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1.
J Cell Mol Med ; 13(1): 188-201, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18373734

RESUMO

Abstract It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Embrionárias/transplante , Terapia de Imunossupressão , Infarto do Miocárdio/patologia , Animais , Linhagem da Célula , Ciclosporina/metabolismo , Células-Tronco Embrionárias/citologia , Humanos , Imunossupressores/metabolismo , Masculino , Camundongos , Miocárdio/citologia , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Teratoma/metabolismo , Teratoma/patologia , Transplante Heterólogo , Função Ventricular Esquerda
2.
Biomaterials ; 29(18): 2757-66, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18396331

RESUMO

We show that synthetic three-dimensional (3D) matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG)-based hydrogels can direct differentiation of pluripotent cardioprogenitors, using P19 embryonal carcinoma (EC) cells as a model, along a cardiac lineage in vitro. In order to systematically probe 3D matrix effects on P19 EC differentiation, matrix elasticity, MMP-sensitivity and the concentration of a matrix-bound RGDSP peptide were modulated. Soft matrices (E=322+/-64.2 Pa, stoichiometric ratio: 0.8), mimicking the elasticity of embryonic cardiac tissue, increased the fraction of cells expressing the early cardiac transcription factor Nkx2.5 around 2-fold compared to embryoid bodies (EB) in suspension. In contrast, stiffer matrices (E=4,036+/-419.6 Pa, stoichiometric ratio: 1.2) decreased the number of Nkx2.5-positive cells significantly. Further indicators of cardiac maturation were promoted by ligation of integrins relevant in early cardiac development (alpha(5)beta(1,) alpha(v)beta(3)) by the RGDSP ligand in combination with the MMP-sensitivity of the matrix, with a 6-fold increased amount of myosin heavy chain (MHC)-positive cells as compared to EB in suspension. This precisely controlled 3D culture system thus may serve as a potential alternative to natural matrices for engineering cardiac tissue structures for cell culture and potentially therapeutic applications.


Assuntos
Diferenciação Celular/fisiologia , Matriz Extracelular/fisiologia , Polietilenoglicóis/química , Engenharia Tecidual/métodos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Matriz Extracelular/química , Citometria de Fluxo , Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Imuno-Histoquímica , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Eur J Obstet Gynecol Reprod Biol ; 128(1-2): 103-7, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16442204

RESUMO

OBJECTIVE: To examine the Young's modulus of the human amniotic membranes, as well as its relationship to gestational age. To determine whether cellular and material-related parameters affect this modulus. STUDY DESIGN: In a prospective study at the Obstetric outpatient clinic of the University Hospital Zurich Young's modulus, thickness and mesenchymal:epithelial cell ratio of amniotic membranes of preterm (N=23) and term (N=40) placentae were examined. Significance (P<0.05) was calculated with the Mann-Whitney two-sample rank sum test and Wilcoxon signed rank test, while correlations were made using the Spearman's correlation. RESULTS: The Young's modulus of preterm amniotic membranes was significantly higher than that of term membranes. It varied within the same amniotic membrane. The thickness of the amnion in both preterm and term membranes did not differ significantly. The thinner the preterm and term amniotic membranes, the higher the Young's modulus was. There was no relation to the mesenchymal:epithelial cell ratio in the amnion. CONCLUSIONS: Preterm amniotic membranes are stiffer than term amniotic membranes. Tentatively, we hypothesise that there may be a correlation between the extracellular matrix components and the elastic properties of the membrane.


