RESUMEN
Homologous recombination plays a key role in double-strand break repair, stalled replication fork repair, and meiosis. The RecA/Rad51 family recombinases catalyze the DNA strand invasion reaction that occurs during homologous recombination. However, the high sequence differences between homologous groups have hindered the thoroughly studies of this ancient protein family. The dynamic mechanisms of the family, particularly at the residual level, remain poorly understood. In this work, five representative RecA/Rad51 recombinase family members from all major kingdoms of living organisms: prokaryotes, eukaryotes, archaea, and viruses, were selected to explore the molecular mechanisms behind their conserved biological significance. A variety of techniques, including all-atom molecular dynamics simulation, perturbation response scanning, and protein structure network analysis, were used to examine the flexibility and correlation of protein domains, distribution of sensors and effectors and conserved hub residues. Furthermore, the potential communication routes between the ATP-binding region and the DNA-binding region of each recombinase were identified. Our results demonstrate the conserved molecular dynamics of these recombinases in the early stage of homologous recombination, including cooperative motions between regions, conserved sensing and effecting functional residue distribution, and conserved hub residues. Meanwhile, the unique ATP-DNA communication routes of each recombinase was also revealed. These results provide new insights into the mechanism of RecA/Rad51 family proteins, and provide new theoretical guidance for the development of allosteric inhibitors and the application of RecA/Rad51 family proteins.
Asunto(s)
Recombinasa Rad51 , Rec A Recombinasas , Recombinasa Rad51/genética , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN de Cadena Simple , ADN/química , Recombinasas/genética , Recombinasas/metabolismo , Adenosina TrifosfatoRESUMEN
RecA family recombinases are the core enzymes in the process of homologous recombination, and their normal operation ensures the stability of the genome and the healthy development of organisms. The UvsX protein from bacteriophage T4 is a member of the RecA family recombinases and plays a central role in T4 phage DNA repair and replication, which provides an important model for the biochemistry and genetics of DNA metabolism. UvsX shares a high degree of structural similarity and function with RecA, which is the most deeply studied member of the RecA family. However, the detailed molecular mechanism of UvsX has not been resolved. In this study, a comprehensive all-atom molecular dynamics simulation of the UvsX protein dimer complex was carried out in order to investigate the conformational and binding properties of UvsX in combination with ATP and DNA, and the simulation of RecA was synchronized with the property comparison learning for UvsX. This study confirmed the highly conserved molecular structure characteristics and catalytic centers of RecA and UvsX, and also discovered differences in regional conformation, volatility and the ability to bind DNA between the two proteins at different temperatures, which would be helpful for the subsequent understanding and application of related recombinases.
Asunto(s)
Recombinasas , Proteínas Virales , Recombinasas/genética , Recombinasas/metabolismo , Proteínas Virales/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Rec A Recombinasas/genética , Bacteriófago T4/química , ADN de Cadena SimpleRESUMEN
Heavy-metal pollution has been established to affect ginseng quality. However, this effect is still unknown in ginseng of different ages, emphasizing the need to investigate the effects of heavy metals in soils on ginseng growth. Herein, we determined the content of heavy metals (Cu, Cd, Pb, Hg, and As) in ginseng of different ages (2 to 6-year-old) and the corresponding soil samples. Then, the total ginsenosides content of ginseng and rate-limiting enzyme (HMGR, SQE, CYP450) activity in the synthesis of ginsenosides were assessed. Results from 200 differently-aged Chinese ginseng showed that increased ginsenoside content in 3 to 5-year-old ginseng was paralleled by increased heavy metal element content in ginseng and its soil. The activity of rate-limiting enzymes increased in the first four years of ginseng growth and then exhibited a steady or downward trend. Further analysis suggested that heavy metal elements in soils could directly affect ginsenoside content. Moreover, we found that Cu significantly affected the rate-limiting enzyme CYP450 activity. Further principal component analysis and correlation analysis found that heavy metals could obviously inhibit ginseng growth during the 5th and 6th years. Heavy metal content in soils has huge prospects for predicting ginsenoside, Cu and As content in ginseng. This study provided support for ginseng cultivation, quality research and quality assessment.
