RESUMO
Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.
Assuntos
Oligopeptídeos , Proteínas Priônicas , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Domínios Proteicos , Sequências Repetitivas de AminoácidosRESUMO
Oral squamous cell carcinoma (OSCC), an aggressive cancer originating in the oral cavity, is one of the leading causes of cancer deaths in males worldwide. This study investigated the antitumor activity and mechanisms of piperlongumine (PL), a natural compound isolated from Piper longum L., in human OSCC cells. The effects of PL on cell proliferation, the cell cycle, apoptosis, senescence and reactive oxygen species (ROS) levels in human OSCC cells were investigated. PL effectively inhibited cell growth, caused cell cycle arrest and induced apoptosis and senescence in OSCC cells. Moreover, PL-mediated anti-human OSCC behavior was inhibited by an ROS scavenger N-acetyl-l-cysteine (NAC) treatment, suggesting that regulation of ROS was involved in the mechanism of the anticancer activity of PL. These findings suggest that PL suppresses tumor growth by regulating the cell cycle and inducing apoptosis and senescence and is a potential chemotherapy agent for human OSCC cells.
Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dioxolanos/farmacologia , Acetilcisteína/farmacologia , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Humanos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Nasal polyposis is characterised by persistent inflammation of the upper airways. Autophagy has been implicated in many chronic inflammatory diseases. Whether autophagy plays a role in nasal polyp (NP) inflammation is completely unknown and deserves investigation. METHODS: LC3 and COX-2 expression, the common autophagy and inflammation indicators, respectively, was analysed by immunoblotting in fresh tissues of NP and control nasal mucosa (NM). Primary cultures of NP-derived fibroblasts (NPDFs) and NMDFs were established for in vitro studies. Autophagy was induced by amino acid starvation and LC3 ectopic overexpression or inhibited by 3-methyladenine in the fibroblasts. Inflammation was induced by IL1-ß and TNF-α. LC3 and COX-2 expression was confirmed in NP specimens by immunohistochemistry. RESULTS: LC3 expression was decreased while COX-2 expression was significantly increased in fresh NP tissues compared with the NM control. In NMDFs and NPDFs, autophagy induction by starvation and LC3 overexpression downregulated COX-2 expression. Conversely, autophagy inhibition by 3-methyladenine enhanced COX-2 expression. However, IL1-ß and TNF-α had no effect on autophagy. Immunohistochemical studies on the NP specimens showed that most displayed low LC3 expression, whereas COX-2 was highly expressed in >50% of the specimens. Examination of two consecutive NP sections from the same tissue blocks revealed a negative correlation between LC3 and COX-2 expression. CONCLUSION: Autophagy is deficient in NP tissues and COX-2 is negatively regulated by autophagy in NP-derived fibroblasts. Since COX-2 is essential for the production of pro-inflammatory mediators, this study might help interpret persistent mucosal inflammation in NP. Attenuation of inflammation by restoring autophagy might be a therapeutic strategy for treating NP.
Assuntos
Autofagia/fisiologia , Ciclo-Oxigenase 2/metabolismo , Pólipos Nasais/metabolismo , Rinite/etiologia , Estudos de Casos e Controles , Técnicas de Cultura de Células , Fibroblastos/fisiologia , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Pólipos Nasais/patologiaRESUMO
Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.
Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/imunologia , Vírus da Dengue/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Western Blotting , Vírus da Dengue/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Imunofluorescência , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
Microbial fuel cells (MFCs) represent a novel platform for treating wastewater and at the same time generating electricity. Using Pseudomonas putida (BCRC 1059), a wild-type bacterium, we demonstrated that the refinery wastewater could be treated and also generate electric current in an air-cathode chamber over four-batch cycles for 63 cumulative days. Our study indicated that the oil refinery wastewater containing 2213 mg/L (ppm) chemical oxygen demand (COD) could be used as a substrate for electricity generation in the reactor of the MFC. A maximum voltage of 355 mV was obtained with the highest power density of 0.005 mW/cm² in the third cycle with a maximum current density of 0.015 mA/cm² in regard to the external resistor of 1000 Ω. A maximum coulombic efficiency of 6 × 10⻲% was obtained in the fourth cycle. The removal efficiency of the COD reached 30% as a function of time. Electron transfer mechanism was studied using cyclic voltammetry, which indicated the presence of a soluble electron shuttle in the reactor. Our study demonstrated that oil refinery wastewater could be used as a substrate for electricity generation.
