RESUMO
Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin's transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel's conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Bicamadas Lipídicas , Membranas/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Toxinas Biológicas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Quitosana/farmacologia , Humanos , Células Jurkat , Potenciais da Membrana , Membranas/metabolismo , Membranas/patologia , Faloidina/metabolismo , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Propídio/metabolismo , Toxinas Biológicas/toxicidadeRESUMO
Graphene foam holds promise for tissue engineering applications. In this study, graphene foam was used as a three-dimension scaffold to evaluate cell attachment, cell morphology, and molecular markers of early differentiation. The aim of this study was to determine if cell attachment and elaboration of an extracellular matrix would be modulated by functionalization of graphene foam with fibronectin, an extracellular matrix protein that cells adhere well to, prior to the establishment of three-dimensional cell culture. The molecular dynamic simulation demonstrated that the fibronectin-graphene interaction was stabilized predominantly through interaction between the graphene and arginine side chains of the protein. Quasi-static and dynamic mechanical testing indicated that fibronectin functionalization of graphene altered the mechanical properties of graphene foam. The elastic strength of the scaffold increased due to fibronectin, but the viscoelastic mechanical behavior remained unchanged. An additive effect was observed in the mechanical stiffness when the graphene foam was both coated with fibronectin and cultured with cells for 28 days. Cytoskeletal organization assessed by fluorescence microscopy demonstrated a fibronectin-dependent reorganization of the actin cytoskeleton and an increase in actin stress fibers. Gene expression assessed by quantitative real-time polymerase chain reaction of 9 genes encoding cell attachment proteins (Cd44, Ctnna1, Ctnnb1, Itga3, Itga5, Itgav, Itgb1, Ncam1, Sgce), 16 genes encoding extracellular matrix proteins (Col1a1, Col2a1, Col3a1, Col5a1, Col6a1, Ecm1, Emilin1, Fn1, Hapln1, Lamb3, Postn, Sparc, Spp1, Thbs1, Thbs2, Tnc), and 9 genes encoding modulators of remodeling (Adamts1, Adamts2, Ctgf, Mmp14, Mmp2, Tgfbi, Timp1, Timp2, Timp3) indicated that graphene foam provided a microenvironment conducive to expression of genes that are important in early chondrogenesis. Functionalization of graphene foam with fibronectin modified the cellular response to graphene foam, demonstrated by decreases in relative gene expression levels. These findings illustrate the combinatorial factors of microscale materials properties and nanoscale molecular features to consider in the design of three-dimensional graphene scaffolds for tissue engineering applications.
Assuntos
Condrócitos/citologia , Condrogênese , Fibronectinas/metabolismo , Grafite/química , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Animais , Adesão Celular , Moléculas de Adesão Celular , Técnicas de Cultura de Células , Condrócitos/metabolismo , Matriz Extracelular/química , Fibronectinas/química , CamundongosRESUMO
Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD), the exact mechanism by which apoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of apoE4 (nApoE41-151) localizes within the nucleus of microglia in the human AD brain, suggesting a potential role in gene expression. In the present study, we investigated this possibility utilizing BV2 microglia cells treated exogenously with nApoE41-151. The results indicated that nApoE41-151 leads to morphological activation of microglia cells through, at least in part, the downregulation of a novel ER-associated protein, CXorf56. Moreover, treatment of BV2 cells with nApoE41-151 resulted in a 68-fold increase in the expression of the inflammatory cytokine, TNFα, a key trigger of microglia activation. In this regard, we also observed a specific binding interaction of nApoE41-151 with the TNFα promoter region. Collectively, these data identify a novel gene-regulatory pathway involving CXorf56 that may link apoE4 to microglia activation and inflammation associated with AD.