Asunto(s)
Ginsenósidos , Metales Pesados , Panax , Contaminantes del Suelo , China , Monitoreo del Ambiente , Ginsenósidos/análisis , Metales Pesados/análisis , Medición de Riesgo , Suelo , Contaminantes del Suelo/análisisRESUMEN
Effective methods are required to quantify the organochlorine pesticide procymidone due to its potentially harmful effects toward human health and the environment. Here, hydrophilic hollow imprinted microspheres were prepared via a simple method as fluorescent sensors (@MIH-prm) for the sensitive and selective detection of PRM in ginseng. A new method of adsorption efficiency evaluation for @MIH-prm was subsequently introduced (EBS%), the effective binding site, which provided a comprehensive evaluation of the performance compared with conventional methods. The results showed that @MIH-prm could detect PRM in filtered and diluted ginseng juice with high sensitivity (LOD, 0.569 nM) and a rapid detection rate (quantitative detection of PRM within 18 min). Good selectivity was observed in the presence of combinations of different pesticides, and the adsorption of PRM could be described by the pseudo-second-order kinetic model. PRM concentrations exhibited good linearity over 1-40 nM, and the accuracy (recovery rates, 99.2 to 103.1%) and precision (RSD at 1.0 × 10-9 M, 3.14%) indicated that @MIH-prm could be used for the quantitative analysis of PRM in complex matrices. Hence, @MIH-prm has good application potential in pollution control monitoring and enforcement.
Asunto(s)
Impresión Molecular , Panax , Compuestos Bicíclicos con Puentes , Colorantes , Humanos , Impresión Molecular/métodos , Polímeros Impresos Molecularmente , Panax/química , Polímeros/químicaRESUMEN
ABSTRACT: Food additives are widespread in the human diet; however, their excessive intake can have an impact on the quality of health. This study evaluated food additives in bread and pasta products consumed by residents in various regions of Jilin Province, People's Republic of China, from 2019 to 2021. We collected samples of bread and six types of pasta products from farmers' markets and morning markets and used high-performance liquid chromatography, UV-visible spectrophotometry, and graphite furnace atomic absorption spectrometry to detect the content of the following food additives: sodium formaldehyde sulfoxylate, aluminum, and borate compounds. For 836 samples in total, we detected the presence of sodium formaldehyde sulfoxylate, aluminum, and borate compounds in excess rates reaching 3.5, 10, and 4.7%, respectively. Aluminum in fried breadsticks exceeded the standard by 40%. The results of this study can be used to assess the overall pass rate of bread and pasta products sold in Jilin Province and support the detection of possible food safety problems.
Asunto(s)
Aluminio , Pan , Aluminio/análisis , Boratos/análisis , Pan/análisis , China , Aditivos Alimentarios/química , Formaldehído , Humanos , Sodio , Triticum/químicaRESUMEN
ABSTRACT: This study evaluated the microbial contamination status of cold dishes consumed by residents of Jilin Province and investigated to determine the incidence of four pathogenic bacteria in cold dishes. A total of 300 samples of cold dishes, including meat, vegetable, and mixed products, were collected from three purchasing places: supermarkets, farmers' markets, and mobile vendors. Viable bacteria were isolated using conventional culture methods. After separation, a quick and easy PCR was used to detect Listeria monocytogenes, Staphylococcus aureus, enterotoxigenic Escherichia coli, and Salmonella. The results showed that the total number of microbial colonies in the vegetable samples exceeded the standard rate of 8% and the total number of microbial colonies in the meat and mixed samples did not exceed the standard. The total microbial colony count exceeded the standard in all three procurement sites, with the highest exceedance of 7.4% in the mobile vendor sites. The detection rates of enterotoxigenic E. coli, S. aureus, L. monocytogenes, and Salmonella, among the four pathogenic bacteria detected in all samples, were 4.3, 3.3, 3.0, and 1.0%, respectively. This study can be used to qualitatively assess the microbiological quality associated with cold dishes. It provides data to support the detection of possible food safety problems.
