RESUMO
OBJECTIVE: Multiple tubers in patients with tuberous sclerosis complex (TSC) often are responsible for drug-resistant epilepsy. The complexity of the epileptic network formed by multiple tubers complicates localization of the epileptogenic zone that is needed to design a surgical treatment strategy. High frequency oscillations (HFOs) on intracranial video-electroencephalography (IVEEG) may be a valuable surrogate marker for the localization of the epileptogenic zone. The purpose of this study was to test the hypothesis that high occurrence rate (OR) of interictal HFOs can guide the localization of the epileptogenic zone. METHODS: We analyzed the OR of interictal HFOs at 80-200 Hz (ripples) and >200 Hz (fast ripples, FRs). We divided OR of interictal HFOs between high and low rates by thresholding. We analyzed the correlation between seizure outcomes using Engel classification and the resection ratio of the seizure onset zone (SOZ), and high-OR HFOs using ordinal logistic regression analysis. RESULTS: We collected 10 patients. The seizure outcomes resulted in Engel classification I in three patients, II in four, III in one, and IV in two. High-OR ripples (5-57 [mean 29] channels, 1-4 [2.8] lobes) and high-OR FRs (9-66 [mean 27] channels, 1-4 [2.6] lobes) were widely distributed. The resection ratio of SOZ did not show statistically significant correlation with the seizure outcome. The resection ratio of high-OR ripples showed statistically significant correlation with the seizure outcome (p = 0.038). The resection ratio of high-OR FRs showed statistically significant correlation with the seizure outcome (p = 0.048). SIGNIFICANCE: The multiple extensive zones with high-OR HFOs suggest a complex and widespread epileptic network in patients with TSC. In a subset of TSC patients with drug-resistant epilepsy, resection of cortex with both interictal high-OR FRs and ripples on IVEEG correlated with a good seizure outcome.
Assuntos
Encéfalo/fisiopatologia , Epilepsia/etiologia , Convulsões/etiologia , Esclerose Tuberosa/complicações , Adolescente , Encéfalo/patologia , Encéfalo/cirurgia , Mapeamento Encefálico/métodos , Criança , Pré-Escolar , Eletroencefalografia/métodos , Epilepsia/patologia , Epilepsia/fisiopatologia , Epilepsia/cirurgia , Feminino , Humanos , Masculino , Estudos Retrospectivos , Convulsões/patologia , Convulsões/fisiopatologia , Convulsões/cirurgia , Resultado do Tratamento , Esclerose Tuberosa/patologia , Esclerose Tuberosa/fisiopatologia , Esclerose Tuberosa/cirurgiaRESUMO
OBJECTIVE: Rho/Rho-kinase (ROCK) pathway in vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of cardiovascular diseases, including pulmonary arterial hypertension (PAH). Rho-kinase has 2 isoforms, ROCK1 and ROCK2, with different functions in different cells; ROCK1 for circulating inflammatory cells and ROCK2 for the vasculature. In the present study, we aimed to examine whether ROCK2 in VSMC is involved in the pathogenesis of PAH. APPROACH AND RESULTS: In patients with PAH, the expression of ROCK2 was increased in pulmonary arterial media and primary pulmonary arterial smooth muscle cells when compared with controls. To investigate the role of ROCK2 in VSMC, we generated VSMC-specific heterozygous ROCK2-deficient (ROCK2(+/-)) mice and VSMC-specific ROCK2-overexpressing transgenic (ROCK2-Tg) mice. The extent of hypoxia-induced pulmonary hypertension was reduced in ROCK2(+/-) mice and was enhanced in ROCK2-Tg mice compared with respective littermates. The protein expression of ROCK activity and phosphorylated extracellular signal-regulated kinase and the number of Ki67-positive proliferating cells in the lung were reduced in ROCK2(+/-) mice and were increased in ROCK2-Tg mice compared with respective littermates. In cultured mouse aortic VSMC, migration and proliferation activities were reduced in ROCK2(+/-) mice, and migration activity was increased in ROCK2-Tg mice compared with respective littermates. In addition, in primary pulmonary arterial smooth muscle cells from a patient with PAH, ROCK2 was required for migration and proliferation through ROCK and extracellular signal-regulated kinase activation. CONCLUSIONS: ROCK2 in VSMC contributes to the pathogenesis of PAH.