Assuntos
Apolipoproteína E4/metabolismo , Regulação da Expressão Gênica , Microglia/fisiologia , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apolipoproteína E4/genética , Astrócitos/citologia , Astrócitos/fisiologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Camundongos , Microglia/citologia , Fragmentos de Peptídeos/genética , Fatores de Transcrição/genéticaRESUMO
Many promising attributes of ZnO nanoparticles (nZnO) have led to their utilization in numerous electronic devices and biomedical technologies. nZnO fabrication methods can create a variety of intrinsic defects that modulate the properties of nZnO, which can be exploited for various purposes. Here we developed a new synthesis procedure that controls certain defects in pure nZnO that are theorized to contribute to the n-type conductivity of the material. Interestingly, this procedure created defects that reduced the nanoparticle band gap to â¼3.1 eV and generated strong emissions in the violet to blue region while minimizing the defects responsible for the more commonly observed broad green emissions. Several characterization techniques including thermogravimetric analysis, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, Raman, photoluminescence, and inductively coupled plasma mass spectrometry were employed to verify the sample purity, assess how modifications in the synthesis procedure affect the various defects states, and understand how these alterations impact the physical properties. Since the band gap significantly decreased and a relatively narrow visible emissions band was created by these defects, we investigated utilizing these new nZnO for bioimaging applications using traditional fluorescent microscopy techniques. Although most nZnO generally require UV excitation sources to produce emissions, we demonstrate that reducing the band gap allows for a 405 nm laser to sufficiently excite the nanoparticles to detect their emissions during live-cell imaging experiments using a confocal microscope. This work lays the foundation for the use of these new nZnO in various bioimaging applications and enables researchers to investigate the interactions of pure nZnO with cells through fluorescence-based imaging techniques.
Assuntos
Nanopartículas/química , Óxido de Zinco , Humanos , Células Jurkat , Microscopia de Fluorescência , Espectroscopia Fotoeletrônica , Espectroscopia de Infravermelho com Transformada de Fourier , Óxido de Zinco/química , Óxido de Zinco/farmacologiaRESUMO
Ligament wound healing involves the proliferation of a dense and disorganized fibrous matrix that slowly remodels into scar tissue at the injury site. This remodeling process does not fully restore the highly aligned collagen network that exists in native tissue, and consequently repaired ligament has decreased strength and durability. In order to identify treatments that stimulate collagen alignment and strengthen ligament repair, there is a need to develop in vitro models to study fibroblast activation during ligament wound healing. The objective of this study was to measure gene expression and matrix protein accumulation in fibroblast-collagen gels that were subjected to different static stress conditions (stress-free, biaxial stress, and uniaxial stress) for three time points (1, 2 or 3 weeks). By comparing our in vitro results to prior in vivo studies, we found that stress-free gels had time-dependent changes in gene expression (col3a1, TnC) corresponding to early scar formation, and biaxial stress gels had protein levels (collagen type III, decorin) corresponding to early scar formation. This is the first study to conduct a targeted evaluation of ligament healing biomarkers in fibroblast-collagen gels, and the results suggest that biomimetic in-vitro models of early scar formation should be initially cultured under biaxial stress conditions.
Assuntos
Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Ligamentos , Modelos Biológicos , Cicatrização , Animais , Matriz Extracelular/patologia , Fibroblastos/patologia , Géis , Ligamentos/lesões , Ligamentos/metabolismo , Ligamentos/patologia , Camundongos , Células NIH 3T3RESUMO
Ligament mechanical behavior is primarily regulated by fibrous networks of type I collagen. Although these fibrous networks are typically highly aligned, healthy and injured ligament can also exhibit disorganized collagen architecture. The objective of this study was to determine whether variations in the collagen fibril network between neighboring ligaments can predict observed differences in mechanical behavior. Ligament specimens from two regions of bovine fetlock joints, which either exhibited highly aligned or disorganized collagen fibril networks, were mechanically tested in uniaxial tension. Confocal microscopy and FiberFit software were used to quantify the collagen fibril dispersion and mean fibril orientation in the mechanically tested specimens. These two structural parameters served as inputs into an established hyperelastic constitutive model that accounts for a continuous distribution of planar fibril orientations. The ability of the model to predict differences in the mechanical behavior between neighboring ligaments was tested by (1) curve fitting the model parameters to the stress response of the ligament with highly aligned fibrils and then (2) using this model to predict the stress response of the ligament with disorganized fibrils by only changing the parameter values for fibril dispersion and mean fibril orientation. This study found that when using parameter values for fibril dispersion and mean fibril orientation based on confocal imaging data, the model strongly predicted the average stress response of ligaments with disorganized fibrils ([Formula: see text]); however, the model only successfully predicted the individual stress response of ligaments with disorganized fibrils in half the specimens tested. Model predictions became worse when parameters for fibril dispersion and mean fibril orientation were not based on confocal imaging data. These findings emphasize the importance of collagen fibril alignment in ligament mechanics and help advance a mechanistic understanding of fibrillar networks in healthy and injured ligament.
