RESUMO
The physiochemical properties of hydrogels utilized in 3D culture can be used to modulate cell phenotype and morphology with a striking resemblance to cellular processes that occur in vivo. Indeed, research areas including regenerative medicine, tissue engineering, in vitro cancer models, and stem cell differentiation have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this biomimetic milieu. In many models of disease such as Alzheimer's disease (AD) that could benefit from the in vivo-like cell morphology associated with 3D culture, other aspects of the disease such as protein aggregation have yet to be methodically considered in this 3D context. A hallmark of AD is the accumulation of the peptide amyloid-ß (Aß), whose aggregation is associated with neurotoxicity. We have previously demonstrated the attenuation of Aß cytotoxicity when cells were cultured within type I collagen hydrogels versus on 2D substrates. In this work, we investigated the extent to which this phenomenon is conserved when Aß is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated the Aß structure and aggregation kinetics in solution and hydrogels composed of type I collagen, agarose, hyaluronic acid, and polyethylene glycol using fluorescence correlation spectroscopy and thioflavin T assays. Our results reveal that all hydrogels tested were associated with enhanced Aß aggregation and Aß cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing Aß structures and shifting the aggregate equilibrium toward larger species. If this phenomenon of altered protein aggregation in 3D hydrogels can be generalized to other contexts including the in vivo environment, it may be necessary to reevaluate aspects of protein aggregation disease models used for drug discovery.
RESUMO
In the biological milieu of a cell, soluble crowding molecules and rigid confined environments strongly influence whether the protein is properly folded, intrinsically disordered proteins assemble into distinct phases, or a denatured or aggregated protein species is favored. Such crowding and confinement factors act to exclude solvent volume from the protein molecules, resulting in an increased local protein concentration and decreased protein entropy. A protein's structure is inherently tied to its function. Examples of processes where crowding and confinement may strongly influence protein function include transmembrane protein dimerization, enzymatic activity, assembly of supramolecular structures (e.g., microtubules), nuclear condensates containing transcriptional machinery, protein aggregation in the contexts of disease and protein therapeutics. Historically, most protein structures have been determined from pure, dilute protein solutions or pure crystals. However, these are not the environments in which these proteins function. Thus, there has been an increased emphasis on analyzing protein structure and dynamics in more "in vivo-like" environments. Complex in vitro models using hydrogel scaffolds to study proteins may better mimic features of the in vivo environment. Therefore, analytical techniques need to be optimized for real-time analysis of proteins within hydrogel scaffolds.
Assuntos
Hidrogéis , Agregados Proteicos , Dobramento de Proteína , ProteínasRESUMO
Alzheimer's disease (AD) is the most common form of dementia and is associated with the accumulation of amyloid-ß (Aß), a peptide whose aggregation has been associated with neurotoxicity. Drugs targeting Aß have shown great promise in 2D in vitro models and mouse models, yet preclinical and clinical trials for AD have been highly disappointing. We propose that current in vitro culture systems for discovering and developing AD drugs have significant limitations; specifically, that Aß aggregation is vastly different in these 2D cultures carried out on flat plastic or glass substrates vs. in a 3D environment, such as brain tissue, where Aß confinement alters aggregation kinetics and thermodynamics. In this work, we identified attenuation of Aß cytotoxicity in 3D hydrogel culture compared to 2D cell culture. We investigated Aß structure and aggregation in solution vs. hydrogel using Transmission Electron Microscopy (TEM), Fluorescence Correlation Spectroscopy (FCS), and Thioflavin T (ThT) assays. Our results reveal that the equilibrium is shifted to stable extended ß-sheet (ThT positive) aggregates in hydrogels and away from the relatively unstable/unstructured presumed toxic oligomeric Aß species in solution. Volume exclusion imparted by hydrogel confinement stabilizes unfolded, presumably toxic species, promoting stable extended ß-sheet fibrils. STATEMENT OF SIGNIFICANCE: Alzheimer's disease (AD) is a devastating disease and has been studied for over 100 years. Yet, no cure exists and only 5 prescription drugs are FDA-approved to temporarily treat the AD symptoms of declining brain functions related to thinking and memory. Why don't we have more effective treatments to cure AD or relieve AD symptoms? We propose that current culture methods based upon cells cultured on flat, stiff substrates have significant limitations for discovering and developing AD drugs. This study provides strong evidence that AD drugs should be tested in 3D culture systems as a step along the development pathway towards new, more effective drugs to treat AD.