Assuntos
Fontes de Energia Bioelétrica , Indústria Química/métodos , Poluição por Petróleo/prevenção & controle , Pseudomonas putida/fisiologia , Eliminação de Resíduos Líquidos/métodos , Águas Residuárias , Purificação da Água/métodos , Fontes de Energia Bioelétrica/microbiologia , Análise da Demanda Biológica de Oxigênio , Eletricidade , Eletrodos , Desenho de Equipamento , Poluentes Químicos da ÁguaRESUMO
DNA hypermethylation of promoter CpG islands is associated with epigenetic silencing of tumor suppressor genes in oral squamous cell carcinomas (OSCCs). We used a methyl-CpG-binding domain protein capture method coupled with next-generation sequencing (MBDCap-seq) to survey global DNA methylation patterns in OSCCs with and without nodal metastasis and normal mucosa (total n = 58). Of 1462 differentially methylated CpG islands identified in OSCCs relative to normal controls, MBDCap-seq profiling uncovered 359 loci linked to lymph node metastasis. Interactive network analysis revealed a subset of these loci (n = 23), including the anaplastic lymphoma kinase (ALK) gene, are potential regulators and effectors of invasiveness and metastatic progression. Promoter methylation of ALK was preferentially observed in OSCCs without node metastasis, whereas relatively lower methylation levels were present in metastatic tumors, implicating an active state of ALK transcription in the latter group. The OSCC cell line, SCC4, displayed reduced ALK expression that corresponded to extensive promoter CpG island methylation. SCC4 treatment with demethylating agents induced ALK expression and increased invasion and migration characteristics. Inhibition of ALK activity in OSCC cells with high ALK expression (CAL27, HSC3 and SCC25), decreased cell growth and resulted in changes in invasive potential and mesenchymal marker expression that were cell-line dependent. Although ALK is susceptible to epigenetic silencing during oral tumorigenesis, overwriting this default state may be necessary for modulating invasive processes involved in nodal metastases. Given the complex response of OSCC cells to ALK inhibition, future studies are required to assess the feasibility of targeting ALK to treat invasive OSCCs.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Mesoderma/patologia , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma de Células Escamosas/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Progressão da Doença , Epigênese Genética , Humanos , Metástase Linfática , Mesoderma/metabolismo , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Neoplasias Bucais/metabolismo , Invasividade Neoplásica , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/metabolismo , Ativação TranscricionalRESUMO
Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and this membrane-bound protein can be cleaved from the cell membranes by phosphoinositide phospholipase C. Numerous studies have investigated GPI-free recombinant PrP, but the role of GPI on misfolding of PrP is not well known. In this study, we synthesized a GPI analog that was covalently linking to a PrP S230C mutant, resulting in S230C-GPI. The structural changes in S230C-GPI upon binding to lipid vesicles composed of mixtures of the zwitterionic lipid (POPC) and the anionic lipid (POPG) were analyzed by circular dichroism spectroscopy, and the amyloid aggregation of S230C-GPI in the liberation from phospholipid vesicles was monitored by proteinase K-digestion assay. Our results indicate that S230C-GPI in the liberation of lipid vesicles has high tendency to misfold into amyloid fibrils, while the membrane-bound S230C-GPI proteins are highly stable and rarely convert into amyloid forms. In addition, the role of cholesterol in S230C-GPI was studied. The effect of GPI, cholesterol and phospholipid vesicles on misfolding of PrP is further discussed.
Assuntos
Glicosilfosfatidilinositóis/química , Fosfolipídeos/química , Colesterol/química , Dicroísmo Circular , Microscopia Eletrônica de Transmissão , Príons/química , Dobramento de ProteínaRESUMO
Arecoline, the major alkaloid of areca nut, has been shown to cause strong genotoxicity and is considered a potential carcinogen. However, the detailed mechanism for arecoline-induced carcinogenesis remains obscure. In this study, we noticed that the levels of p21 and p27 increased in two oral squamous cell carcinoma cell lines with high confluence. Furthermore, when treated with arecoline, elevated levels of p21 and p27 could be downregulated through the reactive oxygen species/mTOR complex 1 (ROS/mTORC1) pathway. Although arecoline decreased the activity of mTORC1, the amounts of autophagosome-like vacuoles or type II LC3 remained unchanged, suggesting that the downregulation of p21 and p27 was independent of autophagy-mediated protein destruction. Arecoline also caused DNA damage through ROS, indicating that the reduced levels of p21 and p27 might facilitate G (1) /S transition of the cell cycle and subsequently lead to error-prone DNA replication. In conclusion, these data have provided a possible mechanism for arecoline-induced carcinogenesis in subcytolytic doses in vivo.