Assuntos
Âmnio/anatomia & histologia , Ruptura Prematura de Membranas Fetais/patologia , Idade Gestacional , Modelos Teóricos , Âmnio/anormalidades , Âmnio/citologia , Elasticidade , Feminino , Humanos , Placenta/anormalidades , Placenta/anatomia & histologia , Placenta/citologia , Gravidez , Prognóstico , Estudos Prospectivos
4.
Int J Biochem Cell Biol ; 37(3): 493-503, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15618004

RESUMO

During embryogenesis, endothelial progenitor cells participate in the initial processes of primitive blood vessel formation (vasculogenesis). It has become evident that progenitors to vascular endothelial cells also exist in the adult. Endothelial progenitors normally reside in the adult bone marrow but may become mobilized into circulation by cytokine or angiogenic growth factor signals from the periphery, enter extravascular tissue, and promote de novo vessel formation by virtue of physically integrating into vessels and/or supplying growth factors (adult vasculogenesis). For that reason, autologous endothelial progenitors, mobilized in situ or transplanted, has become a major target of therapeutic revascularization approaches to ischemic disease and endothelial injury. Moreover, endothelial progenitors represent a potential target of strategies to block tumor growth.


Assuntos
Células da Medula Óssea/fisiologia , Endotélio Vascular/citologia , Células-Tronco Hematopoéticas/fisiologia , Neovascularização Fisiológica , Adulto , Citocinas/fisiologia , Endotélio Vascular/fisiologia , Previsões , Humanos , Modelos Biológicos , Fatores de Crescimento do Endotélio Vascular/fisiologia
5.
J Control Release ; 101(1-3): 93-109, 2005 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-15588897

RESUMO

A number of vascular therapies could benefit from advanced methods for presentation of angiogenic growth factors, including growth of endothelium on small caliber vascular grafts and revascularization of ischemic tissue through induction of collateral vessels and microvessels. To explore methods to optimize the presentation and release of angiogenic factors in such applications in device integration and tissue repair, we studied three variant forms of vascular endothelial growth factor 121 (VEGF121), each with differential susceptibility to local cellular proteolytic activity, formulated within fibrin matrices. (1) The prototypic variant alpha2PI(1-8)-VEGF121 remains immobilized in fibrin matrices until its liberation by cell-associated enzymes, such as plasmin, that degrade the fibrin network [slow, cell-demanded release; J. Control. Release 72 (2001) 101-113]; the alpha2PI(1-8) domain serves as a site for covalent attachment to fibrin during coagulation. (2) We created a new VEGF variant, alpha2PI(1-8)-Pla-VEGF121 that couples to fibrin via a plasmin-sensitive sequence (Pla). Cleavage of this target site by plasmin enables direct release of alpha2PI(1-8)-Pla-VEGF121 from bulk matrix degradation (accelerated, cell-demanded release). (3) Native VEGF121 (burst, passive release) was considered as a reference. VEGF release profiles were determined experimentally as well as mathematically, alpha2PI(1-8)-Pla-VEGF121 being released ca. fourfold more quickly than alpha2PI(1-8)-VEGF121, both being retained compared to native VEGF121; the differences in release could be accounted for based on knowledge of the plasmin sensitivity of the bound growth factor and the structure of the fibrin network. The bound factors were competent in inducing endothelial cell proliferation, the matrix-bound forms being more effective than native VEGF121; as well as competent in inducing endothelial progenitor cell maturation into endothelial cells. These matrix-bound variants of VEGF121 may be particularly useful where retention in locally applied surgical sites is desired, such as prevention of washout from vascular graft coatings and slowing loss from tissue ingrowth matrices used in local tissue revascularization and repair.


Assuntos
Células Endoteliais/fisiologia , Fibrina/metabolismo , Células-Tronco/fisiologia , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Proliferação de Células , Sistemas de Liberação de Medicamentos , Fibrinolisina/farmacologia , Humanos , Matemática , Modelos Biológicos , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Curr Opin Biotechnol ; 15(5): 430-4, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15464373

RESUMO

New solutions are needed to regenerate hearts damaged by myocardial infarction, to overcome bad prognosis of patients with heart failure, and to address the shortage of heart donors. In the past few years, cardiac tissue engineering has emerged as a new and ambitious approach that combines knowledge from material chemistry with cell biology and medicine. In this short review, we present an overview on the most promising materials and cell-therapy strategies used in the past few years for the regeneration of the wounded heart.