Assuntos
Doença de Alzheimer , Hidrogéis , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Animais , Colágeno , Modelos Animais de Doenças , Hidrogéis/farmacologia , Camundongos , Fragmentos de PeptídeosRESUMO
beta-Amyloid (Aß) is the primary protein component of senile plaques in Alzheimer's disease (AD) and is believed to play a role in its pathology. To date, the mechanism of action of Aß in AD is unclear. We and others have observed that Aß interacts either with or in the vicinity of the α6 sub-unit of integrin, and believe this may be important in its interaction with neuronal cells. In this study, we used confocal microscopy and flow cytometry to explore the residue specific interactions of Aß40 with the cell surface and the α6 integrin receptor sub-unit. We probed the importance of the RHD sequence in Aß40 and found that removal of the residues or their mutation using the Aß8-40 or the D7N early onset AD sequence, respectively, led to a greater interaction between Aß40 and an antibody bound to the α6-integrin sub-unit, as measured by fluorescence resonance energy transfer (FRET). These results suggest that the RHD sequence of Aß40 does not mediate Aß-α6 integrin interactions. However, the cyclic RGD mimicking peptide, Cilengitide, reduced the measured interaction between Aß40 fibrils without the RHD sequence and an antibody bound to the α6-integrin sub-unit. We further probed the role of electrostatic forces on Aß40-cell interactions and observed that the Aß sequence that included the N-terminal segment of the peptide had reduced cellular binding at low salt concentrations, suggesting that its first 7 residues contribute to an electrostatic repulsion for the cell surface. These findings contribute to our understanding of Aß-cell surface interactions and may provide insight into development of novel strategies to block Aß-cell interactions that contribute to pathology in Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/química , Integrina alfa6/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Ligação Proteica , Eletricidade EstáticaRESUMO
Every year, an unacceptably large number of infant deaths occur in developing nations, with premature birth and asphyxia being two of the leading causes. A well-regulated thermal environment is critical for neonatal survival. Advanced incubators currently exist, but they are far too expensive to meet the needs of developing nations. We are developing a thermodynamically advanced low-cost incubator suitable for operation in a low-resource environment. Our design features three innovations: (1) a disposable baby chamber to reduce infant mortality due to nosocomial infections, (2) a passive cooling mechanism using low-cost heat pipes and evaporative cooling from locally found clay pots, and (3) insulated panels and a thermal bank consisting of water that effectively preserve and store heat. We developed a prototype incubator and visited and presented our design to our partnership hospital site in Mysore, India. After obtaining feedback, we have determined realistic, nontrivial design requirements and constraints in order to develop a new prototype incubator for clinical trials in hospitals in India.
Assuntos
Equipamentos Descartáveis , Desenho de Equipamento , Incubadoras para Lactentes , Conservação de Recursos Energéticos/economia , Custos e Análise de Custo , Infecção Hospitalar/economia , Infecção Hospitalar/prevenção & controle , Países em Desenvolvimento , Equipamentos Descartáveis/economia , Pesquisas sobre Atenção à Saúde , Custos Hospitalares , Hospitais Urbanos , Humanos , Incubadoras para Lactentes/economia , Índia , Recém-Nascido , Avaliação das Necessidades , Organizações , Pais , Atenção Primária à Saúde/economia , Estudo de Prova de Conceito , Atenção Secundária à Saúde/economia , Recursos HumanosRESUMO
Like engineered materials, an artery's biomechanical behavior and function depend on its microstructure. Glycation is associated with both normal aging and diabetes and has been shown to increase arterial stiffness. In this study we examined the direct effect of glycation on the mechanical response of intact arteries and on the mechanical response and structure of elastin isolated from the arteries. Samples of intact arteries and isolated elastin were prepared from porcine aortas and glycated. The mechanical response of all samples was completed using a uniaxial material test system. Glycation levels were measured using ELISA. A confocal microscope was used to image differences in the structure of the glycated and untreated elastin fibers. We found that, under the conditions used in this study, glycation led to decreased stiffness of elastin isolated from arteries, which was associated with a thinning of elastin fibers as imaged by confocal microscopy. We observed no effect of glycation on collagen fibers under our treatment conditions. These results suggest that glycation leads to weakening of the elastin component of arteries that could contribute to vascular defects seen in diabetes and aging. Prevention of glycation reactions may be an important consideration for vascular health later in life.