Assuntos
Arecolina/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação para Baixo/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Agonistas Colinérgicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Dano ao DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Microscopia de Fluorescência , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Complexos Multiproteicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genéticaRESUMO
Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor that specifies pancreatic endocrine cell fates during pancreas development. It can also initiate a transdifferentiation program when expressed in pancreatic exocrine and ductal cells. However, how Ngn3 initiates a transcriptional cascade to achieve endocrine differentiation is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which is a transcriptional coactivator for nuclear receptors, also interacts with Ngn3. The association between Ngn3 and CCAR1 was verified by pull-down assays and co-immunoprecipitation analyses. Using gene reporter assays, we found that CCAR1 is essential for Ngn3 to activate the expression of the reporter genes containing the NeuroD promoter. Moreover, down-regulation of endogenous CCAR1 in the PANC-1 pancreatic ductal cell line inhibits the transdifferentiation program initiated by Ngn3. CCAR1 is, therefore, a novel partner of Ngn3 in mediating endocrine differentiation.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Ilhotas Pancreáticas/citologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Reguladoras de Apoptose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas do Tecido Nervoso/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: The effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated. METHODS: Effects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence. RESULTS: The results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy. CONCLUSIONS: Taken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Autofagia , Carcinoma de Células Escamosas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular , Morfolinas/farmacologia , Neoplasias Bucais/tratamento farmacológico , Purinas/farmacologia , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Citometria de Fluxo , Imunofluorescência , Humanos , Masculino , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
PURPOSE: A novel and effective treatment is urgently needed to deal with the current treatment dilemma in incurable differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC). Reversine, a small synthetic purine analogue (2,6-disubstituted purine), has been shown to be effective in tumor suppression. METHODS: We performed in vitro evaluation of anti-tumor effects of reversine on proliferation, cell cycle, and apoptosis in human PDTC, ATC, and follicular thyroid cancer cell lines, respectively. RESULTS: Treatment of these three lines with reversine inhibited proliferation in a time- and dose-dependent manner. G2/M accumulation was demonstrated in cell cycle analysis. Reversine induced apoptosis in PDTC cells with caspase-3 and caspase-8 activation, but not caspase-9. Use of a pan-caspase inhibitor before treatment with reversine attenuated cell death. Reversine also showed in vivo growth inhibitory effects on ATC cells in a xenograft nude mice model. CONCLUSIONS: Data demonstrated that reversine is effective in inhibiting the growth of thyroid cancer cells by cell cycle arrest or apoptosis, especially with the more aggressive ATC and PDTC. Apoptosis was induced by the mitochondria-independent pathway. Reversine is therefore worthy of further investigation in clinical therapeutics.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Purinas/química , Purinas/uso terapêutico , Glândula Tireoide/efeitos dos fármacos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Purinas/farmacologia , Carcinoma Anaplásico da Tireoide , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
Microbial fuel cells (MFCs) represent a novel technology for wastewater treatment with electricity production. Electricity generation with simultaneous nitrate reduction in a single-chamber MFC without air cathode was studied, using glucose (1 mM) as the carbon source and nitrate (1 mM) as the final electron acceptor employed by Bacillus subtilis under anaerobic conditions. Increasing current as a function of decreased nitrate concentration and an increase in biomass were observed with a maximum current of 0.4 mA obtained at an external resistance (R(ext)) of 1 KΩ without a platinum catalyst of air cathode. A decreased current with complete nitrate reduction, with further recovery of the current immediately after nitrate addition, indicated the dependence of B. subtilis on nitrate as an electron acceptor to efficiently produce electricity. A power density of 0.0019 mW/cm(2) was achieved at an R(ext) of 220 Ω. Cyclic voltammograms (CV) showed direct electron transfer with the involvement of mediators in the MFC. The low coulombic efficiency (CE) of 11% was mainly attributed to glucose fermentation. These results demonstrated that electricity generation is possible from wastewater containing nitrate, and this represents an alternative technology for the cost-effective and environmentally benign treatment of wastewater.
