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1.
J Biol Chem ; 294(51): 19486-19497, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31690623

RESUMO

Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαß) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gtα) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαß in its nonactivated state. Following reconstitution of PDE6 and activated Gtα with liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα-binding sites per holoenzyme. Each Gtα interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.

2.
J Mol Biol ; 431(19): 3677-3689, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31394113

RESUMO

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαßγ2) is composed of a catalytic heterodimer (Pαß) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pß) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαß, Pαß-cGMP, Pαßγ2, and Pαßγ2-cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαß catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.

3.
ACS Appl Mater Interfaces ; 10(24): 20296-20305, 2018 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-29808989

RESUMO

Repairing osteochondral defect (OCD) using advanced biomaterials that structurally, biologically, and mechanically fulfill the criteria for stratified tissue regeneration remains a significant challenge for researchers. Here, a multilayered scaffold (MLS) with hierarchical organization and heterogeneous composition is developed to mimic the stratified structure and complex components of natural osteochondral tissues. Specifically, the intermediate compact interfacial layer within the MLS is designed to resemble the osteochondral interface to realize the closely integrated layered structure. Subsequently, macroscopic observations, histological evaluation, and biomechanical and biochemical assessments are performed to evaluate the ability of the MLS of repairing OCD in a goat model. By 48 weeks postimplantation, superior hyalinelike cartilage and sound subchondral bone are observed in the MLS group. Furthermore, the biomimetic MLS significantly enhances the biomechanical and biochemical properties of the neo-osteochondral tissue. Taken together, these results confirm the potential of this optimized MLS as an advanced strategy for OCD repair.


Assuntos
Condrócitos , Materiais Biocompatíveis , Osso e Ossos , Cartilagem Articular , Tecidos Suporte , Cicatrização
4.
J Biosci Bioeng ; 126(3): 389-396, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29685821

RESUMO

Treating full-layer injury of bone and cartilage is currently a significant challenge in orthopedic trauma repair. Joint damage typically includes chondral defects, and the underlying subchondral defect sites are difficult to repair. Tissue engineering technology could potentially be used to treat such injuries; however, results to date been unsatisfactory. The aim of this study was to design a multilayer composite scaffold containing cartilage, bone, and calcified layers to simulate physiological full-thickness bone-cartilage structure. The cartilage layer was created using an improved temperature-gradient thermally induced crystallization technology. The bone and calcified layers were synthesized using 3D printing technology. We examined the scaffold by using scanning electron microscope (SEM), X-ray diffraction (XRD), fluorescence staining, and micro computed tomography (Micro-CT), and observed clearly oriented structures in the cartilage layer, overlapping structures in the bone scaffold, and a compressed calcified layer. Biomechanical performance testing showed that the scaffolds were significantly stronger than scaffolds without a calcified layer (traditional scaffolds) in maximum tensile strength and maximum shear strength (P < 0.05). After inoculating cells onto the scaffolds, we observed similar cell adherence and proliferation to that observed in traditional scaffolds, likely because of the high porosity of the whole scaffold. Our scaffolds could be used in bone and cartilage full-thickness injury repair methods, as well as applications in the field of tissue engineering.


Assuntos
Osso e Ossos/citologia , Cálcio/química , Cartilagem Articular/citologia , Cartilagem/citologia , Técnicas de Cultura de Células , Engenharia Tecidual , Tecidos Suporte/química , Animais , Osso e Ossos/fisiologia , Cartilagem/fisiologia , Cartilagem Articular/fisiologia , Bovinos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Proliferação de Células , Forma Celular , Células Cultivadas , Masculino , Teste de Materiais , Fenômenos Mecânicos , Porosidade , Impressão Tridimensional , Propriedades de Superfície , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Microtomografia por Raio-X
5.
Waste Manag ; 67: 171-180, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28551279

