RESUMO
Fluorescence spectroscopy is a common method for detecting amyloid fibrils in which organic fluorophores are used as markers that exhibit an increase in quantum yield upon binding. However, most of the dyes exhibit enhanced emission only when bound to mature fibrils, and significantly weaker signals are obtained in the presence of amyloid oligomers. In the concept of population inversion, a laser is used as an excitation source to keep the major fraction of molecules in the excited state to create the pathways for the occurrence of stimulated emission. In the case of the proteins, the conformational changes lead to the self-ordering and thus different light scattering conditions that can influence the optical signatures of the generated light. Using this methodology, we show it is possible to optically detect amyloid oligomers using commonly available staining dyes in which population inversion can be induced. The results indicate that rhodamine 6G molecules are complexed with oligomers, and using a laser-assisted methodology, weakly emissive states can be detected. Significant spectral red-shifting of rhodamine 6G dispersed with amyloid oligomers and a notable difference determined by comparison of spectra of the fibrils suggest the existence of specific dye aggregates around the oligomer binding sites. This approach can provide new insights into intermediate oligomer states that are believed to be responsible for toxic seeding in neurodegeneration diseases.
Assuntos
Biopolímeros/química , Lasers , Muramidase/química , Rodaminas/química , Espectrometria de FluorescênciaRESUMO
Thioflavin T (ThT) informed microviscosity changes can be used to monitor protein aggregation. Steady-state, time-resolved and lasing spectroscopy were used to detect transient states in α-synuclein - a protein associated with Parkinson's disease. The major focus was on the nucleation phase, where conventional ThT fluorescence assay lacks appropriate sensitivity to detect early stage oligomers. Instead, lasing spectroscopy and lasing threshold parameters, in particular, were sensitive to detecting protein oligomers. Through lasing spectroscopy, a change in microviscosity correlating with the stages of protein aggregation was observed at two wavelengths 405 and 440 nm. The two wavelengths are associated with free dye molecules and ß-sheet bound ThT molecules. This provides a perspective on elucidating the early formed protein aggregation, a critical aspect in understanding the pathogenesis of neurodegenerative diseases. The insights from the presented study shows the potential of using lasing spectroscopy as a sensitive tool in studying protein aggregation dynamics.
Assuntos
Benzotiazóis , Agregados Proteicos , alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Benzotiazóis/química , Viscosidade , Humanos , Corantes Fluorescentes/química , Espectrometria de FluorescênciaRESUMO
We report that short, synthetic, double- as well as single-stranded DNA can be aligned in stretched humid poly(vinyl alcohol) (PVA) matrix, and the secondary structure (nucleobase orientation) can be characterized with linear dichroism (LD) spectroscopy. Oligonucleotides of lengths varying between 10 (3.4 nm) and 60 bases (20.4 nm) were investigated with respect to structural properties in the gel-like polymer environment. The DNA conformation as a function of relative humidity reveals a strong dependence of helical structure of DNA on PVA hydration level, results of relevance for nanotechnical studies of DNA-based supramolecular systems. Also, the PVA gel could provide possibilities to test models for nucleic acid interactions and distribution in cell contexts, including structural stability of genetic material in the cell and PVA-packaging for gene delivery. A method by which duplex oligonucleotides, with sequences designed to provide specific binding sites, become amenable to polarized-light spectroscopy opens up new possibilities for studying structure in DNA complexes with small adduct molecules as well as proteins.
Assuntos
DNA de Cadeia Simples/química , Oligonucleotídeos/química , Álcool de Polivinil/química , Estrutura MolecularRESUMO
Two-photon excitation of emissive markers with near-infrared (NIR) light is of a particular interest for imaging in biology and medicine because NIR light is relatively weakly absorbed and scattered by tissues. At the same time the mechanism of two-photon absorption allows excitation of molecules located deep inside a scattering medium. In this work we demonstrate that the two-photon excitation combined with the effect of light amplification in the stimulated emission process provides a sensitive method for detecting amyloids of different forms. We investigate the two-photon excited amplified spontaneous emission (ASE) of a fluorescent dye, coumarin 307, in the brain tissue infiltrated with various amyloid phantoms i.e. oligomers, protofibrils and mature fibrils. All these forms of amyloids can be detected by observation of ASE and determination of thresholds for light amplification. On this basis we suggest that a relatively simple extension of currently used emission-based optical spectroscopy techniques can provide key information on pathogenic amyloid structures in tissue.
