RESUMO
The enzyme-like properties of nanozymes may be considerably affected by the structure and surface groups, which thus need to be optimized. Here, through a simple NaOH chemical corrosion method, the chemical structure similar to N-Methylpyrrolidone (NMP), which possessed intrinsic oxidase-like activity, was introduced into polypyrrole (PPy), and then this nanomaterial became oxygen-functionalized PPy (o-PPy) with excellent oxidase-like activity from PPy without this property. Furthermore, after compounding magnetic Fe3O4, the obtained nanocomposites Fe3O4@o-PPy nanoparticles (Fe3O4@o-PPy NPs) showed superiorities in separation during synthesis and real-time control of enzyme catalysis. Studies have found that the enzymatic activity of Fe3O4@o-PPy NPs depended on the amount of functionalized oxygen and the conjugation extent of o-PPy. Fe3O4@o-PPy NPs had efficient oxidase-like activity under a wide range of pH and temperature. Based on the oxidase-like activity of Fe3O4@o-PPy NPs, a colorimetric sensor for glutathione (GSH), which presented rich color changes and satisfactory colorimetric resolution by adding the amaranth, was realized. We believe that the functional modification and structural regulation of PPy can not only realize its wider application but also promote the discovery of novel and efficient nanozymes.
Assuntos
Colorimetria , Glutationa , Oxigênio , Polímeros , Pirróis , Pirróis/química , Colorimetria/métodos , Polímeros/química , Oxigênio/química , Glutationa/química , Glutationa/análise , Oxirredutases/química , Oxirredutases/metabolismo , Concentração de Íons de Hidrogênio , Materiais Biomiméticos/químicaRESUMO
Glycoprotein non-metastatic melanoma protein B (GPNMB) is ubiquitously expressed and has protective effects on the central nervous system. In particular, it is also expressed in the peripheral nervous system (PNS) and upregulated after peripheral nerve injury. However, the role and underlying mechanism of GPNMB in the PNS, especially in peripheral nerve regeneration (PNR), are still unknown and need to be further investigated. In this study, recombinant human GPNMB (rhGPNMB) was injected into a sciatic nerve injury model. It was found that rhGPNMB facilitated the regeneration and functional recovery of the injured sciatic nerve in vivo. Moreover, it was also confirmed that GPNMB activated the Erk1/2 and Akt pathways via binding with Na+/K + -ATPase α1 (NKA α1) and promoted the proliferation and migration of Schwann cells (SCs) and their expression and secretion of neurotrophic factors and neural adhesion molecules in vitro. Our findings demonstrate that GPNMB facilitates PNR through activation of the Erk1/2 and Akt pathways in SCs by binding with NKA α1 and may be a novel strategy for PNR.
Assuntos
Melanoma , Traumatismos dos Nervos Periféricos , Receptores Fc , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Células de Schwann/metabolismo , Regeneração Nervosa/fisiologia , Nervo Isquiático/lesões , ATPase Trocadora de Sódio-Potássio/metabolismo , Glicoproteínas , Traumatismos dos Nervos Periféricos/metabolismo , Glicoproteínas de Membrana/metabolismoRESUMO
The compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the tobacco specific nitrosamines (TSNAs), is widely recognized as a major carcinogen found in tobacco products, environmental tobacco smoke and wastewater. Thus, a selective enrichment and sensitive detection method for monitoring the risk of NNK exposure is highly desirable. In this study, a magnetic molecularly imprinted polymer (MMIP) functionalized with dendritic nanoclusters was synthesized to selectively recognize NNK via the dummy template imprinting strategy, aiming to avoid residual template leakage and increase the imprinting efficiency. The nanocomposites were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, as well as vibrating sample magnetometry (VSM) and nitrogen adsorption/desorption analysis. The resulting MMIPs exhibited high adsorption capacity, fast binding kinetics and good selectivity for trace amounts of NNK. A rapid, low cost and efficient method for detecting NNK in tobacco products was established using magnetic dispersive solid-phase extraction coupled with HPLC-DAD with a good linear range of 0.1-250 µg mL-1. The limit of detection (LOD) and limit of quantification (LOQ) of NNK were 13.5 and 25.0 ng mL-1, respectively. The average recoveries were 87.8-97.3% with RSDs lower than 3%. The results confirmed that the MMIPs could be used as an excellent selective adsorbent for NNK, with potential applications in the pretreatment of tobacco products.
