RESUMEN
Alginates are fairly abundant in nature and possess many interesting properties, including their biocompatibility and ability to absorb large amounts of water. Hence, increasing interest in their derivatization has been observed and the determination of the number of newly introduced functionalities has become a key issue. For this purpose, literature generally reports on conventional 1H-NMR spectra, typically recorded at elevated temperatures and/or after hydrolysis of the alginate to circumvent line broadening effects resulting from the high viscosity. The present work reports on the modification of alginate with methacrylate functionalities and determination of the resulting degree of substitution (DS), i.e. the number of introduced methacrylate moieties relative to the initial amount of hydroxyl groups along the alginate backbone, via NMR spectroscopy. Freeze-drying and low power water presaturation were applied to improve the quality of the 1H NMR spectra. Nevertheless, it remains a qualitative method, to be used only for mutual comparisons of samples. A new and accurate method for DS determination of methacrylated alginates, based on 13C-NMR spectroscopy, is proposed. Quantitative 13C-NMR spectra were recorded with reduced measuring times by addition of a paramagnetic relaxation agent. The proposed method will also be applicable for other water-soluble functionalized alginates and polysaccharides in general.
Asunto(s)
Alginatos/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Compuestos de Vinilo/química , Agua/química , Estructura Molecular , Espectroscopía de Protones por Resonancia Magnética , Solubilidad , Compuestos de Vinilo/síntesis químicaRESUMEN
A combined experimental and first-principles study is performed to study the origin of conductivity in ZnO:Al nanoparticles synthesized under controlled conditions via a reflux route using benzylamine as a solvent. The experimental characterization of the samples by Raman, nuclear magnetic resonance (NMR) and conductivity measurements indicates that upon annealing in nitrogen, the Al atoms at interstitial positions migrate to the substitutional positions, creating at the same time Zn interstitials. We provide evidence for the fact that the formed complex of AlZn and Zni corresponds to the origin of the Knight shifted peak (KS) we observe in 27Al NMR. As far as we know, the role of this complex has not been discussed in the literature to date. However, our first-principles calculations show that such a complex is indeed energetically favoured over the isolated Al interstitial positions. In our calculations we also address the charge state of the Al interstitials. Further, Zn interstitials can migrate from AlZn and possibly also form Zn clusters, leading to the observed increased conductivity.
RESUMEN
Cellular adhesion and growth on solid-state surfaces is the central theme in the development of cell-based biosensors and implantable medical devices. Suitable interface techniques must be applied to construct stable and well-organized thin films of biologically active molecules that would control the development of neuronal cells on chips. Peptides such as RGD fragments, poly-L-lysine (PLL), or basal lamina proteins, such as laminin or fibronectin, are often used in order to promote cellular adhesion on surfaces. In this paper we describe the characterization of several self-assembled monolayers (SAMs) for their ability to anchor a laminin-derived synthetic peptide, PA22-2, a peptide known to promote neuronal attachment and stimulate neurite outgrowth. We have evaluated the immobilization of PA22-2 onto 16-mercaptohexadecanoic acid, 4-maleimide-N-(11-undecyldithio)butanamide, and 2-(maleimide)ethyl-N-(11-hexaethylene oxide-undecyldithio)acetamide SAM functionalized Au substrates. The neuronal attachment and outgrowth have been evaluated in embryonic mouse hippocampal neuron cultures up to 14 days in vitro. Our results show that differences in the cell morphologies were observed on the surfaces modified with various SAMs, despite the minor differences in chemical composition identified using standard characterization tools. These different cell morphologies can most probably be explained when investigating the effect of a given SAM layer on the adsorption of proteins present in the culture medium. More likely, it is the ratio between the specific PA22-2 adsorption and nonspecific medium protein adsorption that controls the cellular morphology. Large amounts of adsorbed medium proteins could screen the PA22-2 sites required for cellular attachment.