RESUMO

Due to the high cost of pure CO2, carbonation of MSWI fly ash has not been fully developed. It is essential to select a kind of reaction gas with rich CO2 instead of pure CO2. The CO2 uptake and leaching toxicity of heavy metals in three typical types of municipal solid waste incinerator (MSWI) fly ash were investigated with simulated oxy-fuel combustion flue gas under different reaction temperatures, which was compared with both pure CO2 and simulated air combustion flue gas. The CO2 uptake under simulated oxy-fuel combustion flue gas were similar to that of pure CO2. The leaching concentration of heavy metals in all MSWI fly ash samples, especially in ash from Changzhou, China (CZ), decreased after carbonation. Specifically, the leached Pb concentration of the CZ MSWI fly ash decreased 92% under oxy-fuel combustion flue gas, 95% under pure CO2 atmosphere and 84% under the air combustion flue gas. After carbonation, the leaching concentration of Pb was below the Chinese legal limit. The leaching concentration of Zn from CZ sample decreased 69% under oxy-fuel combustion flue gas, which of Cu, As, Cr and Hg decreased 25%, 33%, 11% and 21%, respectively. In the other two samples of Xuzhou, China (XZ) and Wuhan, China (WH), the leaching characteristics of heavy metals were similar to the CZ sample. The speciation of heavy metals was largely changed from the exchangeable to carbonated fraction because of the carbonation reaction under simulated oxy-fuel combustion flue gas. After carbonation reaction, most of heavy metals bound in carbonates became more stable and leached less. Therefore, oxy-fuel combustion flue gas could be a low-cost source for carbonation of MSWI fly ash.


Assuntos
Incineração , Metais Pesados , Resíduos Sólidos , Carbono , China , Cinza de Carvão , Material Particulado
6.
Biofabrication ; 9(2): 025021, 2017 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-28462906

RESUMO

Integrative osteochondral repair is a useful strategy for cartilage-defect repair. To mimic the microenvironment, it is necessary that scaffolds effectively mimic the extracellular matrix of natural cartilage and subchondral bone. In this study, biomimetic osteochondral scaffolds containing an oriented cartilage layer, a compact layer, and a three-dimensional (3D)-printed core-sheath structured-bone layer were developed. The oriented cartilage layer was designed to mimic the structural and material characteristics of native cartilage tissue and was fabricated with cartilage matrix-chitosan materials, using thermal-induced phase-separation technology. The 3D-printed core-sheath structured-bone layer was fabricated with poly(L-lactide-co-glycolide)/ß-tricalcium phosphate-collagen materials by low-temperature deposition technology, using a specially designed core-sheath nozzle, and was designed to mimic the mechanical characteristics of subchondral bone and improve scaffold hydrophilicity. The compact layer was designed to mimic the calcified-layer structure of natural cartilage to ensure the presence of different suitable microenvironments for the regeneration of bone and cartilage. A dissolving-bonding process was developed to effectively combine the three parts together, after which the bone and cartilage scaffolds exhibited good mechanical properties and hydrophilicity. Additionally, goat autologous bone mesenchymal stem cells (BMSCs) were isolated and then seeded into the bone and cartilage layers, respectively, and following a 1 week culture in vitro, the BMSC-scaffold constructs were implanted into a goat articular-defect model. Our results indicated that the scaffolds exhibited good biocompatibility, and 24 weeks after implantation, the femoral condyle surface was relatively flat and consisted of a large quantity of hyaloid cartilage. Furthermore, histological staining revealed regenerated trabecular bone formed in the subchondral bone-defect area. These results provided a new method to fabricate biomimetic osteochondral scaffolds and demonstrated their effectiveness for future clinical applications in cartilage-defect repair.


Assuntos
Biomimética/métodos , Osso e Ossos/citologia , Cartilagem Articular/citologia , Células-Tronco Mesenquimais/citologia , Temperatura Ambiente , Tecidos Suporte/química , Animais , Fenômenos Biomecânicos , Cartilagem Articular/fisiologia , Cartilagem Articular/ultraestrutura , Forma Celular , Módulo de Elasticidade , Cabras , Interações Hidrofóbicas e Hidrofílicas , Regeneração , Cicatrização
7.
Nanotechnology ; 28(11): 114003, 2017 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-28103586

RESUMO

Gallium nitride-based nanopyramid light-emitting diodes are a promising technology to achieve highly efficient solid-state lighting and beyond. Here, periodic nanopyramid light-emitting diode arrays on gallium nitride/sapphire templates were fabricated by selective-area metalorganic chemical vapor deposition and multiple-exposure colloidal lithography. The electric field intensity distribution of incident light going through polystyrene microspheres and photoresist are simulated using finite-different time-domain method. Nitrogen as the carrier gas and a low V/III ratio (ratio of molar flow rate of group-V to group-III sources) are found to be important in order to form gallium nitride nanopyramid. In addition, a broad yellow emission in photoluminescence and cathodoluminescence spectra were observed. This phenomena showed the potential of nanopyramid light-emitting diodes to realize long wavelength visible emissions.

