5-Methylcytosine (5-mC) is an important epigenetic mark that plays critical roles in a variety of cellular processes. To properly exert physiological functions, the distribution of 5-mC needs to be tightly controlled in both DNA and RNA. In addition to methyltransferase-mediated DNA and RNA methylation, premethylated nucleotides can be potentially incorporated into DNA and RNA during replication and transcription. To exclude the premodified nucleotides into DNA and RNA, endogenous 5-methyl-2'-deoxycytidine monophosphate (5-Me-dCMP) generated from nucleic acids metabolism can be enzymatically deaminated to thymidine monophosphate (TMP). Therefore, previous studies failed to detect 5-Me-dCMP or 5-methylcytidine monophosphate (5-Me-CMP) in cells. In the current study, we established a method by chemical labeling coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5-Me-dCMP and 5-Me-CMP. As N,N-dimethyl-p-phenylenediamine (DMPA) was utilized for labeling, the detection sensitivities of nucleotides increased by 88-372-fold due to the introduction of a tertiary amino group and a hydrophobic moiety from DMPA. Using this method, we found that endogenous 5-Me-dCMP and 5-Me-CMP widely existed in cultured human cells, human tissues, and human urinary samples. The presence of endogenous 5-Me-dCMP and 5-Me-CMP indicates that deaminases may not fully deaminate these methylated nucleotides. Consequently, the remaining premethylated nucleosides could be converted to nucleoside triphosphates as building blocks for DNA and RNA synthesis. Furthermore, we found that the contents of 5-Me-dCMP and 5-Me-CMP exhibited significant decreases in renal carcinoma tissues and urine samples of lymphoma patients compared to their controls, probably due to more reutilization of methylated nucleotides in DNA and RNA synthesis. This study is, to the best of our knowledge, the first report for detecting endogenous 5-Me-dCMP and 5-Me-CMP in mammals. The detectable endogenous methylated nucleotides indicate the potential deleterious effects of premodified nucleotides on aberrant gene regulation in cancers.
A micelle-mediated extraction technique, i.e. ionic liquid-based microwave-assisted extraction (ILs-MAE) technique has been developed for the effective extraction of podophyllotoxin from three Chinese medicinal plants. Several operating parameters were successively optimized by single-factor and L(9) (3(4)) orthogonal array experiments. 1-Butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)]), 1-decyl-3-methylimidazolium tetrafluoroborate ([demim][BF(4)]) and 1-allyl-3-methylimidazolium tetrafluoroborate ([amim][BF(4)]) were selected as the optimal surfactants for Dysosma versipellis, Sinopodophyllum hexandrum and Diphylleia sinensis, respectively. Compared with other extraction techniques, such as ionic liquids-based maceration extraction (ILs-ME), heat extraction (ILs-HE) and ultrasound-assisted extraction (ILs-UAE), the ILs-MAE technique not only took a shorter time but also afforded a higher extraction rate of podophyllotoxin from the herbs. Reversed phase high performance liquid chromatography was employed for the analysis of podophyllotoxin. The results showed that the linearity for analyzing podophyllotoxin in all three herbs was in the concentration range of 0.005-0.4 mg mL(-1) with the correlation coefficient between 0.9993 and 0.9996. LODs were 2.05-2.58 μg mL(-1) and RSDs of inter-day stability were less than 5.8%. Repeatability and intermediate precision were separately lower than 3.3% and 6.3%. The recoveries for podophyllotoxin extracted with the ILs-MAE technique were in the range of 97.1-102% and all RSDs were lower than 3.0%. Furthermore, the mechanism of ILs-MAE was preliminarily studied by means of kinetic mechanism, surface structures and chemical compositions of samples before and after different extraction techniques. On the basis of the destruction of herb surface microstructures and high solubility of ILs, the ILs-MAE technique eventually got the maximum yield value.
The preparation of magnetic molecularly imprinted polymers (MMIPs) which can be used for the separation and purification of rutin from Chinese medicinal plants has been proposed. By applying the improved co-precipitation method, magnetic Fe(3)O(4) particles were easily prepared, followed by the modification of TEOS and functionalization with -CH=CH(2). Using functionalized Fe(3)O(4) particles as the magnetic cores, rutin as the template, and acrylamide as the functional monomer, MMIPs were synthesized by surface-imprinted polymerization under the protection of nitrogen gas and successive mechanical stirring at 60 °C for 24 h. Magnetic non-molecularly imprinted polymers (MNIPs) were also prepared with the same synthesis procedure as with MMIPs only without the presence of rutin. Magnetic particles were characterized by FT-IR, XRD, and TG analysis. And the selectivity of MMIPs was also investigated in detail. In addition, the performance of the MMIPs for the adsorption of rutin in the analysis of Chinese medicinal plants was assessed. The mean recoveries were 84.33% (RSD: 3.22%, n = 3) for Saururus chinensis (Lour.) Bail and 85.20% (RSD: 3.58%, n = 3) for Flos Sophorae, respectively, which showed that the prepared MMIPs with many advantages possess the value of practical application.
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