramsay sedation score measured immediately after surgery was greater in BD group than BS group. BD patients also shown increased time to the first request of postoperative morphine and decreased total morphine consumption as compared with BS patients.Collectively, intravenous administration of DEX without loading dose promoted the efficacy of spinal bupivacaine anesthesia and postoperative analgesia in patients undergoing lower limb surgery.


Assuntos
Analgésicos não Entorpecentes/administração & dosagem , Anestésicos Locais/administração & dosagem , Bupivacaína/administração & dosagem , Dexmedetomidina/administração & dosagem , Extremidade Inferior/cirurgia , Estudos Prospectivos , Adulto , Analgesia Controlada pelo Paciente , Método Duplo-Cego , Feminino , Humanos , Infusões Intravenosas , Masculino , Dor Pós-Operatória/tratamento farmacológico , Resultado do Tratamento
5.
Chem Commun (Camb) ; 50(94): 14900-3, 2014 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-25325394

RESUMO

Here we demonstrate a methodology, termed protein stapling, for the introduction of covalent constraints into recombinant proteins. Using the azide-alkyne click reaction as the stapling chemistry, we have improved the thermostability of a model leucine zipper protein. Additionally, stapling the core of the small, globular protein G resulted in improved binding to its target, immunoglobulin G.


Assuntos
Alquinos/química , Azidas/química , Proteínas de Bactérias/química , Sequência de Aminoácidos , Imunoglobulina G/metabolismo , Zíper de Leucina , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína
6.
J Pharm Sci ; 103(1): 152-60, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24338750

RESUMO

Approximately 30% of active pharmaceutical ingredients (APIs) experience agglomeration, granulation, and breakage during agitated drying. Currently, there is no small-scale bench tool to help assess and observe granulation behavior of APIs in the laboratory and subsequently lead to the development of a robust drying method. As a result, more conservative drying methods are usually used at scale and much longer drying times are needed. In this work, we build on work reported in the literature and demonstrate that a mixer torque rheometer (MTR) can be a useful small-scale tool to flag potentially problematic granulation behavior of APIs under different conditions. The results from the MTR were confirmed using a second new tool involving the use of an acoustic mixer to verify and observe the granulation behavior on a small scale. We also show consistency between the data collected at the laboratory and the pilot plant scales.


Assuntos
Preparações Farmacêuticas/química , Tecnologia Farmacêutica/instrumentação , Laboratórios , Tamanho da Partícula
7.
Langmuir ; 28(29): 10934-41, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22746532

RESUMO

There is considerable current interest in developing methods to integrate nanoparticles into optical, electronic, and biological systems due to their unique size-dependent properties and controllable shape. We report herein a versatile new approach for covalent immobilization of nanoparticles onto substrates modified with photoactive, phthalimide-functional, self-assembled monolayers. Upon illumination with UV radiation, the phthalimide group abstracts a hydrogen atom from a neighboring organic molecule, leading to radical-based photografting reactions. The approach is potentially "universal" since virtually any polymeric or organic-inorganic hybrid nanoparticle can be covalently immobilized in this fashion. Because grafting is confined to illuminated regions that undergo photoexcitation, masking provides a simple and direct method for nanoparticle patterning. To illustrate the technique, nanoparticles formed from diblock copolymers of poly(styrene-b-polyethylene oxide) and laden with Hostasol Red dye are photografted and patterned onto glass and silicon substrates modified with photoactive phthalimide-silane self-assembled monolayers. Atomic force microscopy and X-ray photoelectron spectroscopy are applied to characterize the grafted nanoparticle films while confocal fluorescence microscopy is used to image patterned nanoparticle deposition.


Assuntos
Nanopartículas/química , Raios Ultravioleta , Vermelho Congo/química , Estrutura Molecular , Tamanho da Partícula , Ftalimidas/química , Polietilenoglicóis/química , Poliestirenos/química , Silanos/química , Silício/química , Propriedades de Superfície
8.
Mol Pharm ; 9(8): 2228-36, 2012 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-22734614

