RESUMEN
Autism Spectrum Disorder (ASD) is characterized by substantial, yet highly heterogeneous abnormalities in functional brain connectivity. However, the origin and significance of this phenomenon remain unclear. To unravel ASD connectopathy and relate it to underlying etiological heterogeneity, we carried out a bi-center cross-etiological investigation of fMRI-based connectivity in the mouse, in which specific ASD-relevant mutations can be isolated and modeled minimizing environmental contributions. By performing brain-wide connectivity mapping across 16 mouse mutants, we show that different ASD-associated etiologies cause a broad spectrum of connectional abnormalities in which diverse, often diverging, connectivity signatures are recognizable. Despite this heterogeneity, the identified connectivity alterations could be classified into four subtypes characterized by discrete signatures of network dysfunction. Our findings show that etiological variability is a key determinant of connectivity heterogeneity in ASD, hence reconciling conflicting findings in clinical populations. The identification of etiologically-relevant connectivity subtypes could improve diagnostic label accuracy in the non-syndromic ASD population and paves the way for personalized treatment approaches.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Animales , Trastorno del Espectro Autista/diagnóstico por imagen , Trastorno del Espectro Autista/genética , Trastorno Autístico/diagnóstico por imagen , Trastorno Autístico/genética , Encéfalo , Mapeo Encefálico , Imagen por Resonancia Magnética , Ratones , Vías NerviosasRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) early diagnosis is⯠crucial â¯and new, cheap and user-friendly techniques for biomarker identification⯠are â¯needed. "Protein corona" (PC) is emerging a new bio-interface potentially useful in tumor early diagnosis. In a previous investigation, we showed that relevant differences between the â¯protein patterns of⯠PCs formed on lipid NPs after exposure to PDAC and non-cancer plasma⯠samples exist. To extend that research, We performed this pilot study to investigate the effect of PDAC tumor size and distant metastases on PC composition. METHODS: Twenty PDACs were clinically staged according to the UICC TNM staging system 8â¯tâ¯hâ¯Edition. Collected plasma samples were let to interact with lipid NPs; resulting PCs were characterized by SDS-PAGE. To properly evaluate changes in the PC, the protein intensity profiles were reduced to four regions of molecular weight: < 25â¯kDa, 25-50â¯kDa, 50-120â¯kDa, > 120â¯kDa.⯠RESULTS: Data analysis allowed toâ¯distinguish T1-T2 cases from T3 andâ¯above all fromâ¯metastatic ones (pâ¯<â¯0.05). Discrimination power was particularly due to a subset of plasma proteins with molecular â¯weight comprised between 25-50â¯kDa â¯and 50-120â¯kDa. CONCLUSIONS: PC composition is critically influenced by tumor size and presence of distant metastases in PDAC. If our findings will be further confirmed, we envision that future developments of cheap and user-friendly PC-based tools will allow to improve the accuracy of PDAC clinical staging, identifying among resectable â¯PDACs with potentially better prognosis (i.e. T1 and T2) thoseâ¯at higher risk of occult distant metastases.
