Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 778
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Eur J Histochem ; 63(3)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31455073

RESUMO

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.


Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Polímeros/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Bases , Carbocianinas/química , Carbocianinas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Inativação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Metabolismo dos Lipídeos , Lipídeos/toxicidade , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/toxicidade , Neoplasias Hepáticas/genética , Polímeros/metabolismo , Polímeros/toxicidade , Interferência de RNA
2.
Spectrochim Acta A Mol Biomol Spectrosc ; 216: 190-201, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30901704

RESUMO

Spectral-fluorescent properties of polymethine dye probes anionic 3,3'-di(sulfopropyl)-4,5,4',5'-dibenzo-9-ethylthiacarbocyanine-betaine (DEC) and cationic 3,3',9-trimethylthiacarbocyanine iodide (Cyan 2) in the presence of biological surfactants, bile salts sodium cholate (NaC), sodium deoxycholate (NaDC) and sodium taurocholate (NaTC), as well as sodium dodecyl sulfate (SDS), have been studied in a wide range of surfactant concentrations. When a surfactant is introduced into a solution of DEC, changes of the spectral-fluorescent properties are observed due to decomposition of dye dimers into cis-monomers and cis-trans conversion of the resulting monomers. In the presence of SDS, both processes occur in parallel, caused by noncovalent interaction of dye monomers with micelles, and mainly occur near the critical micelle concentration (CMC). In contrast, upon the introduction of increasing concentrations of bile salts, decomposition of dye dimers into the monomers begins at lower concentrations than cis-trans conversion. The former process is almost completed at concentrations close to CMC of secondary micelles (CMC2), while the latter process occurs even at concentrations of bile salts much higher than CMC2. Hence, DEC can serve as a probe that permits estimating the value of CMC2 and is indicative of reorganization of secondary micelles upon an increase in bile salt concentration. Aggregation of DEC and Cyan 2 on bile salts is also observed. Since it is observed at relatively low concentrations of bile salts (

Assuntos
Carbocianinas/metabolismo , Ácido Desoxicólico/metabolismo , Indóis/metabolismo , Colato de Sódio/metabolismo , Tensoativos/metabolismo , Ácido Taurocólico/metabolismo , Betaína/análogos & derivados , Betaína/metabolismo , Carbocianinas/química , Ácido Desoxicólico/química , Dimerização , Indóis/química , Micelas , Colato de Sódio/química , Dodecilsulfato de Sódio/química , Dodecilsulfato de Sódio/metabolismo , Espectrometria de Fluorescência , Tensoativos/química , Ácido Taurocólico/química
3.
Iran Biomed J ; 23(3): 200-8, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30797224

RESUMO

Background: Mesenchymal stem cells (MSCs) can be used to treat premature ovarian failure (POF). Different methods have already been applied to detect MSCs in tissues. This study aimed to investigate the quantitative distribution of CM-DiI-labeled human umbilical cord vein MSCs (hUCV-MSCs) in different regions of the ovarian tissue of the cyclophosphamide (CTX)-induced POF in mice. Methods: Adult female C57BL/6 mice (n = 40) were divided into four groups: (1) Mice receiving PBS as control (Ctrl) group; (2) mice receiving hUCV-MSCs intravenously as Ctrl + hUCV-MSCs group; (3) mice receiving CTX intraperitoneally (i.p.) as CTX group; (4) mice receiving CM-DiI-labeled hUCV-MSCs after CTX injection as CTX + hUCV-MSCs group. Histological changes and CM-DiI-labeled hUCV-MSCs distribution were analyzed in the ovarian tissues. Quantitative real-time PCR was performed to detect human mitochondrial cytochrome b (MTCYB) gene in the ovarian tissues of the mice. Results: The mean number of the fluorescent hUCV-MSCs was 20 ± 2.5 (57.1%) in the medulla, 11.3 ± 2.8 (32.2%) in the cortex, and 5.5 ± 1 (15%) in the germinal epithelium of the ovarian tissue (p < 0.05). Moreover, MTCYB gene was detected in the mice ovaries of the CTX + hUCV-MSCs group, but not in other groups. Conclusion: Our findings suggest that the distribution of the transplanted hUCV-MSCs in different regions of the ovarian tissue is not equal, and it is greater in the medulla than the cortex and germinal epithelium. This is the first report of quantitative distribution of MSCs in different regions of ovarian tissue in the POF model.