Assuntos
Materiais Biocompatíveis , Insuficiência Cardíaca/terapia , Infarto do Miocárdio/terapia , Regeneração , Animais , Transplante de Células/métodos , Coração/fisiologia , Humanos , Engenharia Tecidual/métodos
7.
Micron ; 36(4): 293-320, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15857770

RESUMO

Recent developments in the application of Atomic Force Microscopy (AFM) and other biophysical techniques for the study of bacterial interactions and adhesion are discussed in the light of established biological and microscopic approaches. Whereas molecular-biological techniques combined with electron microscopy allow the identification and localization of surface constituents mediating bacterial interactions, with AFM it has become possible to actually measure the forces involved in bacterial interactions. Combined with the flexibility of AFM in probing various types of physical interactions, such as electrostatic interactions, specific ligand-receptor interactions and the elastic forces of deformation and extension of bacterial surface polymers and cell wall, this provides prospects for the elucidation of the biophysical mechanism of bacterial interaction. However, because of the biochemical and a biophysical complexity of the bacterial cell wall, integrated approaches combining AFM with electron microscopy and biophysical techniques are needed to elucidate the mechanism by which a bacterium interacts with a host or material surface. The literature on electron microscopy of the bacterial cell wall is reviewed, with particular emphasis on the staining of specific classes of cell-wall constituents. The application of AFM in the analysis of bacterial surfaces is discussed, including AFM operating modes, sample preparation methods and results obtained on various strains. For various bacterial strains, the integration of EM and AFM data is discussed. Various biophysical aspects of the analysis of bacterial surface structure and interactions are discussed, including the theory of colloidal interactions and Bell's theory of cell-to-cell adhesion. An overview is given of biophysical techniques used in the analysis of the properties of bacterial surfaces and bacterial surface constituents and their integration with AFM. Finally, we discuss recent progress in the understanding of the role of bacterial interactions in medicine within the framework of the techniques and concepts discussed in the paper.


Assuntos
Bactérias/ultraestrutura , Microscopia de Força Atômica/métodos , Microscopia Eletrônica/métodos , Aderência Bacteriana/fisiologia , Fenômenos Fisiológicos Bacterianos , Técnicas Bacteriológicas , Fenômenos Biofísicos , Biofísica , Parede Celular/química , Parede Celular/ultraestrutura , Elasticidade , Técnica de Congelamento e Réplica/métodos , Técnica de Fratura por Congelamento/métodos , Microscopia Imunoeletrônica/métodos , Coloração e Rotulagem/métodos
8.
Ultramicroscopy ; 97(1-4): 199-208, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12801672

RESUMO

The adhesion of lactic acid bacteria to the intestinal epithelium is one of the most important factors determining probiotic ability of a bacterial strain. Studying bacterial adhesion requires knowledge of the structure and properties of the bacterial surface, which can be studied by atomic force microscopy under native conditions. The observation of the surface topography of bacteria from the species Lactobacillus crispatus, L. helveticus and L. johnsonii shows major differences between bacteria having a crystalline-like protein layer as part of the cell wall and those without such layers. Force volume images calculated into elasticity and adhesion force maps of different bacterial strains show that L. crispatus and L. helveticus have a surface with a homogeneous stiffness with no adhesion events. This is most likely caused by the S-layer, which completely covers the surface of the bacteria. We infer that the absence of adhesion peaks is caused by the semi-crystalline character of such protein layers, in agreement with the results obtained from electron microscopy. Analysis of a number of L. johnsonii strains shows that these bacteria have surface properties which strongly differ from the L. crispatus and L. helveticus strains. For L. johnsonii DMS20533 and L. johnsonii ATCC33200 high adhesion forces are observed, which can be related to a surface rich in polysaccharides. L. johnsonii ATCC332 has lower adhesion forces compared to the other two and, furthermore, the surface topography shows depressions. We suppose that this strain has a surface pattern consisting of crystalline-like proteins alternating with polysaccharide-rich domains. The wide variety in surface properties of lactobacilli could well have wide-ranging implications for food processing and for health benefits.