Assuntos
Hipertensão Pulmonar/enzimologia , Hipóxia/complicações , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Quinases Associadas a rho/metabolismo , Adolescente , Adulto , Animais , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipertensão Pulmonar Primária Familiar , Feminino , Regulação Enzimológica da Expressão Gênica , Heterozigoto , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/prevenção & controle , Mediadores da Inflamação/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Estresse Oxidativo , Fosforilação , Artéria Pulmonar/enzimologia , Interferência de RNA , Fatores de Tempo , Transfecção , Adulto Jovem , Quinases Associadas a rho/deficiência , Quinases Associadas a rho/genéticaRESUMO
OBJECTIVE: The pleiotropic effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) independent of cholesterol-lowering effects are thought to be mediated through inhibition of the Rho/Rho-kinase pathway. However, we have previously demonstrated that the pleiotropic effects of regular-dose statins are mediated mainly through inhibition of the Rac1 signaling pathway rather than the Rho/Rho-kinase pathway, although the molecular mechanisms of the selective inhibition of the Rac1 signaling pathway by regular-dose statins remain to be elucidated. In this study, we tested our hypothesis that small GTP-binding protein GDP dissociation stimulator (SmgGDS) plays a crucial role in the molecular mechanisms of the Rac1 signaling pathway inhibition by statins in endothelial cells. APPROACH AND RESULTS: In cultured human umbilical venous endothelial cells, statins concentration-dependently increased SmgGDS expression and decreased nuclear Rac1. Statins also enhanced SmgGDS expression in mouse aorta. In control mice, the protective effects of statins against angiotensin II-induced medial thickening of coronary arteries and fibrosis were noted, whereas in SmgGDS-deficient mice, the protective effects of statins were absent. When SmgGDS was knocked down by its small interfering RNA in human umbilical venous endothelial cells, statins were no longer able to induce Rac1 degradation or inhibit angiotensin II-induced production of reactive oxygen species. Finally, in normal healthy volunteers, statins significantly increased SmgGDS expression with a significant negative correlation between SmgGDS expression and oxidative stress markers, whereas no correlation was noted with total or low-density lipoprotein-cholesterol. CONCLUSIONS: These results indicate that statins exert their pleiotropic effects through SmgGDS upregulation with a resultant Rac1 degradation and reduced oxidative stress in animals and humans.
Assuntos
Cardiomegalia/tratamento farmacológico , Vasos Coronários/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neuropeptídeos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Angiotensina II , Animais , Atorvastatina , Biomarcadores/sangue , Cardiomegalia/induzido quimicamente , Cardiomegalia/enzimologia , Cardiomegalia/genética , Cardiomegalia/patologia , Células Cultivadas , Colesterol/sangue , LDL-Colesterol/sangue , Vasos Coronários/enzimologia , Vasos Coronários/patologia , Estudos Cross-Over , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibrose , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Ácidos Heptanoicos/farmacologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Japão , Masculino , Camundongos , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Pravastatina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirróis/farmacologia , Quinolinas/farmacologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
BACKGROUND: Pulmonary hypertension (PH) is a fatal disease characterized by pulmonary artery (PA) remodeling, elevated PA pressure and right ventricular (RV) failure. It has been previously demonstrated that treatment with a Rho-kinase inhibitor, fasudil, ameliorates PH in animal models. Here, whether combination therapy with fasudil and sildenafil further ameliorates PH in rats was examined. METHODS AND RESULTS: PH was induced in Sprague-Dawley rats by the use of a single subcutaneous monocrotaline (MCT) injection, which caused PA remodeling, elevated RV systolic pressure (RVSP), and RV hypertrophy (RVH). While fasudil and sildenafil monotherapy inhibited the development of MCT-induced PH in the prevention and treatment protocols, their combination therapy further improved RVSP and RVH. Moreover, a histological examination demonstrated significant improvements of PA remodeling in the combination group compared with the monotherapy groups. An echocardiographic examination also revealed significant reduction in RV diameter in the combination group compared with the monotherapy groups. Mechanistic experiments revealed significant inhibition of Rho-kinase activity in PA trunk, lung and RV tissues in the combination group as well as in the monotherapy groups. Finally, the combination therapy markedly improved the long-term survival compared with the monotherapy groups. CONCLUSIONS: These results indicate that the combination therapy with fasudil and sildenafil shows the synergistic effects through the inhibition of Rho-kinase activity for the treatment of PH.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Bloqueadores dos Canais de Cálcio/farmacologia , Hipertensão Pulmonar , Monocrotalina/toxicidade , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Sulfonas/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Quimioterapia Combinada , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Masculino , Purinas/farmacologia , Ratos , Ratos Sprague-Dawley , Citrato de SildenafilaRESUMO
We demonstrate unconditional quantum-noise suppression in a collective spin system via feedback control based on quantum nondemolition measurement. We perform shot-noise limited collective spin measurements on an ensemble of 3.7×10(5) laser-cooled (171)Yb atoms in their spin-1/2 ground states. Correlation between two sequential quantum nondemolition measurements indicates -0.80(-0.12)(+0.11) dB quantum-noise suppression in a conditional manner. Our feedback control successfully converts the conditional quantum-noise suppression into the unconditional one without significant loss of the noise reduction level.