RESUMO
A selective method for the determination of myosmine (a tobacco alkaloid) was developed using molecularly imprinted polymers (MIPs) based on Fe3O4@SiO2 and PAMAM dendrimers. Fe3O4@SiO2 with excellent magnetism and rapid separation performance was chosen as carrier for the MIPs. Moreover, the MIPs modified with PAMAM dendrimers exhibited a regular structure and abundant functional groups, which improve the efficiency of imprinting sites. The myosmine concentration was measured by HPLC with PDA detector, and the UV detection wavelength was set to 200 nm. The linear range of this assay was 13.2-400 mg/L with a correlation coefficient of 0.999, and the detection limit was 4.0 mg/L (S/N = 3). The MIPs have been used for the determination of myosmine in cigarettes, and the recovery range was 84.2-93.5%, with RSD values in the range 0.41-3.1%. In conclusion, our MIPs combine advantages of simple preparation and remarkable selectivity, which gives them excellent application prospects for the sensitive determination of trace myosmine in tobacco products.
Assuntos
Alcaloides , Dendrímeros , Impressão Molecular , Produtos do Tabaco , Polímeros Molecularmente Impressos , Impressão Molecular/métodos , Dióxido de Silício/química , Polímeros/químicaRESUMO
Glycoprotein nonmetastatic melanoma protein B (GPNMB) exerts neuroprotective effects on amyotrophic lateral sclerosis and cerebral ischemia reperfusion injury in the central nervous system. However, the expression and function of GPNMB in the peripheral nervous system, particularly following peripheral nerve injury, remains unknown. In the present study, the mRNAs and long noncoding RNAs of the distal sciatic nerve were profiled via microarray analysis at days 0, 1, 3, 7, 14, 21 and 28 following transection. The results revealed that the expression of GPNMB mRNA was similar to the proliferation tendency of distal acute denervated Schwann cells (SCs), the results of which were further validated by reverse transcription quantitative polymerase chain reaction, western blot analysis and immunohistochemistry. To investigate the function of GPNMB on SCs, recombinant human GPNMB (rhGPNMB) was added to cultured denervated SCs from the distal stumps of transected sciatic nerve. The proliferation, expression and secretion of neurotrophic factors (NTFs) and neural adhesion molecules (NAMs) were subsequently detected. The results demonstrated that GPNMB expression was increased in distal sciatic nerve following transection in vivo, while rhGPNMB promoted the proliferation of SCs as well as expression and secretion of NTFs and NAMs in vitro. Therefore, GPNMB could be a novel strategy for peripheral nerve regeneration.
Assuntos
Glicoproteínas/metabolismo , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Nervo Isquiático/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Sistema Nervoso Central/metabolismo , Humanos , Masculino , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Traumatismo por Reperfusão/metabolismo , Células de Schwann/metabolismoRESUMO
Post-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive depending on specific pathological contexts. However, the role of KDM2 proteins during development remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, we have generated two dKdm2 null alleles using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately 1 h shorter than the control. Interestingly, lengthened circadian periods are observed when dKDM2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of dKDM2 and its orthologs in vertebrates regarding the regulation of circadian rhythms will advance our understanding of the epigenetic regulations of circadian clocks.
RESUMO
Maduramicin is one of the most extensively used anticoccidial drugs for the treatment of Eimeria spp. infections. However, overdosage, misuse and drug interactions have resulted in the development of ionophore toxic syndrome. Heart and skeletal muscles have been identified as the main target organs of toxicity. In the present study, primary chicken myocardial cells were isolated to investigate the toxicity and underlying mechanisms of maduramicin. Our results showed that maduramicin causes morphological changes and a decrease in the viability of chicken myocardial cells. Annexin V-FITC/PI and 4',6-diamidino-2-phenylindole (DAPI) staining showed a significant increase in the number of apoptotic cells. Furthermore, caspases-3/8/9 were activated at the gene and protein levels and this was accompanied by the upregulation of apoptosis-related genes, including bcl-2, bax, and cytochrome C. Treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone (z-VAD-fmk) ameliorated the apoptosis and cytotoxicity. Furthermore, intracellular Ca2+ and reactive oxygen species (ROS) were elevated, whereas mitochondrial membrane potential (MMP) and intracellular glutathione (GSH) decreased with exposure to maduramicin. The antioxidant N-acetyl-cysteine (NAC) had no significant effect on maduramicin-induced cytotoxicity and apoptosis. Taken together, our findings demonstrate that maduramicin is cytotoxic to primary chicken myocardial cells via caspase dependent and independent apoptotic pathways.