Assuntos
Aderência Bacteriana , Lactobacillus/ultraestrutura , Glicoproteínas de Membrana , Microscopia de Força Atômica/métodos , Probióticos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Elasticidade , Lactobacillus/classificação , Lactobacillus/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Propriedades de Superfície
9.
Stem Cells Transl Med ; 1(3): 248-60, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23197784

RESUMO

Pluripotent stem cell-seeded cardiopatches hold promise for in situ regeneration of infarcted hearts. Here, we describe a novel cardiopatch based on bone morphogenetic protein 2-primed cardiac-committed mouse embryonic stem cells, embedded into biodegradable fibrin matrices and engrafted onto infarcted rat hearts. For in vivo tracking of the engrafted cardiac-committed cells, superparamagnetic iron oxide nanoparticles were magnetofected into the cells, thus enabling detection and functional evaluation by high-resolution magnetic resonance imaging. Six weeks after transplantation into infarcted rat hearts, both local (p < .04) and global (p < .015) heart function, as well as the left ventricular dilation (p < .0011), were significantly improved (p < .001) as compared with hearts receiving cardiopatches loaded with iron nanoparticles alone. Histological analysis revealed that the fibrin scaffolds had degraded over time and clusters of myocyte enhancer factor 2-positive cardiac-committed cells had colonized most of the infarcted myocardium, including the fibrotic area. De novo CD31-positive blood vessels were formed in the vicinity of the transplanted cardiopatch. Altogether, our data provide evidence that stem cell-based cardiopatches represent a promising therapeutic strategy to achieve efficient cell implantation and improved global and regional cardiac function after myocardial infarction.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Coração/fisiologia , Infarto do Miocárdio/terapia , Transplante de Células-Tronco , Animais , Células-Tronco Embrionárias/fisiologia , Técnicas Imunoenzimáticas , Imageamento por Ressonância Magnética , Masculino , Camundongos , Ratos
10.
Biomaterials ; 30(26): 4318-24, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19500842

RESUMO

The in vitro potential of a synthetic matrix metalloproteinase (MMP)-responsive poly(ethylene glycol) (PEG)-based hydrogel as a bioactive co-encapsulation system for vascular cells and a small bioactive peptide, thymosin beta4 (Tbeta4), was examined. We show that the physical incorporation of Tbeta4 in this bioactive matrix creates a three-dimensional (3D) environment conducive for human umbilical vein endothelial cell (HUVEC) adhesion, survival, migration and organization. Gels with entrapped Tbeta4 increased the survival of HUVEC compared to gels without Tbeta4, and significantly up-regulated the endothelial genes vascular endothelial-cadherin and angiopoietin-2, whereas von Willebrand factor was significantly down-regulated. Incorporation of Tbeta4 significantly increased MMP-2 and MMP-9 secretion of encapsulated HUVEC. The gel acts as a controlled Tbeta4-release system, as MMP-2 and MMP-9 enzymes trigger the release. In addition, Tbeta4 facilitated HUVEC attachment and induced vascular-like network formation upon the PEG-hydrogels. These MMP-responsive PEG-hydrogels may thus serve as controlled co-encapsulation system of vascular cells and bioactive factors for in situ regeneration of ischemic tissues.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Polietilenoglicóis/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timosina/farmacologia , Veias Umbilicais/citologia
11.
J Thorac Cardiovasc Surg ; 133(5): 1163-70, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17467424