Assuntos
Carbocianinas/metabolismo , Movimento Celular , Células-Tronco Mesenquimais/citologia , Ovário/lesões , Ovário/patologia , Coloração e Rotulagem , Cordão Umbilical/citologia , Animais , Ciclofosfamida , Citocromos b/genética , Citocromos b/metabolismo , Feminino , Humanos , Camundongos Endogâmicos C57BL , Insuficiência Ovariana Primária/patologia
4.
Molecules ; 24(3)2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717191

RESUMO

The bioluminescence resonance energy transfer (BRET) approach involves resonance energy transfer between a light-emitting enzyme and fluorescent acceptors. The major advantage of this technique over biochemical methods is that protein-protein interactions (PPI) can be monitored without disrupting the natural environment, frequently altered by detergents and membrane preparations. Thus, it is considered as one of the most versatile technique for studying molecular interactions in living cells at "physiological" expression levels. BRET analysis has been applied to study many transmembrane receptor classes including G-protein coupled receptors (GPCR). It is well established that these receptors may function as dimeric/oligomeric forms and interact with multiple effectors to transduce the signal. Therefore, they are considered as attractive targets to identify PPI modulators. In this review, we present an overview of the different BRET systems developed up to now and their relevance to identify inhibitors/modulators of protein⁻protein interaction. Then, we introduce the different classes of agents that have been recently developed to target PPI, and provide some examples illustrating the use of BRET-based assays to identify and characterize innovative PPI modulators in the field of GPCRs biology. Finally, we discuss the main advantages and the limits of BRET approach to characterize PPI modulators.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Ensaios de Triagem em Larga Escala , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Anticorpos/química , Anticorpos/farmacologia , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbocianinas/química , Carbocianinas/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/farmacologia , Multimerização Proteica , Pontos Quânticos/química , Pontos Quânticos/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química
5.
Spectrochim Acta A Mol Biomol Spectrosc ; 209: 256-263, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30414574

RESUMO

The interaction of fluorescent dyes with serum proteins has garnered significant interest owing to potential for non-covalent labeling and imaging applications. In this work, dimeric benzothiazole-based trimethine cyanine dyes are synthesized and their interaction with bovine serum albumin studied. The dimeric cyanine dyes mainly exist as H-dimers and H-aggregates in aqueous solution. A combination of absorbance, fluorescence, circular dichroism spectroscopy and atomic force and fluorescence microscopy indicate the formation of dye-BSA complexes. Binding of one of the dimeric dyes on BSA with a Ka of 1.49×105M-1 results in disruption of dye self-aggregates and unfolding of the dyes into the monomeric or open conformation. Fluorescence enhancement experienced by the dimeric dyes upon interaction with BSA is superior to that registered by Thioflavin T. Surfactant SDS has been used to further tune the self-aggregation of the dimeric dye resulting in a 200-fold fluorescence enhancement in presence of BSA. Interaction of a dimeric dye with BSA under conditions favoring protein aggregation is found to result in faster dye binding and the resulting fluorescence enhancement is easily visualized by fluorescence microscopy. The interaction of a dimeric cyanine dye aggregate with BSA is promising for non-covalent labeling applications in sharp contrast to the monomeric dye counterpart.


Assuntos
Carbocianinas/química , Fluorescência , Corantes Fluorescentes/química , Polímeros/química , Soroalbumina Bovina/química , Tensoativos/química , Animais , Carbocianinas/metabolismo , Bovinos , Dicroísmo Circular , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência
6.
Invest Ophthalmol Vis Sci ; 59(15): 5876-5884, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30543343