Asunto(s)
Microbiología de Alimentos , Listeria monocytogenes , Recuento de Colonia Microbiana , Escherichia coli , Contaminación de Alimentos/análisis , Prevalencia , Salmonella , Staphylococcus aureus , Verduras/microbiologíaRESUMEN
ABSTRACT: We evaluated fresh vegetables for residues of 18 pesticides with different chemical structures, including organochlorine pesticides, organophosphorus pesticides, carbamate pesticides, and pyrethroid pesticides and estimated that the potential health risks for consumers. A total of 313 samples were collected from 12 kinds of vegetables in Changchun, the capital of Jilin Province, People's Republic of China. Pesticide residues were analyzed by gas chromatography and mass spectrometry, and the curves were highly linear at 0.01 to 1.00 µg/mL (R2 ≥ 0.99). The mean recovery rate of the pesticides was 62 to 110% (relative standard deviation of <5%). The limit of detection was 0.0001 to 0.0167 mg/kg, the limit of quantification was 0.0002 to 0.0556 mg/kg, and the overall detection rate was 28.43%. The prevalence of pesticides and of samples above the standard limit were highest in celery, the prevalence of pesticides was lowest in potatoes, and the prevalence of samples above the standard limit was lowest in cucumber. Three of the 18 pesticides were not detected: omethoate, chlorpyrifos, and fenvalerate. Among the 15 pesticides detected, the maximum risk factor of six (carbofuran, omethoate, phorate, dicofol, dimethoate, and dichlorvos) is >1, indicating possible harm to human health. Residues of a single pesticide may not adversely affect a person's health, but multiple pesticide residues could present a health risk.
Asunto(s)
Residuos de Plaguicidas , Plaguicidas , China , Contaminación de Alimentos/análisis , Humanos , Residuos de Plaguicidas/análisis , VerdurasRESUMEN
Digital PCR (polymerase chain reaction) is a powerful and attractive tool for the quantification of nucleic acids. However, the multiplex detection capabilities of this system are limited or require expensive instrumentation and reagents, all of which can hinder multiplex detection goals. Here, we propose strategies toward solving these issues regarding digital PCR. We designed and tested a self-priming digital PCR chip containing 6-plex detection capabilities using monochrome fluorescence, which has six detection areas and four-layer structures. This strategy achieved multiplex digital detection by the use of self-priming to preintroduce the specific reaction mix to a certain detection area. This avoids competition when multiple primer pairs coexist, allowing for multiplexing in a shorter time while using less reagents and low-cost instruments. This also prevents the digital PCR chip from experiencing long sample introduction time and evaporation. For further validation, this multiplex digital PCR chip was used to detect five types of EGFR (epidermal growth factor receptor) gene mutations in 15 blood samples from lung cancer patients. We conclude that this technique can precisely quantify EGFR mutations in high-performance diagnostics. This multiplex digital detection chip is a simple and inexpensive test intended for liquid biopsies. It can be applied and used in prenatal diagnostics, the monitoring of residual disease, rapid pathogen detection, and many other procedures.
Asunto(s)
Neoplasias Pulmonares , Reacción en Cadena de la Polimerasa Multiplex , Pruebas Genéticas , Humanos , Neoplasias Pulmonares/genética , Mutación , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
ABSTRACT: Heavy metals are an indispensable part of industrial and agricultural development. As the cradle of China's industry and an important province for agricultural production, Jilin Province has been an area of concern about heavy metal pollution in the local environment and grains. In this study, we focused on four heavy metals that are harmful to humans: arsenic (As), cadmium (Cd), methylmercury (MeHg), and inorganic arsenic (iAs). We determined the contents of these metals in 341 grain samples by using graphite furnace atomic absorption spectrometry and liquid chromatography-atomic fluorescence spectrometry and compared our results with the limit value of national standards. To evaluate the potential risk to human health, we determined the target hazard quotient and hazard index. Heavy metals were detected at these rates, from high to low: Cd (48%) > iAs (20.8%) > MeHg (4.6%) > Pb (3%). Most of these values are far below the limit of national standards. The target hazard quotient and hazard index were both smaller than 1; thus, we conclude that heavy metal pollution in grains in Jilin Province is not serious and that people are not at high risk from heavy metals in grains.