Asunto(s)
Carcinoma Ductal Pancreático/sangre , Carcinoma Ductal Pancreático/patología , Liposomas/sangre , Nanopartículas/análisis , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Anciano , Anciano de 80 o más Años , Diagnóstico Precoz , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Persona de Mediana Edad , Peso Molecular , Metástasis de la Neoplasia , Estadificación de Neoplasias , Proyectos Piloto , PronósticoRESUMEN
The self-assembling processes underlining the capabilities of facially differentiated ("Janus") polycationic amphiphilic cyclodextrins (paCDs) as non-viral gene nanocarriers have been investigated by a pluridisciplinary approach. Three representative Janus paCDs bearing a common tetradecahexanoyl multitail domain at the secondary face and differing in the topology of the cluster of amino groups at the primary side were selected for this study. All of them compact pEGFP-C3 plasmid DNA and promote transfection in HeLa and MCF-7 cells, both in absence and in presence of human serum. The electrochemical and structural characteristics of the paCD-pDNA complexes (CDplexes) have been studied by using zeta potential, DLS, SAXS, and cryo-TEM. paCDs and pDNA, when assembled in CDplexes, render effective charges that are lower than the nominal ones. The CDplexes show a self-assembling pattern corresponding to multilamellar lyotropic liquid crystal phases, characterized by a lamellar stacking of bilayers of the CD-based vectors with anionic pDNA sandwiched among them. When exposed to human serum, either in the absence or in the presence of pDNA, the surface of the cationic CD-based vector becomes coated by a protein corona (PC) whose composition has been analyzed by nanoLC-MS/MS. Some of the CDplexes herein studied showed moderate-to-high transfection levels in HeLa and MCF-7 cancer cells combined with moderate-to-high cell viabilities, as determined by FACS and MTT reduction assays. The ensemble of data provides a detail picture of the paCD-pDNA-PC association processes and a rational base to exploit the protein corona for targeted gene delivery on future in vivo applications.
Asunto(s)
Ciclodextrinas/química , ADN/química , Corona de Proteínas/química , Transfección/métodos , Biofisica , Células HeLa , Humanos , Células MCF-7 , NanopartículasRESUMEN
Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.
Asunto(s)
ADN/química , Técnicas de Transferencia de Gen , Lípidos/química , Nanocompuestos/química , Nanoestructuras/química , Animales , Células CHO , Cationes/química , Cricetinae , Cricetulus , ADN/administración & dosificación , Endosomas/metabolismo , Citometría de Flujo , Terapia Genética , Liposomas/química , Pinocitosis , Protaminas/metabolismoRESUMEN
We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.
Asunto(s)
ADN/química , ADN/genética , Nanopartículas/química , Protaminas/química , Animales , Células CHO , Colesterol/análogos & derivados , Colesterol/química , Cricetulus , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Ácidos Grasos Monoinsaturados/química , Técnicas de Transferencia de Gen , Lípidos/química , Liposomas/metabolismo , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Compuestos de Amonio Cuaternario/química , Transfección/métodosRESUMEN
Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H(2), H(2)O(2), and HO(2). The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Microburbujas , Sonido , Espectroscopía Infrarroja por Transformada de Fourier , Terapia por Ultrasonido , Animales , Tampones (Química) , Línea Celular , Óxidos N-Cíclicos/química , Citocinesis/genética , Fibroblastos/citología , Terapia Genética/métodos , Radical Hidroxilo/química , Radical Hidroxilo/metabolismo , Ratones , Microburbujas/efectos adversos , Pruebas de Micronúcleos , Células 3T3 NIH , Fosfatos/química , Sonido/efectos adversos , Detección de Spin , Terapia por Ultrasonido/efectos adversosRESUMEN
We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm(2)) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spectroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698-705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Jurkat/efectos de la radiación , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Rayos Ultravioleta , Apoptosis/fisiología , Células Cultivadas , Citometría de Flujo/métodos , Humanos , Espectrofotometría Infrarroja/métodosRESUMEN
Homeostatic plasticity is a regulatory feedback response in which either synaptic strength or intrinsic excitability can be adjusted up or down to offset sustained changes in neuronal activity. Although a growing number of evidences constantly provide new insights into these two apparently distinct homeostatic processes, a unified molecular model remains unknown. We recently demonstrated that REST is a transcriptional repressor critical for the downscaling of intrinsic excitability in cultured hippocampal neurons subjected to prolonged elevation of electrical activity. Here, we report that, in the same experimental system, REST also participates in synaptic homeostasis by reducing the strength of excitatory synapses by specifically acting at the presynaptic level. Indeed, chronic hyperactivity triggers a REST-dependent decrease of the size of synaptic vesicle pools through the transcriptional and translational repression of specific presynaptic REST target genes. Together with our previous report, the data identify REST as a fundamental molecular player for neuronal homeostasis able to downscale simultaneously both intrinsic excitability and presynaptic efficiency in response to elevated neuronal activity. This experimental evidence adds new insights to the complex activity-dependent transcriptional regulation of the homeostatic plasticity processes mediated by REST.