Assuntos
Antibacterianos/toxicidade , Lactonas/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspases/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Citocromos c/genética , Glutationa/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
The use of neurons induced from stem cells has been introduced as an effective strategy for promoting peripheral nerve regeneration (PNR). The evolution and role of native denervated Schwann cells (SCs) were often ignored when exploring the mechanisms underlying neural transplantation therapy for PNR. The aim of this study was to understand if following injury, native denervated SCs could be reactivated by transplanting of neurons induced from bone marrow-derived mesenchymal stem cells (NI-BMSCs) to promote PNR. We co-cultured denervated SCs with NI-BMSCs in vitro, tested the proliferation of denervated SCs, and measured the expression and secretion of neurotrophic factors and neural adhesion molecules of the denervated SCs. Concurrently, 48 adult male Sprague-Dawley rats were randomly divided into 4 even groups of 12 rats each: normal group, phosphate-buffered saline (PBS) injection group, BMSCs transplantation group and NI-BMSCs transplantation group. PBS injection and cells transplantation were performed 4 weeks post-injury. After 4 weeks of NI-BMSCs transplantation, the survival of seeded NI-BMSCs was examined, proliferation and ultrastructure of native denervated SCs were detected, and myelination, axonal regeneration and the sciatic functional index measurements were also determinated. Our results demonstrated that NI-BMSCs reactivated denervated SCs both in vitro and in vivo and promoted sciatic nerve regeneration.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Células de Schwann/fisiologia , Neuropatia Ciática/cirurgia , Animais , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Transmissão , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Recuperação de Função Fisiológica/fisiologia , Células de Schwann/ultraestrutura , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Bone marrowderived mesenchymal stem cells (BMSCs) and adipose tissuederived mesenchymal stem cells (ADSCs) are able to differentiate into neuronlike cells when exposed to small molecule compounds, however the specific differences in their neuronal differentiation abilities remain to be fully elucidated. The present study aimed to compare the neuronal differentiation abilities of BMSCs and ADSCs. BMSCs and ADSCs from the same Sprague Dawley rats were isolated and cultured for use. The proliferation capacity was revealed using a cell counting method. Following BMSCs and ADSCs induction by four types of smallmolecular compounds, the expression of various neuronal markers and the secretion of several neurotrophic factors were detected by immunofluorescence, western blotting, reverse transcriptionquantitative polymerase chain reaction and ELISA. It was demonstrated that the ADSCs exhibited an increased proliferation capacity compared with BMSCs, according to cumulative population doubling analyses. Following a 7day neuronal induction period, BMSCs and ADSCs exhibited a neuronlike morphology, and were termed neuronal induced (NI)BMSCs and NIADSCs. They expressed neuronal markers including ßtubulin III, microtubule associated protein 2 and choline acetyltransferase. The number of NIBMSCs that positively expressed the neuronal markers was significantly decreased compared with NIADSCs, and the expression and secretion of the neurotrophic factors nerve growth factor and 3'nucleotidase in NIBMSCs were additionally decreased compared with NIADSCs. The findings of the present study indicated that the neuronal differentiation abilities and neurotrophic factor secretion abilities of ADSCs were increased compared with BMSCs. ADSCs may therefore act as efficient candidates in cell transplantation therapy for diseases and injuries of the nervous system.
Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Neurônios/citologia , Animais , Biomarcadores/metabolismo , Proliferação de Células , Forma Celular , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Masculino , Células-Tronco Mesenquimais/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
BACKGROUND: Aberrant epithelial repair has been observed in chronic rhinosinusitis (CRS) patients; however, the mechanism of epithelial cell repair regulation is unclear. Epidermal growth factor (EGF) plays an important role in regulating epithelial cell repair in lower airway and may be a critical factor in the remodeling processes of CRS. The objective of our research is to evaluate the differences between CRS and normal subjects and between chronic rhinosinusitis without nasal polys (CRSsNP) and chronic rhinosinusitis with nasal polys (CRSwNP) in the regulation of EGF pathways and the regulating proliferative position of classic Ras/Raf/MEK/ERK pathways. METHODS: We evaluated the proliferation rates of ethmoidal mucosal cells before and after stimulation with EGF, epidermal growth factor receptor (EGFR) kinase inhibitor AG1478, and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 using MTT assays. We also analyzed the sinonasal epithelial cells collected from control subjects and patients with CRS subtypes CRSsNP and CRSwNP for the expression of ERK1/2, phosphorylated ERK1/2, P21, P15, and P27 using western blotting analyses. RESULTS: The proliferation rates of sinonasal epithelial cells before and after EGF stimulation were lower in CRS patients than in the controls. AG1478 or PD98059 inhibitor treatment of control epithelial cells did not result in a significant difference in proliferation. Although, AG1478 and PD98059 inhibited the proliferation of CRS cells, the degree of proliferation inhibition was markedly different in CRSsNP. AG1478 suppressed the proliferation of CRSwNP epithelial cells, whereas PD98059 had no effect. The ratio of ERK1/2 phosphorylation in CRS cells was lower than that of the control cells. Cyclin-dependent kinase inhibitors were highly expressed in CRS cells compared with that of control cells. ERK1/2 and P27 showed differential expression in CRSsNP and CRSwNP. CONCLUSIONS: Differences existed in EGF pathways in CRS patients and normal subjects as well as in CRSsNP and CRSwNP. Classical Ras/Raf/MEK/ERK pathway may assume absolute superiority in control cells. Ras/Raf/MEK/ERK classical pathway and other pathways might be active at the same time to stimulate epithelial cell proliferation in CRSsNP. The function of Ras/Raf/MEK/ERK classical pathway was weaker in CRSwNP than in CRSsNP and when the classical pathway was blocked in CRSwNP, some other pathway could have completely compensated the proliferation induced by the Ras/Raf/MEK/ERK pathway.