RESUMO

Purpose: To determine whether cerebrospinal fluid (CSF) entry into the optic nerve is altered in glaucoma. Methods: Fluorescent 10-kDa dextran tracer was injected into the CSF of 2-month-old (n = 9) and 10-month-old DBA/2J glaucoma mice (n = 8) and age-matched controls (C57Bl/6; n = 8 each group). Intraocular pressure (IOP) was measured in all mice before tracer injection into CSF. Tracer distribution was assessed using confocal microscopy of optic nerve cross-sections of mice killed 1 hour after injection. Paravascular tracer distribution in the optic nerve was studied in relation to isolectin-stained blood vessels. Tracer intensity and cross-sectional area in the laminar optic nerve were quantitatively assessed in all four groups and statistically compared. Aquaporin 4 (AQP4) and retinal ganglion cell axonal phosphorylated neurofilament (pNF) were evaluated using immunofluorescence and confocal microscopy. Results: IOP was elevated in 10-month-old glaucoma mice compared with age-matched controls. One hour after tracer injection, controls showed abundant CSF tracer in the optic nerve subarachnoid space and within the nerve in paravascular spaces surrounding isolectin-labeled blood vessels. CSF tracer intensity and signal distribution in the optic nerve were significantly decreased in 10-month-old glaucoma mice compared with age-matched controls (P = 0.0008 and P = 0.0033, respectively). AQP4 immunoreactivity was similar in 10-month-old DBA and age-matched control mice. Half of the 10-month-old DBA mice (n = 4/8) showed a decrease in pNF immunoreactivity compared to controls. Altered pNF staining was seen only in DBA mice lacking CSF tracer at the laminar optic nerve (n = 4/5). Conclusions: This study provides the first evidence that CSF entry into the optic nerve is impaired in glaucoma. This finding points to a novel CSF-related mechanism that may help to understand optic nerve damage in glaucoma.


Assuntos
Líquido Cefalorraquidiano/metabolismo , Glaucoma/metabolismo , Doenças do Nervo Óptico/metabolismo , Animais , Aquaporina 4/metabolismo , Axônios/metabolismo , Axônios/patologia , Carbocianinas/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Glaucoma/patologia , Pressão Intraocular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microscopia Confocal , Proteínas de Neurofilamentos/metabolismo , Doenças do Nervo Óptico/patologia , Fosforilação , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia
7.
Talanta ; 188: 66-73, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30029428

RESUMO

Osteosarcoma (OS) is one of most malignant bone tumors in early adolescence, which is a highly metastatic cancer and pulmonary metastasis is the most common cause of death. Thus, the development of efficient approaches to discover potential compounds that target metastasis of OS remains a topic of considerable interest. In this study, subtractive Cell-SELEX was performed to screen OS metastasis specific DNA aptamers by using cell lines with similar tumorigenic potentials but opposite metastatic aggressiveness (highly metastatic 143B cells and non-metastatic U-2 OS cells as the target and negative cells, respectively). This in vitro selection generated an ssDNA aptamer LP-16 that exhibited high binding affinity to 143B cells with an equilibrium dissociation constant (Kd) of 56.73 ±â€¯7.750 nM. However, the aptamer LP-16 did not bind to the non-metastatic U-2 OS and normal hFOB 1.19 cells. We further preliminarily presumed the target molecules of aptamer LP-16 was a membrane protein on the cell surface by proteinase treatment. Furthermore, both in vivo fluorescence imaging and clinical tissue imaging also clearly demonstrated that LP-16 could achieve prominently targeting efficiency. Therefore, the ssDNA aptamer LP-16 generated here could be a promising molecular probe for OS metastasis diagnosis. We have developed subtractive Cell-SELEX to screen osteosarcoma metastasis specific DNA aptamers by using cell lines with similar tumorigenic potentials but opposite metastatic aggressiveness (highly metastatic 143B cells and non-metastatic U-2 OS cells as the target and negative cells, respectively).


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA de Cadeia Simples/metabolismo , Sondas Moleculares/metabolismo , Metástase Neoplásica/diagnóstico por imagem , Osteossarcoma/diagnóstico por imagem , Técnica de Seleção de Aptâmeros/métodos , Animais , Carbocianinas/química , Carbocianinas/metabolismo , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia de Fluorescência , Sondas Moleculares/química , Osteossarcoma/classificação , Osteossarcoma/metabolismo
8.
Int J Mol Sci ; 19(7)2018 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-29986516