Assuntos
Bacillus subtilis/metabolismo , Fontes de Energia Bioelétrica/microbiologia , Glucose/metabolismo , Nitratos/metabolismo , Águas Residuárias/microbiologia , Biomassa , Eletricidade , Eletrodos , Fermentação , Gerenciamento de Resíduos/métodos , Purificação da Água/métodosRESUMO
We developed a fast (<20 min), label-free fiber optic particle plasmon resonance (FOPPR) immunosensing method to detect nervous necrosis virus (NNV), which often infects high-value economic aquatic species, such as grouper. Using spiked NNV particles in a phosphate buffer as samples, the standard calibration curve obtained was linear (R2 = 0.99) and the limit of detection (LOD) achieved was 2.75 × 104 TCID50/mL, which is superior to that obtained using enzyme-linked immunosorbent assay (ELISA). By using an enhancement method called fiber optic nanogold-linked immunosorbent assay (FONLISA), the LOD can be further improved to <1 TCID50/mL, which is comparable to that found by the conventional qPCR method. Employing the larvae homogenate samples of NNV-infected grouper, the results obtained by the FOPPR biosensor agree with those obtained by the quantitative polymerase chain reaction (qPCR) method. We also examined pond water samples from an infected container in an indoor aquaculture facility. The lowest detectable level of NNV coat protein was found to be 0.17 µg/mL, which is one order lower than the LOD reported by ELISA. Therefore, we demonstrated the potential of the FOPPR biosensor as an outbreak surveillance tool, which is able to give warning indication even when the trend of larvae death toll increment is still not clear.
Assuntos
Bass , Técnicas Biossensoriais , Doenças dos Peixes , Nodaviridae , Animais , Larva , Imunoadsorventes , Lagoas , Doenças dos Peixes/diagnóstico , Fosfatos , Necrose , ÁguaRESUMO
In this study the "green chemistry" use of the biosurfactant surfactin for the synthesis of calcium phosphate using the reverse microemulsion technique was demonstrated. Calcium phosphates are bioactive materials that are a major constituent of human teeth and bone tissue. A reverse microemulsion technique with surfactin was used to produce nanocrystalline brushite particles. Structural diversity (analyzed by SEM and TEM) resulted from different water to surfactin ratios (W/S; 250, 500, 1000 and 40,000). The particle sizes were found to be in the 16-200 nm range. Morphological variety was observed in the as-synthesized microemulsions, which consisted of nanospheres (~16 nm in diameter) and needle-like (8-14 nm in diameter and 80-100 nm in length) noncalcinated particles. However, the calcinated products included nanospheres (50-200 nm in diameter), oval (~300 nm in diameter) and nanorod (200-400 nm in length) particles. FTIR and XRD analysis confirmed the formation of brushite nanoparticles in the as-synthesized products, while calcium pyrophosphate was produced after calcination. These results indicate that the reverse microemulsion technique using surfactin is a green process suitable for the synthesis of nanoparticles.
Assuntos
Fosfatos de Cálcio/síntese química , Emulsões/química , Lipopeptídeos/química , Peptídeos Cíclicos/química , Tensoativos/química , Compostos de Cálcio/química , Fosfatos de Cálcio/química , Cristalização , Nanopartículas/química , Nanopartículas/ultraestrutura , Nitratos/química , Tamanho da Partícula , Fosfatos/química , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
The separation of mercury ions from artificially contaminated water by the foam fractionation process using a biosurfactant (surfactin) and chemical surfactants (SDS and Tween-80) was investigated in this study. Parameters such as surfactant and mercury concentration, pH, foam volume, and digestion time were varied and their effects on the efficiency of mercury removal were investigated. The recovery efficiency of mercury ions was highly sensitive to the concentration of the surfactant. The highest mercury ion recovery by surfactin was obtained using a surfactin concentration of 10 × CMC, while recovery using SDS required < 10 × CMC and Tween-80 >10 × CMC. However, the enrichment of mercury ions in the foam was superior with surfactin, the mercury enrichment value corresponding to the highest metal recovery (10.4%) by surfactin being 1.53. Dilute solutions (2-mg L(-1) Hg(2+)) resulted in better separation (36.4%), while concentrated solutions (100 mg L(-1)) enabled only a 2.3% recovery using surfactin. An increase in the digestion time of the metal solution with surfactin yielded better separation as compared with a freshly-prepared solution, and an increase in the airflow rate increased bubble production, resulting in higher metal recovery but low enrichment. Basic solutions yielded higher mercury separation as compared with acidic solutions due to the precipitation of surfactin under acidic conditions.