RESUMO
BACKGROUND: Ezetimibe is an inhibitor of cholesterol absorption in the intestine. We examined whether ezetimibe improves endothelial function, and if so, what mechanisms are involved. METHODS AND RESULTS: Nineteen healthy subjects (male/female 14/5; mean age, 31±3 [SD] years-old) were randomized to receive ezetimibe (10mg/day) or pravastatin (10mg/day) for 4 weeks in a cross-over manner with a 4-week washout interval. Lipid profiles, flow-mediated dilatation (FMD) and Rho-kinase activity of circulating leukocytes (the extent of phosphorylation of myosin binding subunit, a Rho-kinase substrate) were examined. We also evaluated remnant-like particle cholesterol (RLP-C) known as an up-regulator of Rho-kinase and cholesterol absorption status by measuring cholestanol and campesterol/lathosterol ratio (CLR) (both absorption markers). Although ezetimibe and pravastatin equally reduced low-density lipoprotein cholesterol (E: -25% vs. P: -21%), the CLR was reduced by ezetimibe but was rather increased by pravastatin (E: -41% vs. P: +37%; P<0.01). Reduction in RLP-C by ezetimibe was greater compared with pravastatin (E: -33% vs. P: -14%; P<0.05). Importantly, ezetimibe significantly improved FMD (26%, P<0.05) and reduced Rho-kinase activity (-21%, P<0.05), whereas pravastatin had no such effects. A significant correlation was noted between the reduction in cholestanol and the improvement in FMD (P<0.05). CONCLUSIONS: These results indicate that ezetimibe improves endothelial function and inhibits Rho-kinase activity associated with the inhibition of cholesterol absorption, suggesting novel anti-atherogenic effects of the agent in humans.
Assuntos
Anticolesterolemiantes/administração & dosagem , Azetidinas/administração & dosagem , Colesterol/metabolismo , Endotélio Vascular/metabolismo , Absorção Intestinal/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Adulto , Colestanol/sangue , Colesterol/sangue , Estudos Transversais , Ezetimiba , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos/metabolismo , Masculino , Pravastatina/administração & dosagem , Regulação para Cima/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
Electrochemical quartz-crystal nanobalance (EQCN) analysis of the behavior of Pt in aqueous H(2)SO(4) reveals that the interfacial mass reaches a minimum, the potential of minimum mass (E(pmm)), at 0.045 V. A similar behavior is observed for Pt in aqueous HClO(4) and NaOH. E(pmm) is a new parameter describing the electrochemical interface. The value of E(pmm) coincides with the completion of the saturation layer of electroadsorbed H (H(UPD)) and the commencement of H(2)(g) generation or H(2)(g) electro-oxidation. The value of E(pmm) and the structure of the Pt/electrolyte interface are discussed in terms of the interactions of the anions H(3)O(+), H(UPD), H(OPD), and H(2)O with Pt. The layer of H(UPD) embedded in the Pt surface lattice minimizes the surface dipole-water dipole and surface charge-water dipole interactions, thus reduces the wetting ability of Pt. Consequently, the discharge of H(3)O(+) in the electrolytic formation of H(2)(g) or the dissociative adsorption of H(2)(g) that precedes its electro-oxidation to H(3)O(+) proceed easily on Pt, because the species do not have to displace H(2)O molecules. Effective and inexpensive non-platinum electrocatalysts for the electrolytic H(2)(g) generation in water electrolyzers or H(2)(g) electro-oxidation in polymer electrolyte membrane fuel cells should mimic the interfacial behavior of Pt by minimizing the interaction of H(2)O molecules with the electrode.