Asunto(s)
Hipocampo/metabolismo , Homeostasis/fisiología , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Terminales Presinápticos/fisiología , Proteínas Represoras/metabolismo , Animales , Ratones , Proteínas Represoras/genética , Vesículas Sinápticas/metabolismoRESUMEN
Pancreatic cancer is a very aggressive malignancy that is often diagnosed in the advanced stages, with the implication that long-term survivors are extremely rare. Thus, developing new methods for the early detection of pancreatic cancer is an urgent task for current research. To date, nanotechnology offers unprecedented opportunities for cancer therapeutics and diagnosis. The aim of this study is the development of a new pancreatic cancer diagnostic technology based on the exploitation of the nano-bio-interactions between nanoparticles and blood samples. In this study, blood samples from 20 pancreatic cancer patients and 5 patients without malignancy were allowed to interact with designed lipid nanoparticles, leading to the formation of a hard "protein corona" at the nanoparticle surface. After isolation, the protein patterns were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). We found that the protein corona of pancreatic cancer patients was much more enriched than that of healthy individuals. Statistical analysis of SDS-PAGE results allowed us to discriminate between healthy and pancreatic cancer patients with a total discriminate correctness rate of 88%.
Asunto(s)
Detección Precoz del Cáncer , Pruebas Hematológicas , Nanopartículas , Corona de Proteínas/análisis , Anciano , Estudios de Casos y Controles , Electroforesis en Gel de Poliacrilamida , Humanos , Liposomas , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/diagnósticoRESUMEN
Today, liposomes are an advanced technology of drug carriers with a dozen drugs in clinical practice and many more in clinical trials. A bottleneck associated with the clinical translation of liposomes has long been 'opsonization', i.e. the adsorption of plasma proteins at the liposome surface resulting in their rapid clearance from circulation. For decades, the most popular way to avoid opsonization has been grafting polyethylene glycol (PEG) onto the liposome surface. Recent studies have clarified that grafting PEG onto the liposome surface reduces, but does not completely prevent protein binding. In this work, we employed dynamic light scattering, zeta-potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), semi-quantitative densitometry and cell imaging to explore the bio-nano-interactions between human plasma (HP) and Onivyde, a PEGylated liposomal drug that has recently been approved by the Food and Drug Administration (FDA) for the treatment of metastatic pancreatic ductal adenocarcinoma (PDAC). To properly evaluate the role of PEGylation, an unPEGylated variant of Onivyde was used as a reference. Collectively, our findings suggest that: (i) although PEGylated, Onivyde is not "stealth" in HP; (ii) surface chemistry is more important than PEGylation in controlling the bio-nano-interactions between Onivyde and plasma components. Of note is that the PC was found to boost the cellular uptake of Onivyde in the pancreas ductal adenocarcinoma cell line (PANC-1) thus suggesting its prominent role in its indication for PDAC treatment. Relevant implications for drug delivery and drug design are discussed.
Asunto(s)
Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Polietilenglicoles/química , Corona de Proteínas , Línea Celular Tumoral , Humanos , LiposomasRESUMEN
Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.