Assuntos
Fracionamento Químico/métodos , Lipopeptídeos/química , Mercúrio/isolamento & purificação , Peptídeos Cíclicos/química , Tensoativos/química , Bacillus subtilis/química , Concentração de Íons de Hidrogênio , Íons/químicaRESUMO
Betel quid (BQ) is a widely accepted etiological factor for oral squamous cell carcinoma (OSCC) in Southeast Asia, but how BQ chewing leads to oral carcinogenesis remains to be elucidated. We have previously demonstrated that the activation of Src family kinases (SFKs) is critical for BQ-induced oral cancer cell motility. Here we investigate whether this biological effect is mediated by specific membrane receptors in oral cancer cells. We found that BQ-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cell migration could be inhibited by atropine, suggesting the involvement of the muscarinic receptor family. The enhanced activities of ERK1/2 and cell migration were significantly counteracted by PD102807, the selective antagonist of muscarinic M4 receptor. Moreover, cold BQ extract effectively competed with a known ligand, [(3)H]-N-methyl scopolamine, for binding to muscarinic M4 receptor in vitro, thereby implying that BQ could activate motility-promoting signaling pathways through direct interaction with the receptor. The requirement of muscarinic M4 receptor for BQ-induced oral cancer cell migration was demonstrated by knockdown of the receptor using RNA interference (RNAi). Remarkably, ectopic expression of muscarinic M4 receptor in two oral cancer cell lines, Ca9-22 and SCC-9, further augmented BQ-induced cell migration by 83% and 99%, respectively. Finally, we verified that BQ-induced oral cancer cell migration was mediated through a muscarinic M4 receptor-->SFKs-->ERK1/2 signaling pathway. Thus, our findings have identified a novel signaling cascade mediating BQ-induced oral cancer cell motility, which could be a therapeutic target for BQ-related oral malignancies.
Assuntos
Compostos de Cálcio/efeitos adversos , Movimento Celular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Bucais/induzido quimicamente , Óxidos/efeitos adversos , Piper/efeitos adversos , Extratos Vegetais/efeitos adversos , Receptor Muscarínico M4/agonistas , Linhagem Celular Tumoral , Humanos , Mastigação , Neoplasias Bucais/enzimologia , Neoplasias Bucais/patologia , Transdução de SinaisRESUMO
Biological synthesis of gold and silver nanoparticles was carried out using the bacteria Bacillus subtilis. The reduction processes of chloroaurate and silver ions by B. subtilis were found to be different. Gold nanoparticles were synthesized both intra- and extracellularly, while silver nanoparticles were exclusively formed extracellularly. The gold nanoparticles were formed after 1 day of addition of chloroaurate ions, while the silver nanoparticles were formed after 7 days. The nanoparticles were characterized by X-ray diffraction, UV-vis spectra and transmission electron spectroscopy. X-ray diffraction revealed the formation of face-centered cubic (fcc) crystalline gold nanoparticles in the supernatant, broth solution and bacterial pellet. Silver nanoparticles also exhibited diffraction peaks corresponding to fcc metallic silver. UV-vis spectra showed surface plasmon vibrations for gold and silver nanoparticles centered at 530 and 456 nm, respectively. TEM micrographs depicted the formation of gold nanoparticles intra- and extracellularly, which had an average size of 7.6 +/- 1.8 and 7.3 +/- 2.3 nm, respectively, while silver nanoparticles were exclusively formed extracellularly, with an average size of 6.1 +/- 1.6 nm. The bacterial proteins were analyzed by sodium dodecyl sulfonate-polyacrylamide electrophoresis (SDS-PAGE) before and after the addition of metal ion solutions. We believe that proteins of a molecular weight between 25 and 66 kDa could be responsible for chloroaurate ions reduction, while the formation of silver nanoparticles can be attributed to proteins of a molecular weight between 66 and 116 kDa. We also believe that the nanoparticles were stabilized by the surface-active molecules i.e., surfactin or other biomolecules released into the solution by B. subtilis.