RESUMO
2-NBDG is a widely used fluorescent tracer for monitoring d-glucose uptake into single living cells. However, 2-NBDG alone is not sufficient for monitoring the net stereoselective uptake of d-glucose, unless its possible non-stereoselective uptake is properly evaluated. l-Glucose derivatives, which emit fluorescence distinct from that of 2-NBDG, should provide valuable information on the stereoselective uptake, when used with 2-NBDG in combination. In the present study, we synthesized Texas Red (sulforhodamine 101 acid)-coupled and [2-(benz-2-oxa-1,3-diazol-4-yl)amino]-coupled 2-deoxy-D-glucose, referred to as [2-TRG] and [2-BDG], respectively. These derivatives showed emission wavelength longer and shorter than that of 2-NBDG, respectively. 2-TRLG, an antipode of 2-TRG, proved to be an effective tracer for evaluating the extent of non-stereoselective uptake of 2-NBDG when used simultaneously with 2-NBDG. On the other hand, 2-BDG exhibited very weak fluorescence, but the application of a novel cross coupling in the presence of a benzoxadiazole group may be useful for the future development of effective glucose tracers.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Desoxiglucose/análogos & derivados , Glucose/metabolismo , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animais , Encéfalo/metabolismo , Desoxiglucose/síntese química , Desoxiglucose/química , Desoxiglucose/metabolismo , Glucose/química , Camundongos , Microscopia Confocal , Estrutura Molecular , EstereoisomerismoRESUMO
We report successful manipulation of nonclassical atomic spin states. We apply an off-resonant noncircularly-polarized light pulse to a measurement-induced squeezed spin state of a cold atomic ensemble. By changing the pulse duration, we clearly observe a rotation of the anisotropic quantum-noise distribution in good contrast with the case of manipulation of a coherent spin state where the quantum-noise distribution is always isotropic. This is an important step for quantum state tomography, quantum swapping, and precision spectroscopic measurement.
RESUMO
Overexpression of human epidermal growth factor receptor 2 (HER2) is associated with more frequent cancer recurrence and metastasis. Sensitive sensing of HER2 in living breast cancer cells is crucial in the early stages of cancer and to further understand its role in cells. Biomedical imaging has become an indispensable tool in the fields of early cancer diagnosis and therapy. In this study, we designed and synthesized platinum (Pt) nanocluster bionanoprobes with red emission (Ex/Em = 535/630 nm) for fluorescence imaging of HER2. Our Pt nanoclusters, which were synthesized using polyamidoamine (PAMAM) dendrimer and preequilibration, exhibited approximately 1% quantum yield and possessed low cytotoxicity, ultrasmall size, and excellent photostability. Furthermore, combined with ProteinA as an adapter protein, we developed Pt bionanoprobes with minimal nonspecific binding and utilized them as fluorescent probes for highly sensitive optical imaging of HER2 at the cellular level. More importantly, molecular probes with long-wavelength emission have allowed visualization of deep anatomical features because of enhanced tissue penetration and a decrease in background noise from tissue scattering. Our Pt nanoclusters are promising fluorescent probes for biomedical applications.
RESUMO
The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23â¼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells.
RESUMO
An efficient protocol of dehydration was developed under microfluidic conditions. The method was applied to a multikilogram synthesis of pristane, a biologically important natural product, which is now widely used as an adjuvant for monoclonal antibody production. [reaction: see text].