Asunto(s)
Apolipoproteínas/química , Endocitosis , Lípidos , Nanopartículas/metabolismo , Corona de Proteínas , Sistemas de Liberación de Medicamentos , Células HeLa , Humanos , Pinocitosis , Distribución TisularRESUMEN
Sixty-seven cases of Hodgkin's disease (HD) occurring in São Paulo, Brazil, were studied. Males with HD predominated over females 2.3 to 1. Sixty-six percent of the cases occurred in patients under 30 years of age, 31.7% under 20 years of age, and only 7.5% after 50 years of age. Lymphocyte predominance and mixed cellularity histologic types were most common in patients less than 15 years old, and nodular sclerosis was most common in the 15- to 19-year-old group. Sera from all patients had antibody to the viral capsid antigen (VCA) of Epstein-Barr virus (EBV). The geometric mean titer (GMT) of VCA antibody with the use of Jijoye cells as antigen was 1:162, and 31.3% of patients had titers of 1:320 or more; in controls, the GMT was 1:67 and 3.8% had titers of 1:320 or more. Similar results were obtained when EB-3 cells were used as antigen. The highest titers occurred in males, in mixed cellularity and lymphocyte depletion forms, and in stage 2 of illness. EBV-specific IgM antibody and heterophile antibody levels were not elevated, but 20.5% of the HD patients had antibody to the early antigen of EBV present in their sera. Antibody levels for herpes simplex virus, cytomegalovirus, rubella, measles, parainfluenza viruses, and papovavirus were not significantly elevated over those in matched controls.
Asunto(s)
Anticuerpos Antivirales/análisis , Herpesvirus Humano 4/inmunología , Enfermedad de Hodgkin/inmunología , Adolescente , Adulto , Factores de Edad , Anciano , Anticuerpos Heterófilos/análisis , Especificidad de Anticuerpos , Brasil , Cápside/inmunología , Niño , Preescolar , Métodos Epidemiológicos , Femenino , Enfermedad de Hodgkin/etiología , Humanos , Inmunidad Celular , Inmunoglobulina M , Lactante , Masculino , Persona de Mediana EdadRESUMEN
Exposure of nanoparticles (NPs) to biological fluids (e.g., plasma, interstitial fluid, and cytoplasm) leads to the absorption of proteins on the NP surface, forming a protein corona (PC) that drastically influences the NP physicochemical properties. Herein, we highlight the emerging applications of PC towards its use in therapeutics and diagnostics. In particular, special emphasis is given to the exploitation of PC for targeted delivery of nanomaterials and early cancer detection. By highlighting such recent applications of PC, we hope to demonstrate that this bio-entity has the potential to determine the success of NPs in biomedicine beyond their currently envisioned purposes.
RESUMEN
In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a "protein corona" layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø≈ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.
Asunto(s)
Proteínas Sanguíneas/análisis , Nanopartículas , Corona de Proteínas/química , Células HeLa , Humanos , Distribución TisularRESUMEN
Infection of Epstein Barr virus (EBV) to its host cells is initiated by the attachment of the glycoprotein gp 350/220 to the CR2 molecule. We used the sensitivity at the polar environment of the fluorescent probe Laurdan to study the membrane viscosity distribution from single leaving cells on two lymphoid cell lines Raji and Akata. Lipid analysis on both cells line demonstrated a lower cholesterol to phospholipid molar ratio on Akata than Raji cells. Cell fluidity analysis by Laurdan generalized emission polarization (GP) or by DPH polarization, indicated that membrane viscosity of Akata was lower than Raji cells. This difference was correlated to the increased susceptibility of Akata cells in expressing EBV early antigens (EA) after EBV superinfection.