Assuntos
Colágenos Fibrilares/metabolismo , Ligamentos/fisiologia , Modelos Biológicos , Animais , Fenômenos Biomecânicos , Bovinos , Imageamento Tridimensional , Ligamentos/anatomia & histologia , Ratos , Estresse Mecânico , Resistência à TraçãoRESUMO
Zinc oxide nanoparticles (nZnO) are one of the most highly produced nanomaterials and are used in numerous applications including cosmetics and sunscreens despite reports demonstrating their cytotoxicity. Dissolution is viewed as one of the main sources of nanoparticle (NP) toxicity; however, dissolution studies can be time-intensive to perform and complicated by issues such as particle separation from solution. Our work attempts to overcome some of these challenges by utilizing new methods using UV/vis and fluorescence spectroscopy to quantitatively assess nZnO dissolution in various biologically relevant solutions. All biological buffers tested induce rapid dissolution of nZnO. These buffers, including HEPES, MOPS, and PIPES, are commonly used in cell culture media, cellular imaging solutions, and to maintain physiological pH. Additional studies using X-ray diffraction, FT-IR, X-ray photoelectron spectroscopy, ICP-MS, and TEM were performed to understand how the inclusion of these nonessential media components impacts the behavior of nZnO in RPMI media. From these assessments, we demonstrate that HEPES causes increased dissolution kinetics, boosts the conversion of nZnO into zinc phosphate/carbonate, and, interestingly, alters the structural morphology of the complex precipitates formed with nZnO in cell culture conditions. Cell viability experiments demonstrated that the inclusion of these buffers significantly decrease the viability of Jurkat leukemic cells when challenged with nZnO. This work demonstrates that biologically relevant buffering systems dramatically impact the dynamics of nZnO including dissolution kinetics, morphology, complex precipitate formation, and toxicity profiles.
Assuntos
Meios de Cultura/química , Nanopartículas Metálicas/química , Óxido de Zinco/química , Soluções Tampão , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Células Jurkat , Cinética , Espectrometria de Massas , Nanopartículas Metálicas/toxicidade , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Espectroscopia Fotoeletrônica , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Although harboring the apolipoprotein E4 (APOE4) allele is a well known risk factor in Alzheimer's disease (AD), the mechanism by which it contributes to disease risk remains elusive. To investigate the role of proteolysis of apoE4 as a potential mechanism, we designed and characterized a site-directed cleavage antibody directed at position D151 of the mature form of apoE4 and E3. Characterization of this antibody indicated a high specificity for detecting synthesized recombinant proteins corresponding to the amino acid sequences 1-151 of apoE3 and E4 that would generate the 17 kDa (p17) fragment. In addition, this antibody also detected a ~17 kDa amino-terminal fragment of apoE4 following incubation with collagenase and matrix metalloproteinase-9 (MMP-9), but did not react with full-length apoE4. Application of this amino-terminal apoE cleavage-fragment (nApoECFp17) antibody, revealed nuclear labeling within glial cells and labeling of a subset of neurofibrillary tangles in the human AD brain. A quantitative analysis indicated that roughly 80% of labeled nuclei were microglia. To confirm these findings, cultured BV2 microglia cells were incubated with the amino-terminal fragment of apoE4 corresponding to the cleavage site at D151. The results indicated efficient uptake of this fragment and trafficking to the nucleus that also resulted in significant cell death. In contrast, a similarly designed apoE3 fragment showed no toxicity and primarily localized within the cytoplasm. These data suggest a novel cleavage event by which apoE4 is cleaved by the extracellular proteases, collagenase and MMP-9, generating an amino-terminal fragment that is then taken up by microglia, traffics to the nucleus and promotes cell death. Collectively, these findings provide important mechanistic insights into the mechanism by which harboring the APOE4 allele may elevate dementia risk observed in AD.