8.
Phys Chem Chem Phys ; 18(19): 13186-95, 2016 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-26852874

RESUMO

It is known that the combination of TiO2 and graphene and the control of TiO2 crystal facets are both effective routes to improve the photocatalytic performance of TiO2. Here, we report the synthesis and the photocatalytic CO2 reduction performance of graphene supported TiO2 nanocrystals with coexposed {001} and {101} facets (G/TiO2-001/101). The combination of TiO2 and graphene enhanced the crystallinity of TiO2 single nanocrystals and obviously improved their dispersion on graphene. The "surface heterojunction" formed by the coexposed {001} and {101} facets can promote the spatial separation of photogenerated electrons and holes toward different facets and the supports of graphene can further enhance the separation through accelerated electron migration from TiO2 to graphene. The G/TiO2-001/101 exhibited high photocatalytic CO2-reduction activity with a maximum CO yield reaching 70.8 µmol g(-1) h(-1). The enhanced photocatalytic activity of the composites can be attributed to their high surface area, good dispersion of TiO2 nanoparticles, and effective separation of excited charges due to the synergy of graphene supports and the co-exposure of {001} and {101} facets.

9.
Opt Express ; 24(2): A44-51, 2016 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-26832596

RESUMO

Ni/Au electrodes with single, twined and triplet hole array patterns light-emitting diodes have been fabricated by multiple-exposure colloidal lithography. It is found that 45.6%, 83.6% and 15.5% improvement in light output at 350 mA has been achieved by patterning Ni/Au electrodes with single, twined, triplet hole arrays. In addition, patterned Ni/Au LEDs possess much larger view angles than non-patterned ones due to scattering effects of light around the holes, especially for triplet hole array patterned Ni/Au LEDs. Our proposed method for fabricating multiple holes structure would be very promising to improve light output power of LEDs when using advanced electrodes.

10.
PLoS One ; 10(12): e0145667, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26695629

RESUMO

Tissue engineering (TE) has been proven usefulness in cartilage defect repair. For effective cartilage repair, the structural orientation of the cartilage scaffold should mimic that of native articular cartilage, as this orientation is closely linked to cartilage mechanical functions. Using thermal-induced phase separation (TIPS) technology, we have fabricated an oriented cartilage extracellular matrix (ECM)-derived scaffold with a Young's modulus value 3 times higher than that of a random scaffold. In this study, we test the effectiveness of bone mesenchymal stem cell (BMSC)-scaffold constructs (cell-oriented and random) in repairing full-thickness articular cartilage defects in rabbits. While histological and immunohistochemical analyses revealed efficient cartilage regeneration and cartilaginous matrix secretion at 6 and 12 weeks after transplantation in both groups, the biochemical properties (levels of DNA, GAG, and collagen) and biomechanical values in the oriented scaffold group were higher than that in random group at early time points after implantation. While these differences were not evident at 24 weeks, the biochemical and biomechanical properties of the regenerated cartilage in the oriented scaffold-BMSC construct group were similar to that of native cartilage. These results demonstrate that an oriented scaffold, in combination with differentiated BMSCs can successfully repair full-thickness articular cartilage defects in rabbits, and produce cartilage enhanced biomechanical properties.


Assuntos
Cartilagem Articular/lesões , Cartilagem Articular/metabolismo , Teste de Materiais , Regeneração , Engenharia Tecidual , Tecidos Suporte , Animais , Cartilagem Articular/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Coelhos
11.
Opt Express ; 23(15): A957-65, 2015 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-26367696

RESUMO

In this paper, the high performance GaN-based light-emitting diodes (LEDs) on carbon-nanotube-patterned sapphire substrate (CNPSS) by metal-organic chemical vapor deposition (MOCVD) are demonstrated. By studying the mechanism of nucleation, we analyze the reasons of the crystal quality improvement induced by carbon nanotubes (CNTs) in different growth process. Combining with low temperatures photoluminescence (PL) measurements and two-dimensional (2D) finite difference time-domain (FDTD) simulation results, we conclude that the improvement of optical properties and electrical properties of CNPSS mainly originates from the improvement of the internal quantum efficiency (IQE) due to decreased dislocation density during nano-epitaxial growth on CNPSS. Additionally, in order to reduce the light absorption characteristics of CNTs, different time annealing under the oxygen environment is carried out to remove part of CNTs. Under 350 mA current injections, the light output power (LOP) of CNPSS-LED annealed 2 h and 10 h exhibit 11% and 6% enhancement, respectively, compared to that of the CNPSS-LED without annealing. Therefore, high temperature annealing can effectively remove parts of CNTs and further increase the LOP, while overlong annealing time has caused degradation of the quantum well resulting in the attenuation of optical power.