RESUMO

OBJECTIVE: Intimal hyperplasia and surface thrombogenicity are major factors in the high failure rate of synthetic small-diameter bypass grafts. Vascular endothelial growth factor is a potent stimulus for endothelial growth, and its provision in a fibrin matrix coating at the luminal graft surface may hold a key to spontaneous graft endothelialization and improved graft patency. METHODS: Pigs underwent bilateral carotid artery interposition of expanded polytetrafluoroethylene grafts either impregnated with fibrin (n = 11)--engineered to locally release vascular endothelial growth factor121 (vascular endothelial growth factor-fibrin; n = 11)--or left uncoated (n = 12). Graft patency was assessed by quantitative carotid angiography followed by graft histomorphometry at the 1-month experimental end point. RESULTS: Patency rates were not significantly different between study groups. Grafts coated with fibrin or vascular endothelial growth factor-fibrin exhibited significantly increased angiographic narrowing at the proximal anastomosis (for both P < .05 vs uncoated) and no difference at the distal anastomosis and the grafts' middle. Histological analysis showed 80% to 90% endothelial coverage and buildup of intima throughout the lengths of all grafts. Examination of the grafts' midportion revealed significantly enlarged neointimal layers of smooth muscle actin-positive cells in grafts coated with vascular endothelial growth factor-fibrin (242 +/- 47 microm2/micron) and fibrin (177 +/- 41 microm2/micron), compared with uncoated grafts (131 +/- 39 microm2/micron) (for both P < .05 vs uncoated). This thickening could not be explained by enhanced inflammation or vessel wall angiogenesis, which were minimal at the experimental end point. CONCLUSIONS: Fibrin and vascular endothelial growth factor produced effects deleterious to graft healing, by increasing the narrowing at proximal anastomosis and neointimal growth beyond that seen in uncoated grafts. It may reflect direct activation by exogenous vascular endothelial growth factor of vascular smooth muscle cells.


Assuntos
Prótese Vascular , Artérias Carótidas/patologia , Materiais Revestidos Biocompatíveis , Fibrina , Politetrafluoretileno , Túnica Íntima/patologia , Fator A de Crescimento do Endotélio Vascular , Animais , Implante de Prótese Vascular , Capilares/patologia , Artérias Carótidas/cirurgia , Fibrina/farmacologia , Oclusão de Enxerto Vascular/patologia , Imuno-Histoquímica , Macrófagos/patologia , Sus scrofa , Fator A de Crescimento do Endotélio Vascular/farmacologia , Grau de Desobstrução Vascular
12.
Appl Environ Microbiol ; 71(12): 8165-73, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16332799

RESUMO

The effect of the simple and complex basic components of a fermentation medium on the surface properties of Lactobacillus acidophilus NCC2628 is studied by physicochemical methods, such as electrophoresis, interfacial adhesion, and X-ray photonelectron spectroscopy, and by transmission electron microscopy. Starting from an optimized complete medium, the effect of carbohydrates, peptones, and yeast extracts on the physicochemical properties of the cell wall is systematically investigated by consecutively omitting one of the principal components from the fermentation medium at the time. The physicochemical properties and structure of the bacterial cell wall remain largely unchanged if the carbohydrate content of the fermentation medium is strongly reduced, although the concentration of surface proteins increases slightly. Both peptone and yeast extract have a considerable influence on the bacterial cell wall, as witnessed by changes in surface charge, hydrophobicity, and the nitrogen-to-carbon ratio. Both zeta potential and the cell wall hydrophobicity show a positive correlation with the nitrogen-to-carbon ratio of the bacterial surfaces, indicative of the important role of surface proteins in the overall surface physical chemistry. The hydrophobicity of the cell wall, which is low for the cultures grown in the complete medium and in the absence of carbohydrates, becomes fairly high for the cultures grown in the medium without peptones and the medium without yeast extract. UV spectrophotometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with liquid chromatography-tandem mass spectrometry are used to analyze the effect of medium composition on LiCl-extractable cell wall proteins, confirming the major change in protein composition of the cell wall for the culture fermented in the medium without peptones. In particular, it is found that expression of the S-layer protein is dependent on the protein source of the fermentation medium.


Assuntos
Lactobacillus acidophilus/fisiologia , Aderência Bacteriana , Parede Celular/efeitos dos fármacos , Parede Celular/fisiologia , Meios de Cultura , Eletrofisiologia , Fermentação , Lactobacillus acidophilus/efeitos dos fármacos , Lactobacillus acidophilus/crescimento & desenvolvimento , Lactobacillus acidophilus/ultraestrutura , Cloreto de Lítio/farmacologia , Propriedades de Superfície
13.
Biophys J ; 85(6): 4076-92, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14645095