RESUMO
The mechanical behavior of soft connective tissue is governed by a dense network of fibrillar proteins in the extracellular matrix. Characterization of this fibrous network requires the accurate extraction of descriptive structural parameters from imaging data, including fiber dispersion and mean fiber orientation. Common methods to quantify fiber parameters include fast Fourier transforms (FFT) and structure tensors; however, information is limited on the accuracy of these methods. In this study, we compared these two methods using test images of fiber networks with varying topology. The FFT method with a band-pass filter was the most accurate, with an error of [Formula: see text] in measuring mean fiber orientation and an error of [Formula: see text] in measuring fiber dispersion in the test images. The accuracy of the structure tensor method was approximately five times worse than the FFT band-pass method when measuring fiber dispersion. A free software application, FiberFit, was then developed that utilizes an FFT band-pass filter to fit fiber orientations to a semicircular von Mises distribution. FiberFit was used to measure collagen fibril organization in confocal images of bovine ligament at magnifications of [Formula: see text] and [Formula: see text]. Grayscale conversion prior to FFT analysis gave the most accurate results, with errors of [Formula: see text] for mean fiber orientation and [Formula: see text] for fiber dispersion when measuring confocal images at [Formula: see text]. By developing and validating a software application that facilitates the automated analysis of fiber organization, this study can help advance a mechanistic understanding of collagen networks and help clarify the mechanobiology of soft tissue remodeling and repair.
Assuntos
Tecido Conjuntivo/anatomia & histologia , Software , Animais , Bovinos , Análise de Fourier , Processamento de Imagem Assistida por Computador , Reprodutibilidade dos Testes , Estresse MecânicoRESUMO
INTRODUCTION: Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology, but also maintain the gene expression characteristics of primary articular chondrocytes. METHODS: Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. RESULTS: Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 d. DISCUSSION: Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Condrócitos/citologia , Animais , Bovinos , Expressão Gênica , Microscopia de Fluorescência , RotaçãoRESUMO
Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer's disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick's disease, is not well established. To examine a possible role of apoE in Pick's disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick's disease.
Assuntos
Apolipoproteínas E/metabolismo , Região CA1 Hipocampal , Giro Denteado , Doença de Pick , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Giro Denteado/metabolismo , Giro Denteado/patologia , Humanos , Masculino , Doença de Pick/metabolismo , Doença de Pick/patologiaRESUMO
Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen α1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen α1(XI). MS was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen α1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration.
Assuntos
Cartilagem/química , Colágeno Tipo XI/análise , Colágeno Tipo XI/química , Proteômica/métodos , Sequência de Aminoácidos , Animais , Cartilagem/metabolismo , Cartilagem/fisiologia , Bovinos , Colágeno Tipo XI/metabolismo , Matriz Extracelular/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
The Beclin-1 protein is essential for the initiation of autophagy, and recent studies suggest this function may be compromised in Alzheimer's disease (AD). In addition, in vitro studies have supported a loss of function of Beclin-1 due to proteolytic modification by caspases. In the present study, we examined whether caspase-cleavage of Beclin-1 occurs in the AD brain by designing a site-directed caspase-cleavage antibody based upon a known cleavage site within the protein at position D149. We confirmed that Beclin-1 is an excellent substrate for caspase-3 and demonstrates cleavage led to the formation of a 35-kDa C-terminal fragment labeled by our novel antibody following Western blot analysis. Application of this antibody termed Beclin-1 caspase-cleavage product antibody or BeclinCCP in frontal cortex tissue sections revealed strong immunolabeling within astrocytes that localized with plaque regions and along blood vessels in all AD cases examined. In addition, weaker, more variable BeclinCCP labeling was also observed within neurofibrillary tangles that colocalized with the early tau conformational marker, MC-1 as well as the late tangle marker, PHF-1. Collectively, these data support a depletion of Beclin-1 in AD following caspase-cleavage. This article is part of a Special Issue entitled "Autophagy and protein degradation in neurological diseases."