12.
Small ; 11(37): 4910-21, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26179658

RESUMO

Large-area polystyrene (PS) colloidal monolayers with high mechanical strength are created by a combination of the air/water interface self-assembly and the solvent vapor annealing technique. Layer-by-layer (LBL) stacking of these colloidal monolayers leads to the formation of (2+1)D photonic crystal superlattice with enhanced crystalline integrity. By manipulating the diameter of PS spheres and the repetition period of the colloidal monolayers, flexible control in structure and stop band position of the (2+1)D photonic crystal superlattice has been realized, which may afford new opportunities for engineering photonic bandgap materials. Furthermore, an enhancement of 97.3% on light output power of a GaN-based light emitting diode is demonstrated when such a (2+1)D photonic crystal superlattice employed as a back reflector. The performance enhancement is attributed to the photonic bandgap enhancement and good angle-independence of the (2+1)D photonic crystal superlattice.

13.
Opt Express ; 22 Suppl 5: A1284-91, 2014 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-25322183

RESUMO

We reported that the peak efficiency together with the efficiency droop in InGaN-based light emitting diodes could be effectively modified through a simple and low-cost etch-regrown process in n-GaN layer. The etched n-GaN template contained pyramid arrays with inclined side planes. The following lateral overgrowth process from the etched n-GaN template substantially reduced the edge dislocation density and residential compressive strain in epilayers. The efficiency droop of LED samples thus could be modified due to the reduced polarization field, resulting from the strain relaxation in epilayers. What is more, the peak efficiency and reverse current leakage were also modified due to the reduction of dislocations.

14.
J Mol Biol ; 426(22): 3713-3728, 2014 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-25149264

RESUMO

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αßγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αß), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for differential binding of the γ-subunit to the GAFa domains of the α- and ß-subunits are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Retina/enzimologia , Animais , Domínio Catalítico , Bovinos , Cromatografia Líquida , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Proteínas do Olho/genética , Modelos Moleculares , Fragmentos de Peptídeos/análise , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Retina/efeitos dos fármacos , Espectrometria de Massas em Tandem
15.
Artigo em Chinês | MEDLINE | ID: mdl-23879085

RESUMO

OBJECTIVE: To investigate the feasibility of fabricating an oriented scaffold combined with chondrogenic-induced bone marrow mesenchymal stem cells (BMSCs) for enhancement of the biomechanical property of tissue engineered cartilage in vivo. METHODS: Temperature gradient-guided thermal-induced phase separation was used to fabricate an oriented cartilage extracellular matrix-derived scaffold composed of microtubules arranged in parallel in vertical section. No-oriented scaffold was fabricated by simple freeze-drying. Mechanical property of oriented and non-oriented scaffold was determined by measurement of compressive modulus. Oriented and non-oriented scaffolds were seeded with chondrogenic-induced BMSCs, which were obtained from the New Zealand white rabbits. Proliferation, morphological characteristics, and the distribution of the cells on the scaffolds were analyzed by MTT assay and scanning electron microscope. Then cell-scaffold composites were implanted subcutaneously in the dorsa of nude mice. At 2 and 4 weeks after implantation, the samples were harvested for evaluating biochemical, histological, and biomechanical properties. RESULTS: The compressive modulus of oriented scaffold was significantly higher than that of non-oriented scaffold (t=201.099, P=0.000). The cell proliferation on the oriented scaffold was significantly higher than that on the non-oriented scaffold from 3 to 9 days (P < 0.05). At 4 weeks, collagen type II immunohistochemical staining, safranin O staining, and toluidine blue staining showed positive results in all samples, but negative for collagen type I. There were numerous parallel giant bundles of densely packed collagen fibers with chondrocyte-like cells on the oriented-structure constructs. Total DNA, glycosaminoglycan (GAG), and collagen contents increased with time, and no significant difference was found between 2 groups (P > 0.05). The compressive modulus of the oriented tissue engineered cartilage was significantly higher than that of the non-oriented tissue engineered cartilage at 2 and 4 weeks after implantation (P < 0.05). Total DNA, GAG, collagen contents, and compressive modulus in the 2 tissue engineered cartilages were significantly lower than those in normal cartilage (P < 0.05). CONCLUSION: Oriented extracellular matrix-derived scaffold can enhance the biomechanical property of tissue engineered cartilage and thus it represents a promising approach to cartilage tissue engineering.