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Mucosa Nasal/citologia , Sinusite/metabolismo , Adulto , Idoso , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Post-translational modification of histones plays essential roles in the transcriptional regulation of genes in eukaryotes. Methylation on basic residues of histones is regulated by histone methyltransferases and histone demethylases, and misregulation of these enzymes has been linked to a range of diseases such as cancer. Histone lysine demethylase 2 (KDM2) family proteins have been shown to either promote or suppress tumorigenesis in different human malignancies. However, the roles and regulation of KDM2 in development are poorly understood, and the exact roles of KDM2 in regulating demethylation remain controversial. Since KDM2 proteins are highly conserved in multicellular animals, we analyzed the KDM2 ortholog in Drosophila. We have observed that dKDM2 is a nuclear protein and its level fluctuates during fly development. We generated three deficiency lines that disrupt the dKdm2 locus, and together with 10 transposon insertion lines within the dKdm2 locus, we characterized the developmental defects of these alleles. The alleles of dKdm2 define three phenotypic classes, and the intragenic complementation observed among these alleles and our subsequent analyses suggest that dKDM2 is not required for viability. In addition, loss of dKDM2 appears to have rather weak effects on histone H3 lysine 36 and 4 methylation (H3K36me and H3K4me) in the third instar wandering larvae, and we observed no effect on methylation of H3K9me2, H3K27me2 and H3K27me3 in dKdm2 mutants. Taken together, these genetic, molecular and biochemical analyses suggest that dKDM2 is not required for viability of flies, indicating that dKdm2 is likely redundant with other histone lysine demethylases in regulating normal development in Drosophila.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Histona Desmetilases com o Domínio Jumonji/genética , Animais , Animais Geneticamente Modificados , Deleção Cromossômica , Sequência Conservada , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiência , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Genes de Insetos , Teste de Complementação Genética , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/deficiência , Mutação , Filogenia , Estrutura Terciária de ProteínaRESUMO
The E2F transcription factors are important regulators of the cell cycle whose function is commonly misregulated in cancer. To identify novel regulators of E2F1 activity in vivo, we used Drosophila to conduct genetic screens. For this, we generated transgenic lines that allow the tissue-specific depletion of dE2F1 by RNAi. Expression of these transgenes using Gal4 drivers in the eyes and wings generated reliable and modifiable phenotypes. We then conducted genetic screens testing the capacity of Exelixis deficiencies to modify these E2F1-RNAi phenotypes. From these screens, we identified mutant alleles of Suppressor of zeste 2 [Su(z)2] and multiple Polycomb group genes as strong suppressors of the E2F1-RNA interference phenotypes. In validation of our genetic data, we find that depleting Su(z)2 in cultured Drosophila cells restores the cell-proliferation defects caused by reduction of dE2F1 by elevating the level of dE2f1. Furthermore, analyses of methylation status of histone H3 lysine 27 (H3K27me) from the published modENCODE data sets suggest that the genomic regions harboring dE2f1 gene and certain dE2f1 target genes display H3K27me during development and in several Drosophila cell lines. These in vivo observations suggest that the Polycomb group may regulate cell proliferation by repressing the transcription of dE2f1 and certain dE2F1 target genes. This mechanism may play an important role in coordinating cellular differentiation and proliferation during Drosophila development.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Fator de Transcrição E2F1/genética , Proteínas do Grupo Polycomb/genética , Alelos , Animais , Animais Geneticamente Modificados/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/metabolismo , Histonas/metabolismo , Metilação , Fenótipo , Proteínas do Grupo Polycomb/metabolismo , Interferência de RNARESUMO
The characteristics of the bioleaching of realgar by Acidithiobacillus ferrooxidans BY-3 (A. ferrooxidans) were investigated in this work. We examined the effects of using ferrous iron and elemental sulfur as the sole and mixed energy sources on the bioleaching of realgar. Under all experimental conditions, A. ferrooxidans BY-3 significantly enhanced the dissolution of realgar. Moreover, arsenic was more efficiently leached using A. ferrooxidans BY-3 in the presence of ferrous iron than in other culture conditions. A high concentration of arsenic was observed in the absence of alternative energy sources. This concentration was higher than that in cultures with sulfur only and lower than that in cultures with ferrous iron and sulfur. Linear or nonlinear models best fit the experimental data; the nonlinear model exhibited the dual effects of dissolution and removal on the bioleaching of realgar, whereas the linear model only applied to situations of slow bioleaching rather than removal.