Assuntos
Fatores Biológicos/síntese química , Técnicas Analíticas Microfluídicas/métodos , Terpenos/síntese química , Fatores Biológicos/química , Desidratação , Técnicas Analíticas Microfluídicas/instrumentação , Estrutura Molecular , Sensibilidade e Especificidade , Terpenos/químicaRESUMO
AIMS: The pleiotropic effects of HMG-CoA reductase inhibitors (statins) independent of cholesterol-lowering effects have attracted much attention. We have recently demonstrated that the pleiotropic effects of statins are partly mediated through up-regulation of small GTP-binding protein dissociation stimulator (SmgGDS) with a resultant Rac1 degradation and reduced oxidative stress. However, it remains to be elucidated what molecular mechanisms are involved. METHODS AND RESULTS: To first determine in what tissue statins up-regulate SmgGDS expression, we examined the effects of two statins (atorvastatin 10 mg/kg per day and pravastatin 50 mg/kg per day for 1 week) on SmgGDS expression in mice in vivo. The two statins increased SmgGDS expression especially in the aorta. Atorvastatin also increased SmgGDS expression in cultured human umbilical venous endothelial cells (HUVEC) and human aortic endothelial cells, but not in human aortic vascular smooth muscle cells. Furthermore, Akt phosphorylation was transiently enhanced only in HUVEC in response to atorvastatin. Then, to examine whether Akt is involved for up-regulation of SmgGDS by statins, we knocked out Akt1 by its siRNA in HUVEC, which abolished the effects by atorvastatin to up-regulate SmgGDS. Furthermore, when we knocked down ß1-integrin to elucidate the upstream molecule of Akt1, the effect of atorvastatin to up-regulate SmgGDS was abolished. Finally, we confirmed that Akt activator, SC79, significantly up-regulate SmgGDS in HUVEC. CONCLUSION: These results indicate that statins selectively up-regulate SmgGDS in endothelial cells, for which the ß1-integrin/Akt1 pathway may be involved, demonstrating the novel aspects of the pleiotropic effects of statins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Integrina beta1/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologia , Animais , Atorvastatina/farmacologia , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Pravastatina/farmacologia , Regulação para CimaRESUMO
Non-defect Poly(dG).Poly(dC) of 500 bp (170 nm) has been synthesized by using enzymatic reactions and was characterized by its UV spectrum, showing that conjugated pi-electrons between base pairs are spread over the DNA molecule suggesting the absence of structural defects.
Assuntos
DNA Ligases/química , DNA/síntese química , Polidesoxirribonucleotídeos/síntese química , Elétrons , Espectrofotometria UltravioletaRESUMO
Poly(dA).Poly(dT) molecules up to 1000 base pairs (bp) have been synthesized using enzymatic reaction, and characterization by STM observation shows that the DNA has no defects in the duplex structure.
Assuntos
Poli dA-dT/síntese química , DNA Polimerase Dirigida por DNA/química , Eletroforese em Gel de Ágar , Microscopia Eletrônica de Transmissão e Varredura , Conformação de Ácido Nucleico , Poli dA-dT/química , Polinucleotídeo 5'-Hidroxiquinase/química , RNA Ligase (ATP)/química , Endonucleases Específicas para DNA e RNA de Cadeia Simples/químicaRESUMO
The formation of microporous oxide layers on titanium (Ti) by anodization in sulfuric acid (H2SO4) solution and the influence of prior hydrogen charging on their properties are examined using electrochemical techniques, scanning electron microscopy, grazing incident X-ray diffraction, and X-ray photoelectron spectroscopy. When Ti is anodized in 1 M aqueous H2SO4 solution at a high direct current (DC) potential (>150 V) for 1 min, a porous surface layer develops, and the process takes place with spark-discharge. Under these conditions, oxygen evolution at the Ti electrode proceeds vigorously and concurrently with the formation of anodic oxide. The oxygen gas layer adjacent to the Ti surface acts as an insulator and triggers spark-discharge; the latter stimulates the development of pores. In the absence of spark-discharge, the oxide layer has extended surface roughness but low porosity. A porous oxide layer can be prepared by applying a lower DC voltage (130 V) and without spark-discharge, but Ti requires prior hydrogen charging by cathodic polarization in 1 M aqueous H2SO4 solution. Mott-Schottky measurements indicate that the oxide layers are n-type semiconductors and that the charge carrier density in the anodic oxide layer on the hydrogen-charged Ti is lower than in the case of untreated Ti. The hydrogen charging also affects the flat band potential of the anodic oxide layers on Ti by increasing its value. The reduced charge carrier density brought about by hydrogen charging decreases the oxide layer conductivity and creates favorable conditions for its electrical breakdown that stimulates the development of pores. The porous layer on the hydrogen-charged Ti consists of anatase and rutile phases of TiO2; it has the same chemical composition as the porous layer obtained on untreated Ti. X-ray photoelectron spectroscopy measurements show that prior hydrogen charging does not affect the thickness of anodic oxides on Ti. The porous oxide layer on Ti enables the growth of hydroxyapatite, thus revealing good bioactivity in simulated body fluids.