Asunto(s)
Arsénico , Metales Pesados , Contaminantes del Suelo , Arsénico/análisis , Cadmio/análisis , China , Monitoreo del Ambiente , Humanos , Metales Pesados/análisis , Medición de Riesgo , Suelo , Contaminantes del Suelo/análisisRESUMEN
Massive viral outbreaks draw attention to viruses that have not been thoroughly studied or understood. In recent decades, microfluidic chips, known as "lab-on-a-chip", appears as a promising tool for the detection of viruses. Here, we review the development of microfluidic chips that could be used in response to viral detection, specifically for viruses involved in more recent outbreaks. The advantages as well as the disadvantages of microfluidic systems are discussed and analyzed. We also propose ideas for future development of these microfluidic chips and we expect this advanced technology to be used in the future for viral outbreaks.
Asunto(s)
Técnicas Biosensibles , Técnicas Analíticas Microfluídicas , Virus , Dispositivos Laboratorio en un Chip , MicrofluídicaRESUMEN
Point-of-care (POC) testing offers rapid diagnostic results. However, the quantification of current methods is performed using standard curves and external references, and not direct and absolute quantification. This paper describes an integrated multiplex digital recombinase polymerase amplification (ImdRPA) microfluidic chip which combines DNA extraction, multiplex digital RPA and fluorescence detection together in one chip, creating a "sample-in-multiplex-digital-answer-out" system. Multi-layer soft lithography technology was used, with polydimethylsiloxane (PDMS) as the chip material and a glass slide as the substrate. This microfluidic chip has a six-layer structure and screw microvalve control function. The sample preparation for the chip involved magnetic bead-based nucleic acid extraction, which was completed within 15 min without any instrument dependence. The dRPA region was divided into 4 regions (3 positive detection areas and 1 negative control area) and included a total of 12 800 chambers, with each chamber being able to contain a volume of 2.7 nL. The screw valve allowed for the reaction components of each specific goal to be pre-embedded in different regions of the chambers. The reagents were passively driven into the dRPA region using vacuum-based self-priming introduction. Furthermore, we successfully demonstrated that the chip can simultaneously detect three species of pathogenic bacteria within 45 min and give digital quantitative results without the need to establish a standard curve in contaminated milk. Moreover, the detection limit of this ImdRPA microfluidic chip was found to be 10 bacterial cells for each kind of pathogen. These characteristics enhance its applicability for rapid detection of foodborne bacteria at the point-of-care (POC). We envision that the further development of this integrated chip will lead to rapid, multiplex and accurate detection of foodborne bacteria in a feasible manner.
Asunto(s)
Microfluídica , Recombinasas , Dispositivos Laboratorio en un Chip , Técnicas de Amplificación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Atención de Punto , Pruebas en el Punto de AtenciónRESUMEN
The purpose of this study was to investigate the status of organochlorine pesticide residues in 186 ginseng samples collected in Jilin, People's Republic of China. Based on the Chinese Pharmacopoeia 2015 method for detection of organochlorine pesticide residues in ginseng, 22 organochlorine pesticide residues were identified. Chlordane, aldrin, epichlorohydrin, and dieldrin and their isomers were not detected in ginseng from this region. Heptachlor was detected in only one ginseng sample, and the concentration did not exceed the maximum residual limit (MRL) prescribed in the Pharmacopoeia (0.05 mg/kg). Benzene hexachloride was detected in two samples, one of which was above the MRL. Hexachlorobenzene and pentachloronitrobenzene (quintozene) were found in 11.8 and 52.1% of the samples, respectively, and the residues in these samples exceeded the MRL by 4.3 and 8.6%, respectively.
