RESUMO
Intracellular viscosity is a physicochemical factor that determines the outcomes of various biological processes, while nitric oxide (NO) is an essential signaling molecule that controls many cellular processes, including oxidative stress. Anticipating that both may be interrelated with a variety of pathologies, their simultaneous measurement would be highly valuable for the investigation of the pathological condition of cells. However, the development of a sensor for such simultaneous detection has not been attempted yet. Herein, we present the synthesis of naphthalimide-4-(4-nitrophenyl)thiosemicarbazide, probe 1, and its application to living cells under conditions of lipopolysaccharide or nystatin treatment, adopted as oxidative stress and altered intracellular viscosity models, respectively. The probe showed increased fluorescence in response to elevation of viscosity and NO levels at 470 and 550 nm, respectively, in the solution studies. When the probe was used for a confocal microscopic study of HeLa cells under stressed conditions, simultaneous monitoring of viscosity and NO level elevations was possible through fluorescence imaging using band-pass filters of 420-475 and 505-600 nm, respectively, upon excitation at a wavelength of 405 nm. Interestingly, both the cellular viscosity and NO levels increased together under lipopolysaccharide or nystatin treatment. Therefore, we suggest that probe 1 is a fluorescent chemical probe that enables the monitoring of alterations in intracellular viscosity and NO levels in living cells, which would be valuable in studies of various cellular damage models.
Assuntos
Corantes Fluorescentes , Naftalimidas , Células HeLa , Humanos , Microscopia de Fluorescência , Óxido Nítrico , Nitrofenóis , Semicarbazidas , ViscosidadeRESUMO
DNA repair mechanisms maintain genomic integrity upon exposure to various types of DNA damage, which cause either single- or double-strand breaks in the DNA. Here, we propose a strategy for the functional study of single nucleotide polymorphisms (SNPs) in the human DNA repair genes XPD/ERCC2, RAD18, and KU70/XRCC6 and the checkpoint activation gene ATR that are essentially involved in the cell cycle and DNA damage repair. We analyzed the mutational effects of the DNA repair genes under DNA-damaging conditions, including ultraviolet irradiation and treatment with genotoxic reagents, using a Saccharomyces cerevisiae system to overcome the limitations of the human cell-based assay. We identified causal variants from selected SNPs in the present analyses. (i) R594C SNP in RAD3 (human XPD/ERCC2) caused severe reductions in the growth rate of mutant cells upon short-wavelength UV irradiation or chemical reagent treatment. (ii) The growth rates of the selected variants in RAD18, YKU70, and MEC1 were similar to those of wild-type cells on methyl methanesulfonate and hydroxyurea treated media. (iii) We also assessed the structural impact of the SNPs by analyzing differences in the structural conformation and calculating the root mean square deviation, which is a measure of the discordance of the Cα atoms between protein structures. Based on the above results, we propose that these analytical approaches serve as efficient methods for the identification of causal variants of human disease-causing genes and elucidation of yeast-cell based molecular mechanisms.
Assuntos
Reparo do DNA/genética , Técnicas Genéticas , Polimorfismo de Nucleotídeo Único , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Biologia Computacional , Simulação por Computador , Meios de Cultura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Humanos , Hidroxiureia/toxicidade , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Metanossulfonato de Metila/toxicidade , Modelos Moleculares , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Raios Ultravioleta/efeitos adversos , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismoRESUMO
Premature ovarian failure (POF) is characterized by heterogeneous genetic causes such as chromosomal abnormalities and variants in causal genes. Recently, development of techniques made next generation sequencing (NGS) possible to detect genome wide variants including chromosomal abnormalities. Among 37 Korean POF patients, XY karyotype with distal part deletions of Y chromosome, Yp11.32-31 and Yp12 end part, was observed in two patients through NGS. Six deleterious variants in POF genes were also detected which might explain the pathogenesis of POF with abnormalities in the sex chromosomes. Additionally, the two POF patients had no mutation in SRY but three non-synonymous variants were detected in genes regarding sex reversal. These findings suggest candidate causes of POF and sex reversal and show the propriety of NGS to approach the heterogeneous pathogenesis of POF.