RESUMO
The plasmon resonant wavelength for a pair of gold nanoparticles, or gold nanodimer, increases inversely with the gap distance between the two nanoparticles. Taking advantage of this property, we performed nanoscale measurements of DNA conformation changes induced by transcription factor binding. Gold nanoparticles were bridged by double-stranded DC5 DNA that included binding sequences for the transcription factors SOX2 and PAX6, which interact on the DC5 enhancer sequence and activate transcription. The gold nanodimers bound by SOX2 shifted the plasmon resonant wavelength from 586.8 to 604.1 nm, indicating that SOX2 binding induces DNA bending. When the SOX2 formed a ternary complex with PAX6 on DC5, the plasmon resonant wavelength showed a further shift to 611.6 nm, indicating additional bending in the DC5 sequence. Furthermore, we investigated DNA conformation changes induced by SOX2 and PAX6 on the DC5-con sequence, which is a consensus sequence of DC5 for the PAX6 binding region that strengthens the PAX6 binding but at the same time disrupts SOX2-PAX6-dependent transcriptional activation. When the PAX6 binding sequence in DC5 was altered to DC5-con, the plasmon resonant wavelength shifted much less to 606.5 nm, which is more comparable to the 603.9 nm by SOX2 alone. These results demonstrate that SOX2-PAX6 cobinding induces a large conformation change in DC5 DNA.
Assuntos
DNA/química , Nanopartículas/química , Fatores de Transcrição/química , Animais , Dimerização , Proteínas do Olho/química , Ouro/química , Proteínas de Homeodomínio/química , Insetos , Nanopartículas Metálicas/química , Conformação de Ácido Nucleico , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/química , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/química , Fatores de Transcrição SOXB1/química , Eletricidade Estática , Ressonância de Plasmônio de SuperfícieRESUMO
To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.
Assuntos
Anticorpos/metabolismo , Imageamento Tridimensional/métodos , Pontos Quânticos , Receptores de Superfície Celular/metabolismo , Proteína Estafilocócica A/metabolismo , Linhagem Celular , Sobrevivência Celular , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Glutationa/metabolismo , Humanos , Microscopia de Força Atômica , Microscopia Confocal , Espectrometria de FluorescênciaRESUMO
Microporous layers on titanium (Ti) are formed by chemical treatment in highly concentrated alkaline media, and their properties and growth mechanism are examined using electrochemical techniques, in situ resistometry, scanning electron microscopy (SEM), grazing-incident X-ray diffraction (GIXRD), and glow discharge optical emission spectroscopy (GD-OES). Chemical treatment in a 5 M aqueous KOH solution yields results superior to those from the same treatment in a 5 M aqueous NaOH solution, while a 3 M aqueous LiOH solution does not produce porous layers. The cation constituting the solution plays a vital role in the process. An SEM analysis reveals that the KOH solution is the most effective in forming microporosity and that the longer the treatment time, the more porous the near-surface layer. The results of GIXRD analysis show the presence of Na(2)Ti(5)O(11) and K(2)Ti(6)O(13) in the layers formed in the NaOH and KOH solutions, respectively; in the case of the LiOH solution, TiO(2) is formed. Chemical treatment in the NaOH and KOH solutions resembles a general corrosion process with the existence of local cathodic and anodic sites. The reduction reaction produces H(2), some of which becomes absorbed in the near-surface region of Ti, while the oxidation reaction produces the above-mentioned compounds and/or an oxide layer. The presence of hydrogen (H) within the solid is detected using GD-OES. The H-containing near-surface layer partially dissolves, yielding a microporous structure. The development and dissolution of the H-containing near-surface layer of Ti upon chemical treatment in the NaOH and KOH solutions are confirmed by resistometry measurements. They point to the formation of a compact passive layer on Ti upon exposure to the LiOH solution.