Asunto(s)
Contaminación de Alimentos , Hidrocarburos Clorados , Panax , Residuos de Plaguicidas , China , Contaminación de Alimentos/análisis , Hidrocarburos Clorados/análisis , Panax/química , Residuos de Plaguicidas/análisisRESUMEN
Autophagy is an important mechanism for cell death regulation. To improve the anticancer effect during the treatment of leukemia and promote the apoptosis of leukemic cells, it is important to define the relationship between autophagy and apoptosis. A key bioactive compound in traditional Chinese medicine, 20(s)-Ginsenoside (GRh2), demonstrated an advancement in leukemia treatment. Blue LED therapy (BL) is a physical treatment method that can induce leukemic cell death. In this study, we tested the effect of 20(s)-GRh2, BL, and their combination (BL-GRh2) on the activation of leukemic cell apoptosis and autophagy. Both treatments, whether used individually or simultaneously, induce apoptosis through the induction of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP) and regulated the expression of apoptosis-related genes and proteins. Furthermore, using western blotting to analyze the autophagy markers LC3B and P62, we detected the activation of autophagy. In cells treated with autophagy inhibitor 3-MA, both autophagy and apoptosis were inhibited, either by BL alone or by BL-GRh2. However, apoptosis in 20(s)-GRh2-treated cells was enhanced. In cells treated with apoptosis suppressor Z-VAD-FMK, autophagy was inhibited in the BL and BL-GRh2-treated cells, although it was enhanced in cells treated with 20(s)-GRh2 alone. Moreover, we observed a stronger induction of apoptosis by BL-GRh2 in myeloid leukemia cells. Our data indicate that autophagy induced by different factors can play diverse roles on the same cells. Our results also indicate that the combination of traditional Chinese medicine with physical therapy may be a new strategy for anti-cancer therapy.
RESUMEN
In this study, we designed and synthesized two novel fluorescent magnetic nanoparticles. Fe3O4@SiO2-NH-GSH-CdTe (FSGC) (GSHâ¯=â¯glutathione) nanoparticles were synthesized using amino-functionalized Fe3O4@SiO2 nanoparticles and GSH-stabilized CdTe quantum dots (CdTe QDs), while flexible Fe3O4@SiO2-NH-GSH-CdTe-NH-NH2 (FSGCN) nanoparticles were synthesized using the FSGC precursor and 1,6-hexamethylenediamine. These two kinds of nanoprobes exhibited excellent magnetic and fluorescent properties. By comparing the fluorescence quenching effect of folic acid (FA) on FSGC and FSGCN, we found that the quenching effect of FA on FSGC was acute and the process was too fast to determine the FA content. However, the quenching effect of FA on flexible FSGCN was mild and hence it could be used as a nanoprobe to determine FA concentration. At physiological pH, the fluorescence quenching effect of FA on the FSGCN nanoprobes was fitted according to the Stern-Volmer equation with a linear response in the concentration range of 0.14 to 4.20⯵gâ¯mL-1 with a detection limit of 15.1â¯×â¯10-9â¯gâ¯mL-1 (S/Nâ¯=â¯3) under optimized experimental conditions. The proposed flexible nanoprobe was successfully used to determine the content of FA in folic acid tablets. Recovery was found to be in the range of 92.7%-105.6% with a relative standard deviation of 1.12%-3.84%. Owing to their good stability, environment-friendly characteristics, high selectivity, and good optical properties and biocompatibility, these nanoprobes have potential for usage in practical applications.
Asunto(s)
Compuestos de Cadmio/química , Óxido Ferrosoférrico/química , Colorantes Fluorescentes/química , Puntos Cuánticos/química , Telurio/químicaRESUMEN
Excessive reactive oxygen species (ROS) levels are harmful to the body. The peroxidase, GPx, and the superoxide dismutase, SOD, are important antioxidant enzymes for preventing ROS-induced damage. Se-CuZn-65P is an enzyme mimetic with dual GPx and SOD antioxidant function. However, currently, its production is mainly based on the cysteine auxotrophic expression technique, which is inefficient, expensive, and time consuming. In this study, we combined protein engineering and the chemical mutation method to synthesize Se-CuZn-65P. The DNA sequence encoding the 65 amino acid peptide with the desired sequence transformations to incorporate the SOD and the GPx catalytic sites was cloned and expressed in a soluble protein expression vector. The protein yield increased up to 152 mg/L, which is 10 times higher than in previous studies. The SOD and GPx activity of Se-CuZn-65P was high (1181 U/mg and 753 U/µmol, respectively). The binding constant of glutathione was 5.6 × 104 L·mol-1 , which shows that Se-CuZn-65P efficiently catalyzed hydrogen peroxide reduction by glutathione. Mitochondrial damage experiments confirmed the double protective role of the Se-CuZn-65P peptide and demonstrated functional synergy between the SOD and the GPx domains, which indicates its potential to be used in the treatment of ROS-related diseases. Our research may give a new thought to increase the yield of mimic.