RESUMO

JC-1, a cationic fluorescent dye when added to living cells, is known to be localized exclusively in mitochondria, particularly in good physiological conditions characterized by sufficient mitochondrial membrane potential (ΔΨ). The accumulation of JC-1 in these organelles leads to the formation J-aggregates (with a specific red fluorescence emission maximum at 590 nm), which is in addition to the typical green fluorescence of J-monomers (emission maximum of ∼529 nm). The lack of mitochondrial ΔΨ leads to the depression of JC-1 mitochondrial accumulation and a decrease in J-aggregate formation. Therefore, the ratio between the red and green fluorescence of cells loaded with JC-1 is often used for the detection of the mitochondrial membrane potential. However, JC-1 represents a suitable substrate of the multidrug transporter P-glycoprotein (P-gp). Therefore, the depression of the JC-1 content in intracellular space and particularly in the mitochondria to a level that is inefficient for J-aggregate formation could be expected in P-gp-positive cells. In the current paper, we proved this behavior on parental P-gp-negative L1210 (S) cells and their P-gp-positive variants obtained by either selection with vincristine (R) or transfection with the human gene encoding P-gp (T). P-glycoprotein inhibitors cyclosporine A and verapamil fail to restore JC-1 loading of the R and T cells to an extent similar to that observed in S cells. In contrast, the noncompetitive high affinity P-gp inhibitor tariquidar fully restored JC-1 accumulation and the presence of the typical red fluorescence of J-aggregates. In the presence of tariquidar, measurement of the JC-1 fluorescence revealed similar levels of mitochondrial membrane potential in P-gp-negative (S) and P-gp-positive cells (R and T).


Assuntos
Benzimidazóis/metabolismo , Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Ciclosporina/farmacologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Quinolinas/farmacologia , Verapamil/farmacologia
10.
Proc Natl Acad Sci U S A ; 115(25): E5706-E5715, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29866842

RESUMO

The stability of organic dyes against photobleaching is critical in single-molecule tracking and localization microscopy. Since oxygen accelerates photobleaching of most organic dyes, glucose oxidase is commonly used to slow dye photobleaching by depleting oxygen. As demonstrated here, pyranose-2-oxidase slows bleaching of Alexa647 dye by ∼20-fold. However, oxygen deprivation may pose severe problems for live cells by reducing mitochondrial oxidative phosphorylation and ATP production. We formulate a method to sustain intracellular ATP levels in the presence of oxygen scavengers. Supplementation with metabolic intermediates including glyceraldehyde, glutamine, and α-ketoisocaproate maintained the intracellular ATP level for at least 10 min by balancing between FADH2 and NADH despite reduced oxygen levels. Furthermore, those metabolites supported ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate and internalization of PAR2 receptors. Our method is potentially relevant to other circumstances that involve acute drops of oxygen levels, such as ischemic damage in the brain or heart or tissues for transplantation.


Assuntos
Trifosfato de Adenosina/metabolismo , Oxigênio/metabolismo , Carbocianinas/metabolismo , Linhagem Celular , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/metabolismo , Fluorescência , Corantes Fluorescentes/metabolismo , Glucose Oxidase/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Cetoácidos/metabolismo , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , NAD/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fotodegradação , Receptor PAR-2/metabolismo
11.
Nat Commun ; 9(1): 1882, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29760422

RESUMO

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.


Assuntos
Aminoácidos/genética , Toxinas Bacterianas/química , Materiais Biomiméticos/química , Proteínas de Fluorescência Verde/genética , Proteínas Hemolisinas/química , Bicamadas Lipídicas/química , RNA de Transferência/genética , Aminoácidos/metabolismo , Toxinas Bacterianas/metabolismo , Transporte Biológico , Materiais Biomiméticos/metabolismo , Carbocianinas/química , Carbocianinas/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hemolisinas/metabolismo , Lasers , Bicamadas Lipídicas/metabolismo , Fusão de Membrana , Pinças Ópticas , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , RNA de Transferência/metabolismo , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo , Cloreto de Sódio/química
12.
Chem Commun (Camb) ; 54(49): 6252-6255, 2018 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-29736504

RESUMO

Fluorescent unimolecular micelles (FUMs) with multicolor emission acting as fluorescent nanoagents for optical fluorescence imaging have, for the first time, been reported. The FUMs show good water-solubility, ultra-small size, and enhanced biocompatibility, which endow the FUMs with versatile applications including organelle labeling, multicolor markers and high tumor accumulation, revealing that our design can serve as a rational strategy for the development of UM-based fluorescent nanoagents for bioprocess monitoring.