Asunto(s)
Neuronas/metabolismo , Péptidos/química , Compuestos de Sulfhidrilo/química , Adsorción , Animales , Adhesión Celular , Química/métodos , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Hipocampo/metabolismo , Ratones , Modelos Químicos , Oligopéptidos/química , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de SuperficieRESUMEN
Extracellular, high signal-to-noise ratio recordings from electrogenic cells require a tight coupling between the cellular membrane and the recording electrode. Self assembled monolayers (SAMs) of alkanethiols functionalized with peptides were used in combination with micro- and nano-structured features on the sensor surface. This combination of surface chemistry and topography triggers a phagocytosis-like engulfment and ensures tight coupling. In this paper we report the results concerning usage of different SAMs and the influence of the peptide concentration towards cell adhesion and outgrowth. Later on, the optimized peptide functionalized SAMs were applied on micro- and nano-structured sensor surfaces. As a result, phagocytosis-like events could be shown using focused ion beam SEM and confocal fluorescence imaging.
Asunto(s)
Técnicas Biosensibles , Neuronas/citología , Péptidos , Alcanos , Animales , Ingeniería Biomédica , Adhesión Celular , Línea Celular , Proliferación Celular , Células Cultivadas , Electrodos , Ratones , Microscopía Electrónica de Rastreo , Neuronas/metabolismo , Compuestos de Sulfhidrilo , Propiedades de SuperficieRESUMEN
Syntenin is a PDZ protein that binds the cytoplasmic C-terminal FYA motif of the syndecans. Syntenin is widely expressed. In cell fractionation experiments, syntenin partitions between the cytosol and microsomes. Immunofluorescence microscopy localizes endogenous and epitope-tagged syntenin to cell adhesion sites, microfilaments, and the nucleus. Syntenin is composed of at least three domains. Both PDZ domains of syntenin are necessary to target reporter tags to the plasma membrane. The addition of a segment of 10 amino acids from the N-terminal domain of syntenin to these PDZ domains increases the localization of the tags to stress fibers and induces the formation of long, branching plasma membrane extensions. The addition of the complete N-terminal region, in contrast, reduces the localization of the tags to plasma membrane/adhesion sites and stress fibers, and reduces the morphotypical effects. Recombinant domains of syntenin with the highest plasma membrane localization display the lowest nuclear localization. Syndecan-1, E-cadherin, beta-catenin, and alpha-catenin colocalize with syntenin at cell-cell contacts in epithelial cells, and coimmunoprecipitate with syntenin from extracts of these cells. These results suggest a role for syntenin in the composition of adherens junctions and the regulation of plasma membrane dynamics, and imply a potential role for syntenin in nuclear processes.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Proteoglicanos/metabolismo , Secuencia de Aminoácidos , Animales , Cadherinas/metabolismo , Proteínas Portadoras/genética , Adhesión Celular/fisiología , Comunicación Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Secuencia Conservada , Citosol/metabolismo , Células Epiteliales/metabolismo , Humanos , Mamíferos , Ratones , Ratones Endogámicos BALB C , Microsomas/metabolismo , Datos de Secuencia Molecular , Mutación , Conejos , Homología de Secuencia de Aminoácido , Sindecano-1 , Sindecanos , SinteninasRESUMEN
Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked syndrome characterized by pre- and postnatal overgrowth (gigantism), which clinically resembles the autosomal Beckwith-Wiedemann syndrome (BWS). Deletions and translocations involving the glypican-3 gene ( GPC3 ) have been shown to be associated with SGBS. Occasionally, these deletions also include the glypican-4 gene ( GPC4 ). Glypicans are heparan sulfate proteoglycans which have a role in the control of cell growth and cell division. We have examined the mutational status of the GPC3 and GPC4 genes in one patient with Perlman syndrome, three patients with overgrowth without syndrome diagnosis, ten unrelated SGBS-patients and 11 BWS patients. We identified one SGBS patient with a deletion of a GPC3 exon. Six SGBS patients showed point mutations in GPC3. One frameshift, three nonsense, and one splice mutation predict a loss-of-function of the glypican-3 protein. One missense mutation, W296R, changes an amino acid that is conserved in all glypicans identified so far. A GPC3 protein that reproduces this mutation is poorly processed and fails to increase the cell surface expression of heparan sulfate, suggesting that this missense mutation is also a loss-of-function mutation. In three SGBS patients and in all non-SGBS patients, no mutations could be identified. We found three single nucleotide polymorphisms in the GPC4 gene but no evidence for loss-of-function mutations in GPC4 associated with SGBS.