Asunto(s)
Antioxidantes/química , Glutatión Peroxidasa/química , Péptidos/química , Superóxido Dismutasa/química , Animales , Antioxidantes/metabolismo , Dominio Catalítico , Glutatión , Glutatión Peroxidasa/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Péptidos/genética , Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/química , Superóxido Dismutasa/genéticaRESUMEN
Acute myeloid leukemia (AML) and Chronic myelogenous leukemia (CML) are common leukemia in adults. 20(S)-GRh2 is an important bioactive substance that is present in Panax ginseng. However, there are no investigations that deal with the comparison of apoptosis, the occurrence of autophagy, and the relationship between apoptosis and autophagy after being treated with 20(S)-GRh2 in AML and CML. In this study, we explored the effect of 20(S)-GRh2 on the AML and CML (U937 and K562). Fluorescence microscopy, CCK-8, Quantitative realtime PCR, Western blot, transmission electron microscopy (TEM), and flow cytometric analysis were used to detect the occurrence of cell proliferation inhibition, apoptosis, and autophagy. By using the above methods, it was determined that apoptosis induced by 20(S)-GRh2 was more obvious in K562 than U937 cells and 20(S)-GRh2 could generate autophagy in K562 and U937 cells. When pretreated by a specific inhibitor of autophagy, (3-methyladenine), the 20(S)-GRh2-induced apoptosis was enhanced, which indicated that 20(S)-GRh2-induced autophagy may protect U937 and K562 cells from undergoing apoptotic cell death. On the other hand, pretreated by an apoptosis suppressor (Z-VAD-FMK), it greatly induced the autophagy and partially prevented 20(S)-GRh2 induced apoptosis. This phenomenon indicated that 20(S)-GRh2-induced autophagy may serve as a survival mechanism and apoptosis and autophagy could act as partners to induce cell death in a cooperative manner. These findings may provide a rationale for future clinical application by using 20(S)-GRh2 combined autophagy inhibitors for AML and CML.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ginsenósidos/farmacología , Proliferación Celular , Humanos , Células K562 , Especies Reactivas de Oxígeno/metabolismo , Células U937RESUMEN
Panax ginseng is a traditional medicine. Fresh ginseng is one of the most important industries related to ginseng development, and fresh ginseng of varying ages has different medicinal properties. Previous research has not systematically reported the correlation between changes in key enzyme activity with changes in ginsenoside content in fresh ginseng over time. In this study, for the first time, we use ginseng samples of varying ages in Ji'an and systematically reported the changes in the activity of seven key enzymes (HMGR, FPS, SS, SE, DS, CYP450, and GT). We investigated the content of ginsenoside and gene expression of these key enzymes. Ginsenoside content was measured using HPLC. HPLC, GC-MS, and LC-MS were combined to measure the enzyme activity of the key enzymes. Quantitative PCR was used in the investigation of gene expression. By analyzing the correlation between the enzyme activity and the transcription level of the key enzymes with ginsenoside content, we found that DS and GT enzyme activities are significantly correlated with the ginsenoside content in different ages of ginseng. Our findings might provide a new strategy to discriminate between ginseng of different years. Meanwhile, this research provides important information for the in-depth study of ginsenoside biosynthesis.
Asunto(s)
Expresión Génica , Ginsenósidos/biosíntesis , Panax/crecimiento & desarrollo , Proteínas de Plantas/genética , Vías Biosintéticas , Cromatografía Líquida de Alta Presión , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ginsenósidos/análisis , Panax/genética , Panax/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Espectrometría de Masas en TándemRESUMEN
Glutathione peroxidase (GPx) is an antioxidant protein containing selenium. Owing to the limitations of native GPx, considerable efforts have been made to develop GPx mimics. Here, a short 5-mer peptides (5P) was synthesized and characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Enzyme coupled assays were used to evaluate GPx activity. The cell viability and apoptosis of H22 cells were tested, and mice bearing H22 cell-derived tumors were used to determine the effects of 5P on tumor inhibition. In comparison with other enzyme models, 5P provided a suitable substrate with proper catalytic site positions, resulting in enhanced catalytic activity. In our mouse model, 5P showed excellent inhibition of tumor growth and improved immunity. In summary, our findings demonstrated the design and synthesis of the small 5P molecule, which inhibited tumor growth and improved immunity. Notably, 5P could inhibit tumor growth without affecting normal growth. Based on these advantages, the novel mimic may have several clinical applications.