Assuntos
Artérias/anatomia & histologia , Artérias/fisiologia , Animais , Aorta Torácica , Fenômenos Biomecânicos , Cães , Elastina/isolamento & purificação , Glicosilação , Técnicas In Vitro , Microscopia Confocal , Estresse Mecânico , Sus scrofa , Rigidez VascularRESUMO
Reactive oxygen species (ROS), a product of many cellular functions, has been implicated in many age-related pathophysiological processes, including cardiovascular disease. The arterial proteins collagen and elastin may also undergo structural and functional changes due to damage caused by ROS. This study examined the effect of oxidation on the mechanical response of porcine aortas and aorta elastin and the associated changes in structural protein ultrastructure as a step in exploring the role of molecular changes in structural proteins with aging on elastic artery function. We examined the change in mechanical properties of aorta samples after various oxidation times as a first step in understanding how the oxidative environment associated with aging could impact mechanical properties of arterial structural proteins. We used confocal microscopy to visualize how the microstructure of isolated elastin changed with oxidation. We find that short term oxidation of elastin isolated from aortas leads to an increase in material stiffness, but also an increase in the fiber diameter, increase in void space in the matrix, and a decrease in the fiber orientation, possibly due to fiber cross-linking. The short term effects of oxidation on arterial collagen is more complex, with increase in material stiffness seen in the collagen region of the stress stretch curve at low extents of oxidation, but not at high levels of oxidation. These results may provide insight into the relationship between oxidative damage to tissue associated with aging and disease, structure of the arterial proteins elastin and collagen, and arterial mechanical properties and function.
Assuntos
Aorta/fisiologia , Elastina/metabolismo , Envelhecimento , Animais , Fenômenos Biomecânicos , Elasticidade , Elastina/ultraestrutura , Oxirredução , Estresse Oxidativo , SuínosRESUMO
The accumulation of aggregated ß-Amyloid (Aß) in the brain is a hallmark of Alzheimer's disease and is thought to play a role in the neurotoxicity associated with the disease. The mechanism by which Aß aggregates induce toxicity is uncertain. Nonetheless, several small molecules have been found to interact with Aß fibrils and to prevent their toxicity. In this paper we studied the binding of these known toxicity inhibitors to Aß fibrils, as a means to explore surfaces or loci on Aß aggregates that may be significant in the mechanism of action of these inhibitors. We believe knowledge of these binding loci will provide insight into surfaces on the Aß fibrils important in Aß biological activity. The program DOCK was used to computationally dock the inhibitors to an Aß fibril. The inhibitors docked at two shared binding loci, near Lys28 and at the C-termini near Asn27 and Val39. The docking predictions were experimentally verified using lysine specific chemical modifications and Aß fibrils mutated at Asn27. We found that both Congo red and Myricetin, despite being structurally different, bound at the same two sites. Additionally, our data suggests that three additional Aß toxicity inhibitors may also bind in one of the sites. Identification of these common binding loci provides targets on the Aß fibril surface that can be tested in the future for their role in Aß biological activity.
Assuntos
Amiloide/antagonistas & inibidores , Amiloide/química , Amiloide/ultraestrutura , Sítios de Ligação , Vermelho Congo/química , Vermelho Congo/farmacologia , Curcumina/química , Curcumina/farmacologia , Flavonoides/química , Flavonoides/farmacologia , Microscopia Eletrônica de Transmissão , Nicotina/química , Nicotina/farmacologia , Ligação ProteicaRESUMO
beta-Amyloid (Abeta) peptide is believed to play a key role in the mechanism of Alzheimer's disease (AD). Abeta tends to aggregate to form amyloid fibrils. A variety of evidence indicates that Abeta aggregates are toxic in vitro and in vivo. An early "Abeta hypothesis" postulated that AD was the consequence of neuron death induced by insoluble deposits of large Abeta fibrils. Newer findings indicate that small soluble Abeta oligomers are the neurotoxic species, yet their structure is still unknown. Many researchers have tried to probe the differences in molecular structure between Abeta oligomers, protofibrils, and fibrils that give rise to their unique toxicities, but with limited success. In this report, we examine the hypothesis that differences in the toxicity of different aggregated Abeta species are the result of differences in species concentration and diffusivity. Using a simple mathematical analysis based on the assumption of a diffusion-limited reaction, we demonstrate that near 10-fold differences in toxicity between spherical oligomers and fibrils can be explained from size and concentration arguments. While this work does not suggest that Abeta oligomers and fibrils have identical molecular structures, it highlights the possibility that simple physical phenomena may contribute to the biological processes induced by Abeta.