Assuntos
Cumarínicos , Fótons , Encéfalo , Corantes Fluorescentes/químicaRESUMO
There is currently no definitive test for early detection of neurodegeneration which is linked with protein aggregation. Finding methods capable of detecting intermediate states of protein aggregates, named oligomers, is critical for the early stage diagnosis of over 30 neurodegenerative diseases including Alzheimer's or Parkinson's. Currently, fluorescence-based imaging using Thioflavin T (ThT) dye is the gold standard for detecting protein aggregation. It is used to detect aggregation in vitro and in various tissues, including the cerebrospinal fluid (CSF), whereby the disease-related protein recombinant is seeded with the patient's fluid. The major drawback of ThT is its lack of sensitivity to oligomeric forms of protein aggregates. Here, we overcome this limitation by transferring a ThT-oligomer mixture into solid state thin films and detecting fluorescence of ThT amplified in the process of stimulated emission. By monitoring the amplified spontaneous emission (ASE) we achieved a remarkable recognition sensitivity to prefibrillar oligomeric forms of insulin and lysozyme aggregates in vitro, to Aß42 oligomers in the human protein recombinants seeded with CSF and to Aß42 oligomers doped into brain tissue. Seeding with Alzheimer patient's CSF containing Aß42 and Tau aggregates revealed that only Aß42 oligomers allowed generating ASE. Thus, we demonstrated that, in contrast to the current state-of-the-art, ASE of ThT, a commonly used histological dye, can be used to detect and differentiate amyloid oligomers and evaluate the risk levels of neurodegenerative diseases to potential patients before the clinical symptoms occur.
RESUMO
Amyloid fibrils are a well-recognized hallmark of neurodegeneration. A common approach to detect amyloid fibrils is staining with organic molecules and monitoring optical properties using fluorescence spectroscopy. However, the structural diversity of amyloids necessitates new sensitive methods and probes that can be reliably used to characterize them. Here, Coumarin 307 is applied for lysozyme fibrils detection by observation of laser action in the process of two-photon excited stimulated emission. It is shown that the lasing threshold and spectrum significantly depend on the adopted structure (α-helix or ß-sheet) of the lysozyme protein, whereas fluorescence spectrum is insensitive to the protein structure. The applications of coherent stimulated emission light that can be emitted deep inside a scattering medium can be particularly promising for imaging and therapeutic purposes in the neurodegeneration field. Two-photon excitation with the near-infrared light, which allows the deepest penetration of tissues, is an important advantage of the method.
Assuntos
Amiloide/análise , Cumarínicos/química , Muramidase/análise , Doenças Neurodegenerativas/diagnóstico por imagem , Antioxidantes/química , Etanol/química , Corantes Fluorescentes/química , Humanos , Luz , Dinâmica não Linear , Fótons , Estrutura Secundária de Proteína , Espalhamento de Radiação , Espectrometria de FluorescênciaRESUMO
Protein aggregation is associated with numerous devastating diseases such as Alzheimer's, Parkinson's, and prion diseases. Development of therapeutics would benefit from knowledge of the structural organization of protein molecules in these amyloid aggregates, particularly in their aqueous biological milieu. However, detailed structural studies to date have been mainly on the solid state and have required large quantities of purified aggregate. Moreover, these conventional methods require the aggregated assembly to remain structurally stable over days or weeks required to perform the experiment, whereas the pathologically relevant species of in vivo aggregates may be shorter lived. Here, we show the organization of protein chains in dissolved amyloid aggregates can be readily determined spectroscopically using minute quantities of fluorescein-labeled protein segments in a matter of minutes. Specifically, we investigated the possibility of using the ultrafast dynamics of fluorescein to distinguish among three categories of ß-sheet geometry: (1) antiparallel in-register, (2) parallel in-register, or (3) antiparallel out-of-register. Fluorescein, the most commonly used staining dye in biology and medicine, was covalently attached to the N-termini of peptide sequences selected from a library of known amyloid crystal structures. We investigated the aggregates in solution using steady-state and time-resolved absorption and fluorescence spectroscopy. We found that the dynamics of fluorescein relaxation from the excited state revealed amyloid structure-specific information. Particularly, the nonfluorescent cation form of fluorescein showed remarkable sensitivity to local environments created during aggregation. We demonstrate that time-resolved absorption is capable of differentiating strand organization in ß-sheet aggregates when strong intermolecular coupling between chromophores occurs. This approach can be useful in optical recognition of specific fibril architectures of amyloid aggregates.
Assuntos
Proteínas Amiloidogênicas/química , Fluoresceína/química , Corantes Fluorescentes/química , Oligopeptídeos/química , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
AIM: To examine the effectiveness of human placental inhibitors, by injecting vitamin E to rats with transplanted Morris-5123 hepatoma, on the expression of cathepsins B and L in tumor, liver, lung and blood sera after transplantation of Morris 5123 hepatoma. METHODS: Animals were divided into 10 groups receiving three different concentrations of vitamin E and inhibitors along or in combination and compared with negative control (healthy rats) and positive control (tumor rats). Effectiveness of treatment was evaluated with regard to survival time, tumor response and determination of the activities of proteolytic enzymes and their inhibitors using flurogenic substrates. RESULTS: Cathepsins B and L activities were elevated by 16-fold in comparison with negative control tissues, and their endogenous inhibitor activity decreased by 1.2-fold before treatment. In several cases, tumors completely disappeared following vitamin E plus human placental cyteine protease inhibitor (CPI) compared with controls. The number of complete tumor responses was higher when 20 m/kg vitamin E plus 400 microg of CPI was used, i.e. 7/10 rats survived more than two mo. Cathepsins B and L were expressed significantly in tumor, liver, lung tissues and sera in parallel to the increasing of the endogenous inhibitor activity compared with the controls after treatment (P<0.0001). CONCLUSION: The data indicate formation of metastasis significantly reduced in treated rats, which might provide a therapeutic basis for anti-cancer therapy.