Asunto(s)
Gigantismo/genética , Proteoglicanos de Heparán Sulfato/genética , Mutación , Cromosoma X , Secuencia de Aminoácidos , Secuencia de Bases , Síndrome de Beckwith-Wiedemann/genética , Western Blotting , Línea Celular , Análisis Mutacional de ADN , Exones , Citometría de Flujo , Eliminación de Gen , Glipicanos , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Mutación Missense , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Homología de Secuencia de Aminoácido , Síndrome , Translocación GenéticaRESUMEN
Syntenin is an adaptor-like molecule that binds to the cytoplasmic domains of all four vertebrate syndecans. Syntenin-syndecan binding involves the C-terminal part of syntenin that contains a tandem of PDZ domains. Here we provide evidence that each PDZ domain of syntenin can interact with a syndecan. Isolated or combined mutations of the carboxylate binding lysines in the inter-betaAbetaB loops and of the alphaB1 residues in either one or both the PDZ domains of syntenin all reduce syntenin-syndecan binding in yeast two-hybrid, blot-overlay, and surface plasmon resonance assays. PDZ2 mutations have more pronounced effects on binding than PDZ1 mutations, but complete abrogation of syntenin-syndecan binding requires the combination of both the lysine and the alphaB1 mutations in both the PDZ domains of syntenin. Isothermal calorimetric titration of syntenin with syndecan peptide reveals the presence of two binding sites in syntenin. Yet, unlike a tandem of two PDZ2 domains and a reconstituted PDZ1+PDZ2 tandem, a tandem of two PDZ1 domains and isolated PDZ1 or PDZ2 domains do not interact with syndecan bait. We conclude to a co-operative binding mode whereby neither of these two PDZ domains is sufficient by itself but where PDZ2 functions as a "major" or "high affinity" syndecan binding domain, and PDZ1 functions as an "accessory" or "low affinity" syndecan binding domain. The paired, but not the isolated PDZ domains of syntenin bind also strongly to the immobilized cytoplasmic domains of neurexin and B-class ephrins. By inference, these data suggest a model whereby recruitment of syntenin to membrane surfaces requires two compatible types of bait that are in "synteny" (occurring together in location) and engages both PDZ domains of syntenin. The synteny of compatible bait may result from the assemblies and co-assemblies of syndecans and other similarly suited partners in larger supramolecular complexes. In general, an intramolecular combination of PDZ domains that are weak, taken individually, would appear to be designed to detect rather than drive the formation of specific molecular assemblies.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteoglicanos/química , Proteoglicanos/metabolismo , Secuencia de Aminoácidos , Calorimetría , Proteínas Portadoras/genética , Citoplasma/metabolismo , ADN Complementario/metabolismo , Efrina-B1 , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Ligandos , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Mutación Puntual , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteoglicanos/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Sindecanos , Sinteninas , Factores de Tiempo , Técnicas del Sistema de Dos HíbridosRESUMEN
Oligonucleotides that can form a highly stable intramolecular four-stranded DNA structure containing two stacked guanosine-quartets (G-quartets) have been reported to inhibit the replication of the human immunodeficiency virus type 1 (HIV-1) in cell culture. Two possible mechanisms for the observed antiviral activity have been proposed: interference with virus adsorption to the cell and/or inhibition of HIV-1 integrase. We investigated the molecular interaction of G-quartet-containing oligonucleotides with HIV-1 integrase in comparison with random oligonucleotides and dextran sulfate. The prototypical G-quartet-containing oligonucleotide, T30177 (Zintevir), inhibited the overall integration reaction with an IC50 value of 80 nM. A random oligonucleotide was 10-fold less potent, but dextran sulfate was more potent, with an IC50 value of 7 nM. We developed novel kinetic assays to dissect the overall integration reaction in three steps: the formation of the initial stable complex (ISC), the 3'-processing reaction, and the DNA strand-transfer step. We then analyzed the kinetics of the ISC formation and 3'-processing. The rate constant determined for the conversion of ISC into the cleaved product was 0.08 +/- 0.01 min-1. T30177 did not inhibit 3'-processing or DNA strand transfer, whereas dextran sulfate inhibited DNA strand transfer to some extent. Binding studies using surface plasmon resonance technology revealed that both T30177 and dextran sulfate were capable of preventing the binding of integrase to specific DNA. We propose a model in which the interaction of HIV-1 integrase with G-quartets results in the inhibition of the formation of the ISC between integrase and substrate DNA. Finally, we selected for an HIV-1 strain that was resistant to T30177 in cell culture. DNA sequence analysis revealed mutations in the envelope glycoprotein gp120 but not in the integrase gene. Although gp120 seems to be the main target for the antiviral activity in cell culture of G-quartets, the study of their specific inhibition of HIV-1 integrase may lead to the development of effective integrase inhibitors.