Assuntos
Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/ultraestrutura , Simulação por Computador , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
Interaction between aggregates of amyloid beta protein (Abeta) and membranes has been hypothesized by many to be a key event in the mechanism of neurotoxicity associated with Alzheimer's disease (AD). Proposed membrane-related mechanisms of neurotoxicity include ion channel formation, membrane disruption, changes in membrane capacitance, and lipid membrane oxidation. Recently, osmolytes such as trehalose have been found to delay Abeta aggregation in vitro and reduce neurotoxicity. However, no direct measurements have separated the effects of osmolytes on Abeta aggregation versus membrane interactions. In this article, we tested the influence of trehalose, sucrose and trimethylamine-N-oxide (TMAO) on Abeta aggregation and fluorescent dye leakage induced by Abeta aggregates from liposomes. In the absence of lipid vesicles, trehalose and sucrose, but not TMAO, were found to delay Abeta aggregation. In contrast, all of the osmolytes significantly attenuated dye leakage. Dissolution of preformed Abeta aggregates was excluded as a possible mechanism of dye leakage attenuation by measurements of Congo red binding as well as hydrogen-deuterium exchange detected by mass spectrometry (HX-MS). However, the accelerated conversion of high order oligomers to fibril caused by vesicles did not take place if any of the three osmolytes presented. Instead, in the case of disaccharide, osmolytes were found to form adducts with Abeta, and change the dissociation dynamics of soluble oligomeric species. Both effects may have contributed to the observed osmolyte attenuation of dye leakage. These results suggest that disaccharides and TMAO may have very different effects on Abeta aggregation because of the different tendencies of the osmolytes to interact with the peptide backbone. However, the effects on Abeta membrane interaction may be due to much more general phenomena associated with osmolyte enhancement of Abeta oligomer stability and/or direct interaction of osmolyte with the membrane surface.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Membranas Artificiais , Metilaminas/química , Fragmentos de Peptídeos/metabolismo , Sacarose/química , Trealose/química , Peptídeos beta-Amiloides/toxicidade , Vermelho Congo/metabolismo , Lipossomos , Peso Molecular , Neurotoxinas/química , Neurotoxinas/metabolismo , Fragmentos de Peptídeos/toxicidade , Sacarose/farmacologia , Trealose/fisiologiaRESUMO
Beta-amyloid peptide (Abeta) is the major protein constituent found in senile plaques in Alzheimer's disease (AD). It is believed that Abeta plays a role in neurodegeneration associated with AD and that its toxicity is related to its structure or aggregation state. In this study, an approach based on chemical modification of primary amines and mass spectrometric (MS) detection was used to identify residues on Abeta peptide that were exposed or buried upon changes in peptide structure associated with aggregation. Results indicate that the N terminus was the most accessible primary amine in the fibril, followed by lysine 28, then lysine 16. A kinetic analysis of the data was then performed to quantify differences in accessibility between these modification sites. We estimated apparent equilibrium unfolding constants for each modified site of the peptide, and determined that the unfolding constant for the N terminus was approximately 100 times greater than that for K28, which was about six times greater than that for K16. Understanding Abeta peptide structure at the residue level is a first step in designing novel therapies for prevention of Abeta structural transitions and/or cell interactions associated with neurotoxicity in Alzheimer's disease.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Alquilação , Peptídeos beta-Amiloides/metabolismo , Espectrometria de Massas/métodos , Oxirredução , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
The aggregation of amyloid-beta protein (Abeta) in vivo is a critical pathological event in Alzheimer's disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimer's disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Abeta(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Abeta peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Amiloide/química , Amiloide/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Artefatos , Medição da Troca de Deutério , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Solventes/metabolismoRESUMO
beta-Amyloid peptide (A beta), the primary protein component in senile plaques associated with Alzheimer's disease (AD), has been implicated in neurotoxicity associated with AD. Previous studies have shown that the A beta-neuronal membrane interaction plays a role in the mechanism of A beta toxicity. More specifically, it is thought that A beta interacts with ganglioside rich and sialic acid rich regions of cell surfaces. In light of such evidence, we have used a number of different sialic acid compounds of different valency or number of sialic acid moieties per molecule to attenuate A beta toxicity in a cell culture model. In this work, we proposed various mathematical models of A beta interaction with both the cell membrane and with the multivalent sialic acid compounds, designed to act as membrane mimics. These models allow us to explore the mechanism of action of this class of sialic acid membrane mimics in attenuating the toxicity of A beta. The mathematical models, when compared with experimental data, facilitate the discrimination between different modes of action of these materials. Understanding the mechanism of action of A beta toxicity inhibitors should provide insight into the design of the next generation of molecules that could be used to prevent A beta toxicity associated with AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neurônios/metabolismo , Peptídeos beta-Amiloides/química , Materiais Biomiméticos/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Humanos , Modelos Biológicos , Ligação ProteicaRESUMO
Trace detection of the conformational transition of beta-amyloid peptide (Abeta) from a predominantly alpha-helical structure to beta-sheet could have a large impact in understanding and diagnosing Alzheimer's disease. We demonstrate how a novel nanofluidic biosensor using a controlled, reproducible surface enhanced Raman spectroscopy active site was developed to observe Abeta in different conformational states during the Abeta self-assembly process as well as to distinguish Abeta from confounder proteins commonly found in cerebral spinal fluid.