RESUMO

Polymeric nanoparticles with multifunctional capabilities, including surface functionalization, hold great promise to address challenges in targeted drug delivery. Here, we describe a concise, robust synthesis of a heterofunctional polyethylene glycol (PEG), HO-PEG-azide. This macromer was used to synthesize polylactide (PLA)-PEG-azide, a functional diblock copolymer. Rapid precipitation of this copolymer with a hydrophobic cargo resulted in the generation of monodisperse nanoparticles with azides in the surface corona. To demonstrate conjugation to these nanoparticles, a regioselectively modified alkyne-folate was employed as a model small molecule ligand, and the artificial protein A1 with an alkyne moiety introduced by unnatural amino acid substitution was selected as a model macromolecular ligand. Using the copper-catalyzed azide-alkyne ligation reaction, both ligands exhibited good conjugation efficiency even when low concentrations of ligands were used.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Polietilenoglicóis/química , Polímeros/química , Química Click/métodos
9.
ACS Synth Biol ; 1(3): 89-98, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-23651073

RESUMO

The Bcl-2 family of proteins regulates apoptosis at the level of mitochondrial permeabilization. Pro-death members of the family, including Bak and Bax, initiate apoptosis, whereas pro-survival members such as Bcl-x(L) and Mcl-1 antagonize the function of Bak and Bax via heterodimeric interactions. These heterodimeric interactions are primarily mediated by the binding of the helical amphipathic BH3 domain from a pro-death protein to a hydrophobic cleft on the surface of the pro-survival protein. Since high levels of pro-survival Bcl-2 proteins are present in many cancers, peptides corresponding to pro-death BH3 domains hold promise as therapeutics. Here we apply a high-throughput flow cytometry assay to engineer the Bak BH3 domain for improved affinity toward the pro-survival proteins Bcl-x(L) and Mcl-1. Two strategies, engineering the hydrophobic face of the Bak BH3 peptide and increasing its overall helicity, are successful in identifying Bak BH3 variants with improved affinity to Bcl-x(L) and Mcl-1. Hydrophobic face engineering of the Bak BH3 peptide led to variants with up to a 15-fold increase in affinity for Bcl-x(L) and increased specificity toward Bcl-x(L). Engineering of the helicity of Bak BH3 led to modest (3- to 4-fold) improvements in affinity with retention of promiscuous binding to all pro-survival proteins. HeLa cell killing studies demonstrate that the affinity matured Bak BH3 variants retain their expected biological function.


Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Apoptose , Sobrevivência Celular , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Biologia Sintética
10.
Integr Biol (Camb) ; 3(8): 823-31, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21713285

RESUMO

Members of the Bcl-2 family of proteins have opposing roles in programmed cell death; family members can play either pro-apoptotic or anti-apoptotic roles. Heterodimeric interactions between pro-apoptotic and anti-apoptotic members of the Bcl-2 family are critical for the regulation of apoptosis and are important targets for cancer therapeutics. Bcl-2 family interactions are mediated by the highly-conserved BH3 domain, corresponding to a single amphipathic α-helix, which binds in a hydrophobic cleft of its Bcl-2 family interaction partner. Here, using a high-throughput peptide-protein interaction assay based on bacterial cell surface display and flow cytometry, we present quantitative data for a near-complete set of 17 BH3 domains from the human genome binding to each of the 5 anti-apoptotic Bcl-2 family members. Biophysical insights into the affinity and specificity of these interactions are provided by analysis of the interactome data. In addition we carried out a truncation study of the Bim BH3 domain to define the core residues responsible for anti-apoptotic protein binding. The interactome data from this study has implications both in basic research on apoptosis and in the design of peptidic cancer therapeutics.


Assuntos
Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Biofísica/métodos , Membrana Celular/metabolismo , Dimerização , Escherichia coli/metabolismo , Citometria de Fluxo/métodos , Humanos , Cinética , Dados de Sequência Molecular , Peptídeos/química , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
11.
Nano Lett ; 11(4): 1723-6, 2011 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-21417430

RESUMO

New sequencing technologies based on massively parallel signature sequencing (MPSS) have been developed to reduce the cost of genome sequencing. In some current MPSS platforms, DNA-modified micrometer-scale beads are used to template the polymerase chain reaction (PCR). Reducing the size of the beads to nanoscale can lead to significant improvements in sequencing throughput. To this end, we have assembled polymeric nanobeads that efficiently template PCR, resulting in DNA-decorated "nanobeads" with a high extent of functionalization.