Asunto(s)
Membrana Celular/fisiología , Herpesvirus Humano 4/fisiología , Fluidez de la Membrana , 2-Naftilamina/análogos & derivados , Línea Celular , Colesterol/metabolismo , Colorantes Fluorescentes , Herpesvirus Humano 4/patogenicidad , Humanos , Lauratos , Fusión de Membrana , Lípidos de la Membrana/metabolismo , Microscopía Fluorescente , Fosfolípidos/metabolismo , Proteína Quinasa C/metabolismo , Sensibilidad y Especificidad , Temperatura , Termodinámica , ViscosidadRESUMEN
Human B lymphoid cells (Raji) were exposed for 72 h to a 50 Hz sinusoidal magnetic field at a density of 2 milliTesla (rms). The results of exposure showed a decrease in membrane fluidity as detected by Laurdan emission spectroscopy and DPH fluorescence polarization. Field exposure also resulted in a reorganization of cytoskeletal components. Scanning electron microscopy (SEM) revealed a loss of microvilli in the exposed cells. This change in plasma membrane morphology was accompanied by a different actin distribution, as detected by phalloidin fluorescence. We also present evidence that EMF exposure of Raji cells can interfere with protein phosphorylation. Our observations confirm the hypothesis that electric and magnetic fields may modify the plasma membrane structure and interfere with the initiation of the signal cascade pathways.
Asunto(s)
Campos Electromagnéticos/efectos adversos , Actinas/ultraestructura , División Celular , Línea Celular/efectos de la radiación , Línea Celular/ultraestructura , Membrana Celular/efectos de la radiación , Citoesqueleto/efectos de la radiación , Citoesqueleto/ultraestructura , Humanos , Fluidez de la Membrana/efectos de la radiación , Microscopía Confocal , Microscopía Electrónica de Rastreo , Faloidina/análogos & derivados , Proteínas Quinasas/metabolismoRESUMEN
The interaction between the carbohydrate and the amino acid residues in human thyroglobulin has been studied. Previous reports showed that the removal of the two terminal carbohydrates of the complex chains leads to an increase in thyroglobulin binding to thyroid membranes. In our study, after enzymatic release with glycosidases of the sugar moieties from thyroglobulin, a time-dependent decrease in tryptophan fluorescence has been observed. This decrease was also associated with a shift in the emission peak from 335 to 340 nm. The strong quenching of tryptophan emission was also accompanied by a decrease in the exposure of tryptophan residues, as shown by a Stern-Volmer analysis with the neutral quencher acrylamide. These data, together with the increase in fluorescence of the dansylated deglycosylated thyroglobulin, strongly suggest that a significant conformational change of thyroglobulin follows the deglycosylation of the protein.
Asunto(s)
Glicoproteínas/metabolismo , Tiroglobulina/metabolismo , Apoproteínas/metabolismo , Centrifugación , Humanos , Técnicas In Vitro , Conformación Proteica , Espectrometría de Fluorescencia , Relación Estructura-Actividad , TriptófanoRESUMEN
The influence of occupancy by ouabain of its specific binding site on the stability and conformation of the Na+/K+-ATPase has been investigated. When native Na+/K+-ATPase is exposed to guanidinium chloride or diluted acid, tryptophanyl fluorescence falls to 50% of the initial value. If ouabain is bound, higher concentrations of GdmCl or acidity are needed to reach the same decrease in fluorescence. The rotational diffusion coefficient (relaxation time), shows higher values for the Na+/K+-ATPase (ouabain) complex compared to the enzyme alone, suggesting an increase in molecular asymmetry. This observation is confirmed by the Stern-Volmer analysis that shows an increase in the accessibility of the fluorophores in the Na+/K+-ATPase (ouabain) (KSV = 15.6 M-1) with respect to the native enzyme (KSV = 12.5 M-1). Iodine perturbation of the enzyme labelled with FITC, demonstrates a decrease in the accessibility of the fluorescein probe in the Na+/K+-ATPase(ouabain) (KSV = 4 M-1) compared to the Na+/K+-ATPase (KSV = 7 M-1) indicating that after ouabain binding this site of the enzyme is less exposed to the solvent. These data, in agreement with other reports, suggest an allosteric effect of ouabain binding on the Na+/K+-ATPase conformation.
Asunto(s)
Ouabaína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Acrilamida , Acrilamidas , Animales , Fluorescencia , Guanidina , Guanidinas/farmacología , Concentración de Iones de Hidrógeno , Yoduros , Conformación Proteica , Porcinos , TriptófanoRESUMEN
Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.