Assuntos
Cartilagem Articular/citologia , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Fenômenos Biomecânicos , Células da Medula Óssea/citologia , Cartilagem Articular/metabolismo , Bovinos , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese , Colágeno/metabolismo , Matriz Extracelular/ultraestrutura , Estudos de Viabilidade , Feminino , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Coelhos
16.
PLoS One ; 8(1): e54838, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23382984

RESUMO

The structure of an osteochondral biphasic scaffold is required to mimic native tissue, which owns a calcified layer associated with mechanical and separation function. The two phases of biphasic scaffold should possess efficient integration to provide chondrocytes and osteocytes with an independent living environment. In this study, a novel biphasic scaffold composed of a bony phase, chondral phase and compact layer was developed. The compact layer-free biphasic scaffold taken as control group was also fabricated. The purpose of current study was to evaluate the impact of the compact layer in the biphasic scaffold. Bony and chondral phases were seeded with autogeneic osteoblast- or chondrocyte-induced bone marrow stromal cells (BMSCs), respectively. The biphasic scaffolds-cells constructs were then implanted into osteochondral defects of rabbits' knees, and the regenerated osteochondral tissue was evaluated at 3 and 6 months after surgery. Anti-tensile and anti-shear properties of the compact layer-containing biphasic scaffold were significantly higher than those of the compact layer-free biphasic scaffold in vitro. Furthermore, in vivo studies revealed superior macroscopic scores, glycosaminoglycan (GAG) and collagen content, micro tomograph imaging results, and histological properties of regenerated tissue in the compact layer-containing biphasic scaffold compared to the control group. These results indicated that the compact layer could significantly enhance the biomechanical properties of biphasic scaffold in vitro and regeneration of osteochondral tissue in vivo, and thus represented a promising approach to osteochondral tissue engineering.


Assuntos
Cartilagem Articular , Engenharia Tecidual , Tecidos Suporte , Animais , Materiais Biocompatíveis , Cartilagem Articular/anatomia & histologia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Separação Celular , Teste de Materiais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Coelhos , Radiografia
17.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi ; 30(6): 1283-9, 2013 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-24645613

RESUMO

Hela is the cell line of adenocarcinoma of the uterine cervix, and human papillomavirus (HPV) 18 shows positive. We delivered siRNA with target specifically to HPV18 E7 mRNA into nude mice Hela tumor xenografts by nanopatch to inhibit the HPV gene expression, and further to study the superiority, the best action time and concentration of siRNA of using nanopatch to transfer siRNA in vivo. We designed siRNA that target specifically to HPV18 E7 mRNA (siE7) and checked the effect of siE7 in vitro. Tumor xenografts were transfected with siE7 and GenEscort III by nanopatch. Expression of HPV18 E7 mRNA and protein were detected 0 hours, 24 hours, 48 hours, 72 hours after transfection with PT-PCR and Western blot, and the best action time was analyzed using nanopatch to thansfect siRNA in vivo. We transfected GenEscort III and siE7 of Different concentration into tumor xenografts respectively by nanopatch and intraperitoneal injection. Expression of HPV18 E7 mRNA and protein was detected 72 hours after transfection by PT-PCR and Western blot, to analyze the best action concentration of siRNA and the superiority of using nanopatch to thansfect siRNA in vivo. The results proved that SiE7was efficient to inhibit expression of HPV18 E7 mRNA and to advance Hela apoptosis in vitro. SiE7 transfected by nanopatch into xenografts could inhibit effectively expression of HPV18 E7 mRNA and protein. The best action time and concentration of siRNA of using nanopatch to thansfect siRNA in vivo are 72 hour post-transfection and 2 micromol/L siE7. To compare intraperitoneal injection in delivering siRNA in vivo, the effect of nanopatch is very predominant. It can be well concluded that Nanopatch can effectively transfer siRNA in vivo, which can effectively inhibit the HPV gene expression.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Nanoestruturas/administração & dosagem , Proteínas Oncogênicas Virais/genética , RNA Interferente Pequeno/administração & dosagem , Animais , Apoptose , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Nus , Papillomaviridae/genética , Transfecção , Neoplasias do Colo do Útero/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Biol Chem ; 287(31): 26312-20, 2012 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-22665478