Assuntos
DNA/química , DNA/ultraestrutura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Reação em Cadeia da Polimerase/métodos , Polímeros/química , DNA/análise , Teste de Materiais , Tamanho da Partícula
12.
Biophys J ; 99(9): 3056-65, 2010 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-21044604

RESUMO

The antimicrobial peptide microcin J25 (MccJ25) is posttranslationally matured from a linear preprotein into its native lasso conformation by two enzymes. One of these enzymes cleaves the preprotein and the second enzyme installs the requisite isopeptide bond to establish the lasso structure. Analysis of a mimic of MccJ25 that can be cyclized without the influence of the maturation enzymes suggests that MccJ25 does not spontaneously adopt a near-lasso structure. In addition, we conducted atomistically detailed replica-exchange molecular dynamics simulations of pro-microcin J25 (pro-MccJ25), the 21-residue uncyclized analog of MccJ25, to determine the conformational ensemble explored in the absence of the leader sequence or maturation enzymes. We applied a nonlinear dimensionality reduction technique known as the diffusion map to the simulation trajectories to extract two global order parameters describing the fundamental dynamical motions of the system, and identify three distinct pathways. One path corresponds to the spontaneous adoption of a left-handed lasso, in which the N-terminus wraps around the C-terminus in the opposite sense to the right-handed topology of native MccJ25. Our computational and experimental results suggest a role for the MccJ25 leader sequence and/or its maturation enzymes in facilitating the adoption of the right-handed topology.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bacteriocinas/química , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Fenômenos Biofísicos , Cromatografia Líquida de Alta Pressão , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Espectrometria de Massas em Tandem
13.
Proc Natl Acad Sci U S A ; 107(45): 19207-12, 2010 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-20956332

RESUMO

The development of a robust and portable biosensor for the detection of pathogenic bacteria could impact areas ranging from water-quality monitoring to testing of pharmaceutical products for bacterial contamination. Of particular interest are detectors that combine the natural specificity of biological recognition with sensitive, label-free sensors providing electronic readout. Evolution has tailored antimicrobial peptides to exhibit broad-spectrum activity against pathogenic bacteria, while retaining a high degree of robustness. Here, we report selective and sensitive detection of infectious agents via electronic detection based on antimicrobial peptide-functionalized microcapacitive electrode arrays. The semiselective antimicrobial peptide magainin I--which occurs naturally on the skin of African clawed frogs--was immobilized on gold microelectrodes via a C-terminal cysteine residue. Significantly, exposing the sensor to various concentrations of pathogenic Escherichia coli revealed detection limits of approximately 1 bacterium/µL, a clinically useful detection range. The peptide-microcapacitive hybrid device was further able to demonstrate both Gram-selective detection as well as interbacterial strain differentiation, while maintaining recognition capabilities toward pathogenic strains of E. coli and Salmonella. Finally, we report a simulated "water-sampling" chip, consisting of a microfluidic flow cell integrated onto the hybrid sensor, which demonstrates real-time on-chip monitoring of the interaction of E. coli cells with the antimicrobial peptides. The combination of robust, evolutionarily tailored peptides with electronic read-out monitoring electrodes may open exciting avenues in both fundamental studies of the interactions of bacteria with antimicrobial peptides, as well as the practical use of these devices as portable pathogen detectors.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Bactérias/isolamento & purificação , Técnicas Biossensoriais/métodos , Animais , Escherichia coli/isolamento & purificação , Proteínas Imobilizadas , Limite de Detecção , Microeletrodos , Salmonella/isolamento & purificação , Xenopus , Proteínas de Xenopus
14.
J Am Chem Soc ; 131(47): 17078-9, 2009 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19894713

RESUMO

Engineered aminoacyl-tRNA synthetases have been used to enable the incorporation of many unnatural amino acids into recombinant proteins in vivo. In the majority of these studies, the engineered synthetase is harbored on a plasmid while the host retains a wild-type copy of the synthetase in its genome. Herein, we construct a strain carrying a single genomic copy of a methionyl-tRNA synthetase (MetRS) gene, metG*, engineered to enable the incorporation of azidonorleucine (ANL) into proteins. The resulting strain, M15MA metG*, is capable of both supporting robust cell growth and enabling the production of >20 mg/L culture of a recombinant protein, murine dihydrofolate reductase, containing ANL. The extent of replacement of methionine with ANL in this protein is 90%. Using this strain, we also produce ANL-containing OmpC, an outer membrane protein, and demonstrate that the surface of cells displaying this protein can be covalently modified using copper-catalyzed azide-alkyne cycloaddition. Since this mutant MetRS has been introduced into the genome, as opposed to a plasmid, M15MA metG* is genetically stable.


Assuntos
Escherichia coli/genética , Genômica , Metionina tRNA Ligase/genética , Norleucina/metabolismo , Engenharia de Proteínas , Metionina tRNA Ligase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray
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