Assuntos
Bacillus subtilis/metabolismo , Reatores Biológicos/microbiologia , Ouro/química , Nanopartículas Metálicas/química , Prata/química , Cloretos/metabolismo , Eletroforese em Gel de Poliacrilamida , Ouro/metabolismo , Compostos de Ouro/metabolismo , Microscopia Eletrônica de Transmissão , Peso Molecular , Tamanho da Partícula , Prata/metabolismo , Espectrofotometria Ultravioleta , Difração de Raios XRESUMO
BACKGROUND/PURPOSE: Oral squamous cell carcinoma (OSCC) is an aggressive tumor and its occurrence in Taiwan is closely related to chronic smoking, alcohol consumption, and especially to betel quid chewing. It became the fourth most common malignant tumor of Taiwanese men in 2006. Unfortunately, there are few biomarkers for diagnosis and treatment of this disease. METHODS: To find potential markers, two domestic cell lines (OC2 and OCSL) derived from different grades of OSCC were established and their proteins were compared by global proteomic analysis. The expression differences of GRP78 protein in these two cell lines and clinical samples from OSCC patients were verified. RESULTS: Of the 11 candidate proteins expressed differentially in both cell lines, six [heat shock protein 90 kDa beta member 1 (94 kDa glucose-regulated protein; GRP94), protein disulfide-isomerase precursor, vimentin, tubulin beta-2C chain, 78 kDa glucose-regulated protein precursor (GRP78), and annexin A2] were increased in OC2 cells (low-grade OSCC), and five (heat shock protein 90-beta, annexin A1, stress-induced phosphoprotein 1, elongation factor-2, and integrin alpha-3 precursor) were increased in OCSL cells (high-grade OSCC). Some of these proteins have been previously associated with malignant tumors, but no previous association of GRP78 with OSCC has been reported. GRP78 protein expression in these two OSCC cell lines was confirmed by Western blotting. Immunohistochemical staining of clinical samples from OSCC patients revealed that decreased GRP78 protein expression was significantly correlated with advance tumor stage (p < 0.001) and neck lymph node metastasis (p = 0.001). CONCLUSION: GRP78 protein is a possible biomarker of oral cancer in Taiwan.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Escamosas/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Bucais/metabolismo , Western Blotting , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Eletroforese em Gel Bidimensional , Chaperona BiP do Retículo Endoplasmático , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Estadiamento de Neoplasias , Prognóstico , Proteômica , TaiwanRESUMO
Anionic biosurfactant surfactin-mediated gold nanoparticles were synthesized for the first time in this study. Differing proton concentrations is believed to cause structural changes in the lipopeptide surfactin used to stabilize the gold nanoparticles in aqueous solution, the effects of which on the morphology of the nanoparticles were investigated. Synthesis of gold nanoparticles by borohydrate reduction was performed at three pH levels (pH 5, 7 and 9) and two different temperatures, and the nanoparticles were characterized by UV-visible spectroscopy, X-ray diffraction and transmission electron microscopy. The UV-vis spectra showed a blue shift with increasing pH from 5 to 9 (from 528 to 566 nm) at both 4 degrees C and room temperature. The nanoparticles synthesized at pH 7 and 9 remained stable for 2 months, while aggregates were observed at pH 5 within 24 h. TEM micrographs revealed that the mean particle size was about 13.11, 8.16 and 4.70 nm at pH 5, 9 and 7, respectively, at 4 degrees C. The nanoparticles formed at pH 7 were uniform in shape and size, and polydispersed and anisotropic at pH 5 and 9. The nanoparticles synthesized at room temperature were monodispersed and were more uniform as compared with those formed at 4 degrees C. This report describes the use of a renewable and environmentally green and biodegradable surfactant as a template and stabilizing agent in the synthesis of gold nanoparticles.
Assuntos
Bacillus subtilis/metabolismo , Cristalização/métodos , Ouro/química , Química Verde/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Tensoativos/química , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Microtubule-associated protein 2 (MAP2) has been better known for its well-defined role primarily in neurite outgrowth during neuronal development. However, the biological functions of MAP2 in non-neuronal cells, such as epithelial cells, remain largely unknown. In the present study, we sought to investigate the cellular functions of MAP2 by separately establishing stable expression of two MAP2 isoforms, MAP2A and MAP2C, in oral squamous cell carcinoma, Ca9-22. Ectopic expression of MAP2A or MAP2C results in microtubule bundling predominantly at the cell periphery. Remarkably, overexpression of MAP2A but not MAP2C significantly promotes migration of Ca9-22 cells, whereas knockdown of MAP2A expression by specific siRNA oligos dramatically decreases cell migration of HaCaT, an immortalized keratinocyte cell line with abundant endogenous MAP2A. Furthermore, by immunohistochemical studies, MAP2A was shown to highly and selectively express in invasive oral cancer tissues, consistent with its motility-promoting cellular function revealed through in vitro assays. Thus, our findings have not only identified a novel role of MAP2 in non-neuronal cells, but also provided the first implication of MAP2 in malignant oral cancer tissues.