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Encéfalo/metabolismo , Encéfalo/patologia , Caspase 3/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/enzimologia , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Astrócitos/patologia , Autofagia/genética , Proteína Beclina-1 , Encéfalo/enzimologia , Feminino , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteólise , Especificidade por Substrato/genéticaRESUMO
Minor fibrillar collagens are recognized as the organizers and nucleators during collagen fibrillogenesis but likely serve additional functions. The minor fibrillar collagens include collagens type V and XI. Mutations of collagens type V and XI can cause Ehlers-Danlos, Stickler's, and Marshall's syndromes in human. We have characterized the spatiotemporal expression patterns of Col11a1, Col11a2, Col5a1 as well as Col5a3 in zebrafish embryos by in situ hybridization. Col5a1 is expressed in developing somites, neural crest, the head mesenchyme, developing cranial cartilage, pharyngeal arches and vertebrae. Col5a3 is detected in the notochord, mesenchyme cells in the eyes and lens. Both Col11a1 and Col11a2 have similar expression patterns, including notochord, otic vesicle, and developing cranial cartilages. Zebrafish may therefore serve as a valuable vertebrate model system for the study of diseases associated with collagens type V and XI mutations.
Assuntos
Colágenos Fibrilares/genética , Colágenos Fibrilares/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Região Branquial/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Olho/embriologia , Olho/metabolismo , Colágenos Fibrilares/química , Hibridização In Situ , Mesoderma/citologia , Crista Neural/metabolismo , Notocorda/metabolismo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Somitos/metabolismo , Coluna Vertebral/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Collagen is a major component of the newly synthesized pericellular microenvironment of chondrocytes. Collagen types II, IX, and XI are synthesized and assembled into higher ordered complexes by a mechanism in which type XI collagen plays a role in nucleation of new fibrils, and in limiting fibril diameter. This study utilizes a cell line derived from the Swarm rat chondrosarcoma that allows the accumulation and assembly of pericellular matrix. Immunofluorescence and atomic force microscopy were used to assess early intermediates of fibril formation. Results indicate that this cell line synthesizes and secretes chondrocyte-specific pericellular matrix molecules including types II, IX, and XI collagen and is suitable for the study of newly synthesized collagen matrix under the experimental conditions used. AFM data indicate that small fibrils or assemblies of microfibrils are detectable and may represent precursors of the approximately 20 nm thin fibrils reported in cartilage. Treatment with hyaluronidase indicates that the dimensions of the small fibrils may be dependent upon the presence of hyaluronan within the matrix. This study provides information on the composition and organization of the newly synthesized extracellular matrix that plays a role in establishing the material properties and performance of biological materials such as cartilage.
Assuntos
Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo IX/metabolismo , Colágeno Tipo XI/metabolismo , Matriz Extracelular/metabolismo , Animais , Linhagem Celular Tumoral , Condrócitos/citologia , Colágeno Tipo II/ultraestrutura , Colágeno Tipo IX/ultraestrutura , Colágeno Tipo XI/ultraestrutura , Matriz Extracelular/química , Hialuronoglucosaminidase/metabolismo , Microfibrilas/metabolismo , Microfibrilas/ultraestrutura , Microscopia de Força Atômica , Fenótipo , RatosRESUMO
The amino terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms alpha1(XI) NTD[p7] and alpha1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis.