Assuntos
Corantes Fluorescentes/metabolismo , Metacrilatos/metabolismo , Micelas , Neoplasias/diagnóstico por imagem , Polietilenoglicóis/metabolismo , beta-Ciclodextrinas/metabolismo , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Carbocianinas/síntese química , Carbocianinas/química , Carbocianinas/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Feminino , Fluoresceínas/síntese química , Fluoresceínas/química , Fluoresceínas/metabolismo , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Lisossomos/metabolismo , Metacrilatos/síntese química , Metacrilatos/química , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Tamanho da Partícula , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Rodaminas/síntese química , Rodaminas/química , Rodaminas/metabolismo , Solubilidade , beta-Ciclodextrinas/síntese química , beta-Ciclodextrinas/química
13.
Chemistry ; 24(26): 6727-6731, 2018 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-29505156

RESUMO

As key molecules in most biological pathways, proteins physically contact one or more biomolecules in a highly specific manner. Several driving forces (i.e., electrostatic and hydrophobic) facilitate such interactions and a variety of methods have been developed to monitor these processes both in vivo and in vitro. In this work, a new method is reported for the detection of protein interactions by visualizing a color change of a cyanine compound, a supramolecule complex of 3,3-di-(3-sulfopropyl)-4,5,4',5'-dibenzo-9-methyl-thiacarbocyanine triethylammonium salt (MTC). Nuclear magnetic resonance (NMR) studies suggest that the hydrophobic nature of the protein surfaces drives MTC into different types of aggregates with distinct colors. When proteins interact with other biomolecules, the hydrophobic surface of the complex differs, resulting in a shift in the form of MTC aggregation, which results in a color change. As a result, this in vitro method has the potential to become a rapid tool for the confirmation of protein-biomolecule interactions, without the requirements for sophisticated instrumentation or approaches.


Assuntos
Carbocianinas/química , Colorimetria , Proteínas/química , Carbocianinas/metabolismo , DNA/química , DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Proteínas/metabolismo , Eletricidade Estática
14.
Bioorg Med Chem Lett ; 28(4): 572-576, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29402740

RESUMO

Prostate-specific membrane antigen (PSMA) is an important biological target for therapy and diagnosis of prostate cancer. In this study, novel multivalent PSMA inhibitors with glutamate-urea-lysine structures were designed to improve inhibition characteristics. Precursors of the novel inhibitors were prepared from glutamic acid with di-tert-butyl ester. A near-infrared molecular dye, sulfo-Cy5.5, was introduced into the precursors to generate the final PSMA fluorescent inhibitors, compounds 12-14, to visualize prostate cancer. Biological behaviors of the inhibitors were evaluated using in vitro inhibition assays, in vivo fluorescent imaging, and ex vivo biodistribution assays. Ki values from inhibition studies indicated that dimeric inhibitor 13 with a glutamine linker showed approximately 3-fold more inhibitory activity than monomeric inhibitor 12. According to other biological studies using a mouse model of prostate cancer, dimeric inhibitor compounds 13 and 14 had higher tumor accumulation than the monomer. However, glutamine-based dimeric inhibitor 13 showed lower liver uptake than dimeric inhibitor 14, which had a benzene structure. Thus, these studies suggest that glutamine-based dimeric inhibitor 13 can be a promising optical inhibitor of prostate cancer.


Assuntos
Antineoplásicos/farmacologia , Corantes Fluorescentes/farmacologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Carbocianinas/síntese química , Carbocianinas/metabolismo , Carbocianinas/farmacologia , Feminino , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Distribuição Tecidual
15.
Transl Stroke Res ; 9(1): 74-91, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28766251