Assuntos
Peptídeos beta-Amiloides/análise , Técnicas Biossensoriais/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Nanotecnologia/instrumentação , Análise Espectral Raman/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Técnicas Biossensoriais/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Nanotecnologia/métodos , Análise Espectral Raman/métodos , Ressonância de Plasmônio de Superfície/métodosRESUMO
beta-Amyloid peptide (Abeta), the primary protein component in senile plaques associated with Alzheimer's disease (AD), has been implicated in neurotoxicity associated with AD. Previous studies have shown that the Abeta-neuronal membrane interaction plays a crucial role in Abeta toxicity. More specifically, it is thought that Abeta interacts with ganglioside rich and sialic acid rich regions of cell surfaces. In light of such evidence, we have hypothesized that the Abeta-membrane sialic acid interaction could be inhibited through use of a biomimic multivalent sialic acid compound that would compete with the cell surface for Abeta binding. To explore this hypothesis, we synthesized a series of photocrosslinked sialic acid containing oligosaccharides and tested their ability to bind Abeta and attenuate Abeta toxicity in cell culture assays. We show that a polymer prepared via the photocrosslinking of disialyllacto-N-tetraose (DSLNT) was able to attenuate Abeta toxicity at low micromolar concentrations without adversely affecting the cell viability. Polymers prepared from mono-sialyl-oligosaccharides were less effective at Abeta toxicity attenuation. These results demonstrate the feasibility of using photocrosslinked sialyl-oligosaccharides for prevention of Abeta toxicity in vitro and may provide insight into the design of new materials for use in attenuation of Abeta toxicity associated with AD.
Assuntos
Peptídeos beta-Amiloides/química , Ácido N-Acetilneuramínico/química , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Polímeros/química , Peptídeos beta-Amiloides/toxicidade , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/efeitos da radiação , Estrutura Molecular , Ácido N-Acetilneuramínico/efeitos da radiação , Oligossacarídeos/síntese química , Polímeros/síntese química , Polímeros/farmacologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Increasing evidence indicates that soluble aggregates of amyloid beta protein (Abeta) are neurotoxic. However, difficulty in isolating these unstable, dynamic species impedes studies of Abeta and other aggregating peptides and proteins. In this study, hydrogen-deuterium exchange (HX) detected by mass spectrometry (MS) was used to measure Abeta(1-40) aggregate distributions without purification or modification that might alter the aggregate structure or distribution. Different peaks in the mass spectra were assigned to monomer, low molecular weight oligomer, intermediate, and fibril based on HX labeling behavior and complementary assays. After 1 h labeling, the intermediates incorporated approximately ten more deuterons relative to fibrils, indicating a more solvent exposed structure of such intermediates. HX-MS also showed that the intermediate species dissociated much more slowly to monomer than did the very low molecular weight oligomers that were formed at very early times in Abeta aggregation. Atomic force microscopy (AFM) measurements revealed the intermediates were roughly spherical with relatively homogenous diameters of 30-50 nm. Quantitative analysis of the HX mass spectra showed that the amount of intermediate species was correlated with Abeta toxicity patterns reported in a previous study under the same conditions. This study also demonstrates the potential of the HX-MS approach to characterizing complex, multi-component oligomer distributions of aggregating peptides and proteins.