RESUMO

The cGMP phosphodiesterase (PDE6) involved in visual transduction in photoreceptor cells contains two inhibitory γ-subunits (Pγ) which bind to the catalytic core (Pαß) to inhibit catalysis and stimulate cGMP binding to the GAF domains of Pαß. During visual excitation, interaction of activated transducin with Pγ relieves inhibition. Pγ also participates in a complex with RGS9-1 and other proteins to accelerate the GTPase activity of activated transducin. We studied the structural determinants for these important functions of Pγ. First, we identified two important sites in the middle region of Pγ (amino acids 27-38 and 52-54) that significantly stabilize the overall binding affinity of Pγ with Pαß. The ability of Pγ to stimulate noncatalytic cGMP binding to the GAF domains of PDE6 has been localized to amino acids 27-30 of Pγ. Transducin activation of PDE6 catalysis critically depends on the presence of Ile54 in the glycine-rich region of Pγ in order to relieve inhibition of catalysis. The central glycine-rich region of Pγ is also required for transducin to increase cGMP exchange at the GAF domains. Finally, Thr-65 and/or Val-66 of Pγ are critical residues for Pγ to stimulate GTPase activity of transducin in a complex with RGS9-1. We propose that the glycine-rich region of Pγ is a primary docking site for PDE6-interacting proteins involved in the activation/inactivation pathways of visual transduction. This functional mapping of Pγ with its binding partners demonstrates the remarkable versatility of this multifunctional protein and its central role in regulating the activation and lifetime of visual transduction.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Proteínas RGS/química , Transducina/química , Animais , Sítios de Ligação , Bovinos , GMP Cíclico/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Cistina/química , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Isoleucina/química , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Proteínas RGS/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/enzimologia , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Transducina/metabolismo
19.
J Biomed Mater Res A ; 100(4): 1044-50, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22318897

RESUMO

To investigate the potential application of bone marrow stromal cells (BMSCs) and an injectable sodium alginate/gelatin scaffold for bone tissue engineering (BTE). The phenotype of osteogenic BMSCs was examined by mineralized nodules formation and type I collagen expression. Cell proliferation was evaluated by MTT assay. The biocompatibility of scaffold and osteogenic cells were examined by hematoxylin and eosin (H&E) staining. Ectopic bone formation as well as closure of rabbit calvarial critical-sized defects following scaffold-cell implantation were analyzed by histological examination and computed tomography (CT) scanning. Spindle-shaped osteogenic cells of high purity were derived from BMSCs. The osteogenic cells and sodium alginate/gelatin (2:3) scaffold presented fine biocompatibility following cross-linking with 0.6% of CaCl(2). After implantation, the scaffold-cell construct promoted both ectopic bone formation and bone healing in the rabbit calvarial critical-sized defect model. Our data demonstrated that the sodium alginate/gelatin scaffold could be a suitable biomaterial for bone engineering, and the scaffold-osteogenic cells construct is a promising alternative approach for the bone healing process.


Assuntos
Alginatos/química , Células da Medula Óssea/citologia , Osso e Ossos , Gelatina/química , Células Estromais/citologia , Engenharia Tecidual , Animais , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Coelhos
20.
J Biosci Bioeng ; 113(5): 647-53, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22265894

RESUMO

The structure of a cartilage scaffold is required to mimic native articular cartilage, which has an oriented structure associated with its mechanical function. In this study, an oriented cartilage extracellular matrix (ECM)-derived scaffold was fabricated composed of microtubules arranged in parallel in vertical section. The mechanical property was higher than that of a typical non-oriented scaffold (p<0.05). Oriented and non-oriented scaffolds were seeded with chondrogenic-induced bone mesenchymal stem cells and cell-scaffold constructs were implanted subcutaneously in the dorsa of nude mice. At 4 weeks, all samples stained positive for safranin O, toluidine blue, and collagen type II, but negative for collagen type I. Oriented-structure constructs contained numerous parallel giant bundles of densely packed collagen fibers with chondrocyte-like cells aligned along the fibers. Total DNA, glycosaminoglycans and collagen contents increased with time and these values were similar in the two groups. Compared with the native articular cartilage, the Young's modulus of the tissue-engineered (TE) cartilage reached 42.9%, 23.0% in oriented and non-oriented scaffolds respectively, at 4 weeks. These results indicate that oriented ECM-derived scaffolds enhance the biomechanical property of TE cartilage and thus represent a promising approach to cartilage tissue engineering.


Assuntos
Matriz Extracelular/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Cartilagem Articular/citologia , Adesão Celular , Colágeno/análise , Colágeno/metabolismo , Matriz Extracelular/ultraestrutura , Glicosaminoglicanos/análise , Glicosaminoglicanos/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Coloração e Rotulagem
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