Assuntos
Processamento Alternativo/fisiologia , Colágeno Tipo XI/metabolismo , Expressão Gênica , Dobramento de Proteína , Animais , Sequência de Bases , Dicroísmo Circular , Colágeno Tipo XI/química , Colágeno Tipo XI/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/fisiologia , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Collagen type XI is a constituent of the pericellular matrix of chondrocytes and plays a role in the regulation of fibrillogenesis. The amino-terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that has been identified on the surface of cartilage collagen fibrils. The biochemical composition of the amino-terminal domain varies due to alternative splicing of the primary transcript. Recombinantly expressed alpha1(XI) aminoterminal domain isoforms were used in this study to investigate potential interactions. Purified products were analyzed for heparan sulfate binding properties. The results demonstrated that two additional binding sites exist within the alpha1(XI) aminoterminal domain, one within the amino propeptide and one within the variable region of the amino-terminal domain. Analysis of relative affinities indicated that the site located within the amino propeptide (site 1) was of similar affinity to sites that exist within the major triple helix of collagen type XI. Substitution of amino acid residues 147 to 152 within the amino propeptide by site-directed mutagenesis resulted in altered affinity for heparan sulfate. The binding site located within the variable region (site 2) demonstrated significantly higher affinity than other sites within the molecule. Displacement of collagen type XI within the pericellular matrix was observed in cell culture in the presence of excess heparan sulfate and by treatment with heparinase. These studies suggest two additional binding sites located within the noncollagenous amino-terminal domain that may play a role in the function of collagen type XI. The localization of collagen type XI within the pericellular matrix may be dependent upon interactions with heparan sulfate proteoglycans, and these are likely to take place in an isoform-specific manner.
Assuntos
Colágeno Tipo XI/química , Heparitina Sulfato/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Colágeno/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Dobramento de Proteína , Isoformas de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RatosRESUMO
The Staphylococcus aureus cidABC and lrgAB operons have been shown to regulate murein hydrolase activity and affect antibiotic tolerance. The cid operon enhances murein hydrolase activity and antibiotic sensitivity, whereas the lrg operon inhibits these processes. Based on these findings and the structural similarities of the cidA and lrgA gene products to the bacteriophage holin family of proteins, we have proposed that the cid and lrg operons encode holin- and antiholin-like proteins, respectively, that function to control the murein hydrolase activity produced by the bacteria. Analysis of cid operon transcription revealed the presence of two transcripts, one spanning all three cid genes and whose expression is induced by growth in the presence of acetic acid and the other spanning cidB and cidC only that is produced in a sigma B-dependent manner. The cidABC operon lies immediately downstream from the cidR gene, encoding a potential LysR-type transcriptional regulator. In this study, we demonstrate that cidR is involved in the regulation of cidABC expression. Northern blot analyses revealed that the cidR gene product positively regulates cidABC expression by increasing transcription in the presence of acetic acid produced as a result of the metabolism of glucose. As expected for an operon that encodes a positive effector of murein hydrolase activity, the upregulation of cidABC expression resulted in increased murein hydrolase activity produced by these cells. Furthermore, it was demonstrated that antibiotic tolerance and stationary-phase survival of S. aureus are affected by the cidR gene. Taken together, these results demonstrate that the cidR gene product functions as a transcriptional activator of cidABC transcription in response to acetic acid accumulation in the growth medium.
Assuntos
Óperon/genética , Staphylococcus aureus/genética , Sequência de Bases , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos , Cinética , Dados de Sequência Molecular , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Regulation of gene expression is critical to the proper development of neuronal cells. The methyl-CpG binding protein 2 (MeCP2) operates as a transcriptional repressor by facilitating histone deacetylation and DNA methylation-dependent transcriptional silencing. This study examined the importance of MeCP2 in the regulation of neurite formation in PC12 cells. Expression of MeCP2 increased in a time-dependent manner after induction of neuronal differentiation. Expression was assessed at both the transcriptional and translation levels, and reached a maximum at 24 h post-induction. In addition, a marked inhibition of neurite extension and proper localization of a marker for synapse formation, synapsin I, were observed when MeCP2 expression was decreased by the addition of an antisense morpholino oligomer directed to the translational initiation site for MeCP2beta. The removal of the antisense oligomer allowed neurite extension to progress. However, the addition of antisense oligomer to previously differentiated PC12 cells did not affect established neurite processes. Taken collectively, our results indicate a role for MeCP2beta early in the events of neurite formation and that the relative levels of MeCP2alpha and MeCP2beta may be different in early differentiating neurons than is found in the adult brain. In addition, unique functions may exist for the two isoforms of MeCP2. Our results indicate that the inhibition of neurite elaboration caused by a reduction in MeCP2 may be reversible.