Assuntos
Amidas/química , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/química , Amiloide/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/ultraestrutura , Medição da Troca de Deutério , Cinética , Espectrometria de Massas , Microscopia de Força Atômica , Peso Molecular , Fragmentos de Peptídeos/ultraestrutura , Estrutura Secundária de ProteínaRESUMO
A novel detection method for organophosphate neurotoxins has been described, based on the fluorescence quenching of a Coumarin derivative. These dyes are similar in structure to some organophosphates (OPs), and they fluoresce in the blue-green region of the spectra. This methodology has been utilized for the detection of organophosphates whose hydrolysis product is p-nitrophenol by using an enzyme, organophosphorus hydrolase (OPH). Coumarin1 in the presence of p-nitrophenol results in a quenching of fluorescence, providing a direct measure of the concentration of p-nitrophenol present in the sample. The decrease in fluorescence intensity is proportional to the paraoxon concentration in the range of 7.0x10(-7)-1.7x10(-4) M. The specificity of this sensing application for p-nitrophenyl substituent OPs has also been demonstrated. OPs are a class of synthetic organic pesticides which generally have a short residual life and can cause numerous acute and chronic health effects. They have been an integral part of the agricultural industry for the past several decades due to their target specificities and selectable toxicities. The toxic nature of these compounds can be attributed to the species-specific inhibition of acetylcholinesterase (AChE), an important enzyme responsible for the regeneration of neural synaptic function. In addition to their wide agricultural and urban usage, they have also been exploited for the development of neurological chemical warfare agents. Currently available technologies for OP detection include sol-gel thin films, screen printed electrodes, acoustic patterning, gas chromatography-mass spectrometry, and various other intricate techniques that have limited field applicabilities. This optically-based approach promises much simpler and more direct detection capabilities.
Assuntos
Técnicas Biossensoriais , Medições Luminescentes/métodos , Neurotoxinas/análise , Nitrofenóis/análise , Compostos Organofosforados/análise , Praguicidas/análise , Arildialquilfosfatase/química , Cumarínicos/química , Fluorescência , Corantes Fluorescentes/química , Hidrólise , Óptica e Fotônica , Paraoxon/químicaRESUMO
beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be associated with neurotoxicity in the disease. We and others have shown that Abeta binds with relatively high affinity to clustered sialic acid residues on cell surfaces and that removal of cell surface sialic acids attenuates Abeta toxicity. We have also shown that sialic acid functionalized dendrimeric polymers can act as mimics of cell surface sialic acid clusters and attenuate Abeta-induced neurotoxicity. In the current study, we prepared sialic-acid-conjugated dendrimers using a physiologically relevant attachment of the sialic acid to the dendrimeric termini, and evaluated the Abeta toxicity attenuation properties of the dendrimers. We compared performance of sialic-acid-conjugated dendrimeric polymers in which the sialic acid moieties were attached to dendrimeric termini via the anomeric hydroxyl group of the sialic acid, a physiological attachment, to polymers in which the attachment was made via the carboxylic acid group on the sialic acid, a non-physiological attachment. This work enhances our understanding of Abeta-cell surface binding and is a step towards the development of new classes of sequestering agents as therapeutics for the prevention of Abeta toxicity in AD.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Dendrímeros/química , Dendrímeros/farmacologia , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacologia , Fragmentos de Peptídeos/toxicidade , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Fator de Crescimento Neural/farmacologia , NeuroblastomaRESUMO
beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.
Assuntos
Peptídeos beta-Amiloides/química , Cromatografia em Gel , Dicroísmo Circular , Microscopia Eletrônica , Estrutura Secundária de ProteínaRESUMO
Beta-amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and is believed to be associated with neurotoxicity in the disease. Abeta-induced neurotoxicity is strongly dependent on its structure. The aggregation of the peptide from monomeric form to fibrils is a function of many variables including time. It is because of this dynamic nature of Abeta structure that there is a necessity for an in vitro toxicity assay that is rapid enough to eliminate or at least lower the possibility of Abeta structural changes during the time required for the assay. Here we describe a fast and sensitive method with which to assess Abeta-induced neurotoxicity in SH-SY5Y neuroblastoma cells. The method employs two-color flow cytometry using annexin-phycoerythrin, a marker for cell surface phosphatidylserine that typically indicates early stages of apoptosis, and 7-amino-actinomycin D, a membrane impermeant nucleic acid dye. We compare results using the two-color assay to those obtained using the propidium iodide toxicity assay and demonstrate comparability of results, but in 2h or less with the two-color assay as opposed to 24-48 h with the propidium iodide assay. The assay described could be a useful tool in evaluating the role of Abeta structure in biological activity of the peptide.