RESUMO

Intracerebral hemorrhage (ICH) is a cerebrovascular disease with high mortality and morbidity, and the effective treatment is still lacking. We designed this study to investigate the therapeutic effects and mechanisms of melatonin on the secondary brain injury (SBI) after ICH. An in vivo ICH model was induced via autologous whole blood injection into the right basal ganglia in Sprague-Dawley (SD) rats. Primary rat cortical neurons were treated with oxygen hemoglobin (OxyHb) as an in vitro ICH model. The results of the in vivo study showed that melatonin alleviated severe brain edema and behavior disorders induced by ICH. Indicators of blood-brain barrier (BBB) integrity, DNA damage, inflammation, oxidative stress, apoptosis, and mitochondria damage showed a significant increase after ICH, while melatonin reduced their levels. Meanwhile, melatonin promoted further increasing of expression levels of antioxidant indicators induced by ICH. Microscopically, TUNEL and Nissl staining showed that melatonin reduced the numbers of ICH-induced apoptotic cells. Inflammation and DNA damage indicators exhibited an identical pattern compared to those above. Additionally, the in vitro study demonstrated that melatonin reduced the apoptotic neurons induced by OxyHb and protected the mitochondrial membrane potential. Collectively, our investigation showed that melatonin ameliorated ICH-induced SBI by impacting apoptosis, inflammation, oxidative stress, DNA damage, brain edema, and BBB damage and reducing mitochondrial membrane permeability transition pore opening, and melatonin may be a potential therapeutic agent of ICH.


Assuntos
Antioxidantes/uso terapêutico , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/etiologia , Hemorragia Cerebral/complicações , Melatonina/uso terapêutico , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Benzimidazóis/metabolismo , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Lesões Encefálicas/patologia , Carbocianinas/metabolismo , Córtex Cerebral/patologia , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Marcação In Situ das Extremidades Cortadas , Inflamação/tratamento farmacológico , Inflamação/etiologia , Masculino , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/etiologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Rodaminas/metabolismo , Fatores de Tempo
16.
Mol Vis ; 23: 832-843, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29259390

RESUMO

Purpose: Subretinal injections are used to deliver agents in experimental studies of retinal diseases, often through viral vectors. However, few studies have investigated the effects of subretinal injections alone on the structure and function of the healthy or diseased retina, particularly in models of oxygen-induced retinopathy (OIR). We report on the effects of subretinal injections in a rat OIR model, which is used to study mechanisms of retinopathy of prematurity. Methods: Within 6 h of birth, neonatal rat pups were exposed to repeated cycles of oxygen between 50% and 10% O2 every 24 h for 14 days and subsequently moved to room air. On postnatal day 8 (P8), animals were treated in both eyes with advancement of the injection needle into the vitreous (pilot-treated) or with a subretinal PBS injection (sPBS-treated) or were left untreated (untreated). Additional control animals were exposed to microscope light after eyelid opening only (light-treated). Retinal fundus images were recorded on P26. Areas of the avascular retina and intravitreal neovascularization were determined in flat mounted retinas stained with isolectin B4 on P32. Retinal function of the respective eyes was assessed with the Ganzfeld electroretinogram (ERG) on P31 or P32 and with focal ERG in the central retina on P28 or P29. The thickness of the retinal layers was measured with spectral domain optical coherence tomography (OCT) on P30 and in opsin- and TO-PRO 3-stained retinal cryosections from pups euthanized on P32. Two sections were analyzed in each pup. For each section, three images of three different locations were analyzed accounting for 18 thickness measurements per pup. Results: Compared to untreated animals, the avascular area of the retina was greater in the pilot-treated (p<0.05) and sPBS-treated eyes (p<0.01), and the sPBS-treated eyes had a greater avascular retinal area compared to the pilot-treated eyes (p<0.01). The intravitreal neovascular area was larger in the sPBS-treated eyes compared to the untreated eyes (p<0.01). The outer nuclear and outer segment layers were thinner in the pilot- (p<0.01) and sPBS-treated eyes (p<0.05) compared to the untreated eyes as measured with OCT and immunohistochemical staining of the retinal cryosections. Compared to the untreated eyes, the amplitudes of the scotopic a- and b-waves in the Ganzfeld ERG were reduced in the pilot-treated eyes (p<0.001 and p<0.01, respectively), but only the a-wave was reduced in the sPBS-treated eyes (p<0.001). The a-wave amplitude in the focal ERG was reduced in the pilot- and sPBS-treated eyes, and no difference was seen in the b-wave amplitude between any of the groups. There was no difference between the light-treated and untreated eyes in the areas of the avascular retina or intravitreal neovascularization or Ganzfeld or focal ERG. Conclusions: Pilot injections alone without injection into the subretinal space resulted in an increased avascular retinal area, reduced thickness of the photoreceptors, and reduced ERG function compared to the untreated animals. Although subretinal PBS injections further increased the areas of avascular retina and intravitreal neovascularization and resulted in similar retinal thinning compared to the pilot treatment, inner retinal function was improved, as evidenced by higher Ganzfeld b-wave amplitudes. Differences in the Ganzfeld and focal ERGs may indicate that the peripheral retina is more susceptible to remote beneficial effects from potential protective mechanisms induced by subretinal injection. This study stresses the importance of appropriate controls in experiments with subretinal delivery of agents.