Assuntos
Antioxidantes/farmacologia , Catepsina B/metabolismo , Catepsinas/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Vitamina E/farmacologia , Animais , Catepsina L , Cisteína Endopeptidases , Modelos Animais de Doenças , Feminino , Humanos , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Pulmão/metabolismo , Masculino , Transplante de Neoplasias , Placenta/metabolismo , Ratos , Ratos WistarRESUMO
A stretched poly(vinyl alcohol) (PVA) film provides a unique matrix that enables also short DNA oligonucleotide duplex to be oriented and studied by linear dichroism (LD). This matrix further allows controlling DNA secondary structure by proper hydration (A or B form), and such humid films could potentially also mimic the molecular crowding in cellular contexts. However, early attempts to study intercalators and groove binders for probing DNA in PVA failed due to competitive matrix binding. Here we report the successful orientation in PVA of DNA oligonucleotide duplex hairpins with thread-intercalated binuclear complex [µ-(11,11'-bidppz)(phen)4Ru2](4+), and how LD depends on oligonucleotide sequence and metal center chirality. Opposite enantiomers of the ruthenium complex, ΔΔ and ΛΛ, were investigated with respect to enantioselectivity toward GC stretches as long as 22 bp. LD, supported by emission kinetics, reveals that threading intercalation occurs only with ΔΔ whereas ΛΛ remains externally bound, probably in either or both of the grooves of the GC-DNA. Enantioselective binding properties of sterically rigid DNA probes such as the ruthenium complexes could find applications for targeting nucleic acids, e.g., to inhibit transcription in therapeutic context such as treatment of malaria or cancer.
Assuntos
Complexos de Coordenação/química , Substâncias Intercalantes/química , Oligonucleotídeos/metabolismo , Rutênio/química , Sequência de Bases , Sítios de Ligação , Complexos de Coordenação/farmacologia , DNA/química , DNA/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Substâncias Intercalantes/farmacologia , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Álcool de Polivinil/química , Rutênio/farmacologia , EstereoisomerismoRESUMO
We report remarkable multiphoton absorption properties of DNA intercalating ruthenium complexes: (1) [Ru(phen)(2)dppz](2+); (2) [(11,11'-bidppz)(phen)(4)Ru(2)](4+); (3) [11,11'-bipb(phen)(4)Ru(2)](4+). Two-photon spectra in the range from 460 to 1100 nm were measured using the Z-scan technique. In particular, complex 2 was found to exhibit very strong two- and three-photon absorption properties, which could be an effect of symmetric charge transfer from the ends towards the middle of the conjugated dimeric orbital system. We propose that these molecules could provide a new generation of DNA binding nonlinear chromophores for wide applications in biology and material science. The combination of a large two-photon cross section and strong luminescence quantum yields for the molecules when intercalated makes the compounds uniquely bright and photo-stable probes for two-photon luminescence imaging and also promising as enhanced photosensitizers in two-photon sensitizing applications.
Assuntos
Sondas de DNA/química , Fótons , Fármacos Fotossensibilizantes/química , Rutênio/química , Substâncias Intercalantes/química , Luminescência , Compostos OrganometálicosRESUMO
We investigate how DNA interacts with drugs in humid polyvinyl alcohol (PVA) films by using a homologous set of cyanine dyes (YO(+), YO-PRO(2+), and YOYO(4+)) known to intercalate into DNA with increasing affinity with increasing charge. UV-vis spectroscopy shows that the PVA matrix destabilizes all three DNA-dye complexes compared to aqueous solution but to a lesser degree as the dye charge increases. The monovalent YO is fully dissociated from DNA within minutes, whereas the dissociation of the divalent YO-PRO takes about one hour and occurs by a two-step mechanism. The tetravalent homodimer YOYO is even less affected by the PVA environment and remains intercalated in the B-form DNA also in the PVA films. The reduced stability of the DNA-dye complexes is discussed in terms of steric and dielectric properties of the PVA matrix. After being kept in dry PVA films for 48 h the DNA-YOYO complexes can be reformed reversibly by rehumidifying the films for 30 min. The ability to store aligned and confined DNA intercalated with ligand complexes may be useful in studies on structural properties of nucleic acids.