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Antineoplásicos/farmacología , Antioxidantes/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Inmunidad Innata/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Oligopéptidos/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Antioxidantes/síntesis química , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Biocatálisis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Glutatión Peroxidasa/química , Glutatión Peroxidasa/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Ratones , Imitación Molecular , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Especificidad de Órganos , Fagocitosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Resultado del Tratamiento , Proteína X Asociada a bcl-2/agonistas , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/inmunologíaRESUMEN
Security issues of nanoparticles on biological toxicity and potential environmental risk have attracted more and more attention with the rapid development and wide applications of nanotechnology. In this work, we explored the effect and probable mechanism of nano-TiO2 on antioxidant activity of copper, zinc superoxide dismutase (Cu, Zn-SOD) under natural light and mixed light at physiological pH. Nano-TiO2 was prepared by sol-hydrothermal method, and then characterized by X-ray Diffraction (XRD) and Transmission electron micrographs (TEM). The Cu, Zn-SOD was purified by sephadex G75 chromatography and qualitatively analyzed by sodium dodecyl sulfate polypropylene amide gel electrophoresis (SDS-PAGE). The effect and mechanism were elucidated base on Fourier Transform Infrared Spectrometer (FT-IR), Circular Dichroism (CD), zeta potential, and electron spin resonance (ESR) methods. Accompanying the results of FT-IR, CD and zeta potential, it could be concluded that nano-TiO2 had no effect on the antioxidant activity of Cu, Zn-SOD by comparing the relative activity under natural light at physiological pH. But the relative activity of Cu, Zn-SOD significantly decreased along with the increase of nano-TiO2 concentration under the mixed light. The results of ESR showed the cause of this phenomenon was the Cu(II) in the active site of Cu, Zn-SOD was reduced to Cu(I) by H2O2 and decreased the content of active Cu, Zn-SOD. The reduction can be inhibited by catalase. Excess O2·- produced by nano-TiO2 photocatalysis under mixed light accumulated a mass of H2O2 through disproportionation reaction in this experimental condition. The results show that nano-TiO2 cannot affect the antioxidant activity of Cu, Zn-SOD in daily life. The study on the effect of nano-TiO2 on Cu, Zn-SOD will provide a valid theory support for biological safety and the toxicological effect mechanism of nanomaterials on enzyme.
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Antioxidantes/metabolismo , Nanopartículas , Procesos Fotoquímicos , Superóxido Dismutasa/metabolismo , Titanio/química , Titanio/farmacología , Animales , Antioxidantes/química , Catálisis , Dominio Catalítico , Bovinos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Superóxido Dismutasa/químicaRESUMEN
A highly selective fluorescent probe 2-(2-(2-aminoethylamino)ethyl)-3',6'-bis(ethylamino)-2',7'-dimethylspiro[isoindoline-1,9'-xanthen]-3-one (ABDO) for Se (IV) had been synthesized in our earlier report. In this study, this fluorescent sensor is applied on analysis fluorescent imaging of Se (IV) in Hela cells. The experiment conditions, such as the MTT assay, different concentration of saline, incubated time of Hela cells with ABDO and Se (IV), and intracellular action position of Se (IV), are investigated. Through a series of experiments, the fluorescent image of Se (IV) in Hela cells can be observed when the cells cultured by 2 µM ABDO and 2 µM Se (IV) for 210 min. And the intracellular action position of Se (IV) is verified after the co-localization experiments are done. It is mitochondria. These experimental results show that ABDO will be an eagerly anticipated sensor for fluorescent imaging analysis of selenium ion in living cells. Besides, we also can use the complexes of ABDO-Se to observe morphology and distribution of mitochondria in cells like JG-B.