Assuntos
Modelos Animais de Doenças , Oxigênio/toxicidade , Retina/efeitos dos fármacos , Retina/fisiopatologia , Neovascularização Retiniana/fisiopatologia , Retinopatia da Prematuridade/fisiopatologia , Animais , Animais Recém-Nascidos , Carbocianinas/metabolismo , Eletrorretinografia , Feminino , Imuno-Histoquímica , Injeções Intraoculares , Masculino , Microscopia Confocal , Opsinas/metabolismo , Oxigênio/administração & dosagem , Ratos , Ratos Sprague-Dawley , Neovascularização Retiniana/metabolismo , Vasos Retinianos/patologia , Retinopatia da Prematuridade/metabolismo , Tomografia de Coerência Óptica
17.
Biophys Chem ; 230: 62-67, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28965786

RESUMO

Cy3 and Cy5 dyes linked to the 5' end of a double stranded DNA molecule are known to attach to both strands in a way that is very similar to an additional base pair and has a stabilizing effect on the oligonucleotide. Here we adapt the Peyrard-Bishop mesoscopic model to incorporate cyanine dyes and use the technique of thermal equivalence to obtain the appropriate parameters from existing melting temperatures. We have found that the stacking parameters are in the same range of ordinary AT and CG base pairs, in particular Cy3-A was found to be most rigidly stacked. While the cyanines stabilize the AT hydrogen bonds quite strongly the CG bonds are mostly unaffected.


Assuntos
Carbocianinas/química , DNA/química , Pareamento de Bases , Carbocianinas/metabolismo , DNA/metabolismo , Ligações de Hidrogênio , Modelos Moleculares , Desnaturação de Ácido Nucleico , Temperatura Ambiente
18.
Int J Pharm ; 534(1-2): 128-135, 2017 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-28982548

RESUMO

Buccal administration route is a promising way for a large number of drugs exhibiting a low oral bioavailability. The present work describes the formulation and evaluation of a mucoadhesive and thermosensitive in situ gelling delivery system based on poloxamer 407, poloxamer 188 and xanthan gum for buccal drug delivery. First, the mucoadhesion properties were evaluated using a tensile test. The effect of xanthan gum on the mucoadhesion force was demonstrated. Then, to assess the buccal residence time which reflects the mucoadhesion properties, the validation of a fluorescence probe for in vivo optical imaging experiment was conducted. Methyl-Cyanine 5 derivative (Me-Cy5) was used to label the hydrogels, dissolution tests and permeation studies through buccal epithelium cells showed that Me-Cy5 release from hydrogels was mainly due to an erosion mechanism and presented a limited penetration across epithelium cells. These results suggest that, Me-Cy5 is a suitable marker for thermosensitive in situ gelling delivery systems as the probe mostly stays entrapped in the hydrogel and do not cross the epithelial barrier. Buccal residence performance of the hydrogel was evaluated for the first time by non-invasive optical imaging after administration to mice. This technique is an interesting alternative compared to visual observations and sacrifice involved experiments, which could also be exploited to various administration routes.