RESUMO

A variety of strains of the genus Lactobacillus was investigated with respect to the structure, softness, and interactions of their outer surface layers in order to construct structure-property relations of the Gram-positive bacterial cell wall. The role of the conformational properties of the constituents of the outer cell-wall layers and their spatial distribution on the cell wall is emphasized. Atomic force microscopy was used to resolve the surface structure, interactions, and softness of the bacterial cell wall at nanometer-length scales and upwards. The pH-dependence of the electrophoretic mobility and a novel interfacial adhesion assay were used to analyze the average physicochemical properties of the bacterial strains. The bacterial surface is smooth when a compact layer of globular proteins constitutes the outer surface, e.g., the S-layer of L. crispatus DSM20584. In contrast, for two other S-layer containing strains (L. helveticus ATCC12046 and L. helveticus ATCC15009), the S-layer is covered by polymeric surface constituents which adopt a much more extended conformation and which confer a certain roughness to the surface. Consequently, the S-layer is important for the overall surface properties of L. crispatus, but not for the surface properties of L. helveticus. Both surface proteins (L. crispatus DSM20584) and (lipo)teichoic acids (L. johnsonii ATCC332) confer hydrophobic properties to the bacterial surface whereas polysaccharides (L. johnsonii DSM20533 and L. johnsonii ATCC 33200) render the bacterial surface hydrophilic. Using the interfacial adhesion assay, it was demonstrated that hydrophobic groups within the cell wall adsorb limited quantities of hydrophobic compounds. The present work demonstrates that the impressive variation in surface properties displayed by even a limited number of genetically-related bacterial strains can be understood in terms of established colloidal concepts, provided that sufficiently detailed structural, chemical, and conformational information on the surface constituents is available.


Assuntos
Parede Celular/química , Ácido Láctico/metabolismo , Lactobacillus/metabolismo , Aderência Bacteriana , Adesão Celular , Divisão Celular , Coloides , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Microscopia de Força Atômica , Microscopia Eletrônica , Peptidoglicano/química , Polímeros/química , Polissacarídeos/química , Conformação Proteica
14.
Langmuir ; 20(24): 10464-73, 2004 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-15544374

RESUMO

Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors. In this study, the very efficient enzyme beta-lactamase is used as a model protein. The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme. The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent. beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate. The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface. Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site. Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency. In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics. All tested enzyme variants turned out to be active upon immobilization at the polymeric surface. However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site. No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers.


Assuntos
Técnicas Biossensoriais/métodos , Biotina/química , Enzimas Imobilizadas/química , Polietilenoglicóis/química , Polilisina/análogos & derivados , beta-Lactamases/química , Adsorção , Cinética , Nióbio/química , Óxidos/química , Tamanho da Partícula , Polietilenoglicóis/síntese química , Polilisina/síntese química , Polilisina/química , Conformação Proteica , Estrutura Secundária de Proteína , Sensibilidade e Especificidade , Propriedades de Superfície , Fatores de Tempo
15.
Anal Chem ; 74(16): 4269-74, 2002 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-12199601

RESUMO

A new method for simultaneous mapping of cell topography and ion fluxes was developed. A highly sensitive ion sensor system was generated by coating atomic force microscopy tips with a PVC layer containing valinomycin, an ionophore for potassium. The activity of specific ions was traced on artificial ion-releasing PVC substrates. A boundary potential was generated owing to the selective exchange of a specific ion between coated tip and ion-releasing substrate. The boundary potential was detectable as a force induced by ion-selective electrostatic interactions. The selectivity coefficient of valinomycin for potassium against sodium (K(K,Na)f) was -2.5 +/- 0.5. Potassium efflux was measured on living MDCK-F1 cells expressing BK(Ca) channels. We could demonstrate localized areas of high potassium concentrations at the cell surface. The potassium efflux could be reversibly inhibited by thapsigargin, which is known to inhibit the efflux of potassium from BK(Ca) channels by suppression of calcium ATPase.


Assuntos
Técnicas Biossensoriais/métodos , Células/química , Microscopia de Força Atômica , Potássio/metabolismo , Linhagem Celular Transformada , Células/metabolismo , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Íons/metabolismo , Valinomicina/farmacologia
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