Asunto(s)
ADN Viral/efectos de los fármacos , Guanosina , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/efectos de los fármacos , VIH-1/enzimología , Oligonucleótidos/farmacología , Integración Viral/efectos de los fármacos , ADN Viral/análisis , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , VIH-1/genética , Cinética , Proteínas Virales/metabolismoRESUMEN
The four vertebrate glypican-related integral membrane proteoglycans identified so far constitute a discrete family of heparan sulfate proteoglycans that are linked to the cell surface via glycosyl phosphatidylinositol. In addition to the GPI anchor and substitution with heparan sulfate, the members of this family show significant sequence homology and share a unique and characteristic cysteine motif. Starting from an EST entry that showed significant sequence similarity to MXR7 and OCI-5 (coding, respectively, for human and rat glypican-3), we have isolated a human cDNA coding for glypican-5, a novel member of this proteoglycan family. The gene for this novel glypican (GPC5) maps to 13q32. In the adult, it is primarily expressed in brain tissue.
Asunto(s)
Cromosomas Humanos Par 13 , Heparitina Sulfato/genética , Proteoglicanos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario , Proteínas de la Matriz Extracelular , Expresión Génica , Glipicanos , Proteoglicanos de Heparán Sulfato , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The syndecans are transmembrane proteoglycans that place structurally heterogeneous heparan sulfate chains at the cell surface and a highly conserved polypeptide in the cytoplasm. Their versatile heparan sulfate moieties support various processes of molecular recognition, signaling, and trafficking. Here we report the identification of a protein that binds to the cytoplasmic domains of the syndecans in yeast two-hybrid screens, surface plasmon resonance experiments, and ligand-overlay assays. This protein, syntenin, contains a tandem repeat of PDZ domains that reacts with the FYA C-terminal amino acid sequence of the syndecans. Recombinant enhanced green fluorescent protein (eGFP)-syntenin fusion proteins decorate the plasmamembrane and intracellular vesicles, where they colocalize and cosegregate with syndecans. Cells that overexpress eGFP-syntenin show numerous cell surface extensions, suggesting effects of syntenin on cytoskeleton-membrane organization. We propose that syntenin may function as an adaptor that couples syndecans to cytoskeletal proteins or cytosolic downstream signal-effectors.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Proteoglicanos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Células CHO , Proteínas Portadoras/química , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Cricetinae , Citoesqueleto/metabolismo , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Unión Proteica , Proteoglicanos/química , Proteoglicanos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Sindecano-2 , SinteninasRESUMEN
Pregastric esterase (PGE) (EC 3.1.1.3) was purified to homogeneity from calf pharyngeal tissue. The enzyme had an apparent molecular mass of 50 kDa, as determined by SDS/PAGE. The serine-binding reagent diethyl p-nitrophenyl phosphate was a potent inhibitor of PGE. This is in accordance with the claim that a functional serine residue is necessary for the lipolytic activity of lipases. PGE was not inhibited by the thiol reagents 5,5'-dithiobis(2-nitrobenzoic acid) or 4,4'-dithiopyridine. A partial inhibition with dodecyldithio-5-(2-nitrobenzoic acid) was observed, but the same degree of inhibition was caused by the non-esterified fatty acid C(12:0). PGE shows a great sequence similarity to gastric lipases. Gastric lipases have three cysteine residues, and two of these form a disulphide bridge. Blocking the remaining free cysteine with thiol reagents inactivates the gastric lipases. The fact that PGE is not inhibited by thiol reagents indicates that PGE has no functional free thiol group. The PGE cDNA codes only for two cysteines, and their involvement in the formation of a disulphide bridge was demonstrated.