Assuntos
Carbocianinas/química , Carbocianinas/metabolismo , Preparações de Ação Retardada/química , Preparações de Ação Retardada/metabolismo , Mucosa Bucal/metabolismo , Administração Bucal , Animais , Disponibilidade Biológica , Preparações de Ação Retardada/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Epitélio/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Poloxâmero/química , Polissacarídeos Bacterianos/química , Temperatura Ambiente
19.
Front Neural Circuits ; 11: 60, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28878629

RESUMO

The activity-regulated cytoskeleton associated protein Arc is strongly and quickly upregulated by neuronal activity, synaptic potentiation and learning. Arc entry in the synapse is followed by the endocytosis of glutamatergic AMPA receptors (AMPARs), and its nuclear accumulation has been shown in vitro to result in a small decline in the transcription of the GluA1 subunit of AMPARs. Since these effects result in a decline in synaptic strength, we asked whether a change in Arc dynamics may temporally correlate with sleep-dependent GluA1 down-regulation. We measured the ratio of nuclear to cytoplasmic Arc expression (Arc Nuc/Cyto) in the cerebral cortex of EGFP-Arc transgenic mice that were awake most of the night and then perfused immediately before lights on (W mice), or were awake most of the night and then allowed to sleep (S mice) or sleep deprived (SD mice) for the first 2 h of the light phase. In primary motor cortex (M1), neurons with high levels of nuclear Arc (High Arc cells) were present in all mice, but in these cells Arc Nuc/Cyto was higher in S mice than in W mice and, importantly, ~15% higher in S mice than in SD mice collected at the same time of day, ruling out circadian effects. Greater Arc Nuc/Cyto with sleep was observed in the superficial layers of M1, but not in the deep layers. In High Arc cells, Arc Nuc/Cyto was also ~15%-30% higher in S mice than in W and SD mice in the superficial layers of primary somatosensory cortex (S1) and cingulate cortex area 1 (Cg1). In High Arc Cells of Cg1, Arc Nuc/Cyto and cytoplasmic levels of GluA1 immunoreactivities in the soma were also negatively correlated, independent of behavioral state. Thus, Arc moves to the nucleus during both sleep and wake, but its nuclear to cytoplasmic ratio increases with sleep in the superficial layers of several cortical areas. It remains to be determined whether the relative increase in nuclear Arc contributes significantly to the overall decline in the strength of excitatory synapses that occurs during sleep. Similarly, it remains to be determined whether the entry of Arc into specific synapses is gated by sleep.


Assuntos
Complexo Relacionado com a AIDS/metabolismo , Núcleo Celular/metabolismo , Córtex Cerebral/citologia , Citoplasma/metabolismo , Neurônios/ultraestrutura , Sono/fisiologia , Complexo Relacionado com a AIDS/genética , Animais , Carbocianinas/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Vigília/fisiologia
20.
J Comp Neurol ; 525(18): 3951-3961, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28857161

RESUMO

Functional deficits in sensory systems are commonly noted in neurodevelopmental disorders, such as the Rett syndrome (RTT). Defects in methyl CpG binding protein gene (MECP2) largely accounts for RTT. Manipulations of the Mecp2 gene in mice provide useful models to probe into various aspects of brain development associated with the RTT. In this study, we focused on the somatosensory cortical phenotype in the Bird mouse model of RTT. We used voltage-sensitive dye imaging to evaluate whisker sensory evoked activity in the barrel cortex of mice. We coupled this functional assay with morphological analyses in postnatal mice and investigated the dendritic differentiation of barrel neurons and individual thalamocortical axon (TCA) arbors that synapse with them. We show that in Mecp2-deficient male mice, whisker-evoked activity is roughly topographic but weak in the barrel cortex. At the morphological level, we find that TCA arbors fail to develop into discrete, concentrated patches in barrel hollows, and the complexity of the dendritic branches in layer IV spiny stellate neurons is reduced. Collectively, our results indicate significant structural and functional impairments in the barrel cortex of the Bird mouse line, a popular animal model for the RTT. Such structural and functional anomalies in the primary somatosensory cortex may underlie orofacial tactile sensitivity issues and sensorimotor stereotypies characteristic of RTT.


Assuntos
Proteína 2 de Ligação a Metil-CpG/deficiência , Síndrome de Rett/genética , Síndrome de Rett/patologia , Córtex Somatossensorial/patologia , Vias Aferentes/fisiologia , Animais , Carbocianinas/metabolismo , Dendritos/patologia , Dendritos/ultraestrutura , Modelos Animais de Doenças , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/ultraestrutura , Coloração pela Prata , Córtex Somatossensorial/citologia , Vibrissas/inervação , Imagens com Corantes Sensíveis à Voltagem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA