胰岛素样生长因子-1受体(IGF-1R)信号转导通路与各种正常生理活动及多种疾病发生密切相关.将IGF-1R来源的未磷酸化多肽序列LYASVNPEY固定于表面等离子体共振(SPR)生物传感器专用的CM5芯片表面,以含有相应蛋白激酶的细胞裂解液进行处理,利用SPR生物传感器和抗酪氨酸磷酸化抗体PY20进行酪氨酸磷酸化水平检测.结果表明:PY20能灵敏地检测多肽酪氨酸磷酸化水平变化,即能够区分不同孵育时间以及不同成分的细胞裂解液所引起的多肽酪氨酸磷酸化水平差异;研究了IGF-1R来源多肽的酪氨酸磷酸化对多肽-蛋白间相互作用的影响,证实了只存在磷酸化的多肽与其下游的胰岛素受体底物(IRS-1)相互作用,测定了此磷酸化的多肽与IRS-1的亲和常数(KA)为 (2.02 ± 0.09)×108 L mol-1、结合速率常数(ka)为(2.30±0.15)×106 L mol-1s-1以及解离速率常数(kd)为(1.14±0.13)×10-2 s-1.这种新型的模拟受体的多肽酪氨酸磷酸化研究方法可用于受体磷酸化信号通路的研究以及基于这些信号通路的药物筛选研究. 相似文献
In this work, a new ultra-performance liquid chromatograph-evaporative light-scattering detector (UPLC-ELSD) method for quantitation of glycidyl esters (GE) contents in edible oils is presented. The method features complete separation of five GE species within 20 min by a C18 column and gradient elution with a mobile phase consisting of 85% and 2.5% methanol aqueous solutions. The coefficients of regression (R2) were all ≥0.9999 for the linear-quadratic regression curves of GE species in a concentration range of 5~80 μg/mL. The intraday and interday recoveries (%) of GE species in solvent were in a range of 81.3~107.3%, and the intraday and interday coefficients of variation (CVs, %) were all ≤8.6%. The average recovery (%) of GE species spiked in extra-virgin olive oil samples ranged from 88.3~107.8% and the intermediate precision (CV, %) of ≤14% indicated acceptable accuracy and precision. The method exhibited limit of quantification (LOQ) for each GE species (0.6 μg glycidol equivalents/g oil). The method was applied to determine GE concentrations of six commercial oil samples, and total glycidol equivalents were consistent with data obtained by GC-MS method. This UPLC-ELSD method could be adopted for precursory screening and research purposes to improve food safety when MS detectors are unavailable. 相似文献
A novel actively targeted polymer carrier for anticancer drugs based on an N‐(2‐hydroxypropyl)methacrylamide copolymer (PHPMA) is proposed. An oligopeptide sequence GE7, attached to the polymer, is a specific ligand for the EGF receptor overexpressed on most tumor cells. Co‐attachment of selected chemotherapeutics will therefore lead to formation of tumor‐specific polymer therapeutics, further enhanced by the EPR effect. FACS measurements prove elevated binding activity of the fluorescently labeled PHPMA/GE7 conjugate in EGFR‐rich cells (FaDu, MCF‐7), compared to conjugates of scrambled peptides. Cell lines with low EGFR level (SW620, B16F10) bind the GE7 conjugate significantly less.
A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity. 相似文献
Genipin (GP), an active metabolite of geniposide (GE), exhibits more potent pharmacological effects than its parent compound. In this paper, a sensitive LC‐MS/MS method was developed and fully validated for the simultaneous determination of GE and GP in rat plasma. We found that GP degraded rapidly in rat plasma at room temperature as a result of irreversible binding with the endogenous nucleophiles in plasma. GP was stable when the sample's pH was ≤4.0. The degradation of GP in rat plasma was well prevented by immediate addition of 5% glacial acetic acid to the freshly collected plasma. The detection was performed on a tandem mass spectrometer coupled with electrospray ionization source in negative mode. Quantification was conducted by multiple reaction monitoring of the transitions [M + CH3COO]−m/z 447.3 → 225.3 for GE and [M − H]−m/z 225.2 → 123.1 for GP. The method exhibited high sensitivity (LLOQ 1 ng/mL for GE and 0.2 ng/mL for GP) by selecting the acetate adduct ions as the precursor ions for GE. The robust developed method was successfully applied to a pharmacokinetic study in rats after oral administration of GE. 相似文献
Triblock copolymer of poly(p-dioxanone) and polyethylene glycol end-capped with pyrene moieties ((Py-PPDO)2-b-PEG) was synthesized and used as modifier for multi-wall carbon nanotubes (MWCNTs). Nano-aggregates ((Py-PPDO)2-b-PEG@MWCNTs) with shish-kebab like partially wrapped morphology and very good stability were obtained by incorporating the copolymer with MWCNTs. The bare MWCNT sections of (Py-PPDO)2-b-PEG@MWCNTs were able to induce π-π interactions with graphene (GE) and resulted in a novel GE/(Py-PPDO)2-b-PEG@MWCNTs hybrid. The dispersity of GE in solution or polymer matrix was therefore greatly improved. The PCL nanocomposite films using GE/(Py-PPDO)2-bPEG@MWCNTs as hybrid nanofiller exhibited obviously improved mechanical properties especially at very low hybrid nanofiller content. The influence of the nanofiller content and feed ratio of GE/MWCNTs on the mechanical properties of composites films was evaluated. When the feed ratio of GE to MWCNTs is 2:8 and the total loading of nanofiller is only 0.01 wt%, the tensile strength of the composite film increased by 163% and the elongation at break increased by 17% compared to those of neat PCL. These results can be attributed to fine dispersion of the nanofillers in PCL matrix and the hybrid interactions between GE and MWCNTs. Therefore, this work provides a novel method for preparing polymer nanocomposites with high mechanical performance and low nanofiller loading. 相似文献
The intracellular kinase domains of the epidermal growth factor receptor (EGFR) in some tumor cells are significant targets for drug discovery. We have developed a new EGFR cell membrane chromatography (EGFR/CMC)-online-high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening anti-EGFR antagonists from medicinal herbs such as Radix Angelicae Pubescentis. In this study, the HEK293 EGFR cells with high expression of EGFR were used to prepare cell membrane stationary phase (CMSP) in the EGFR/CMC model. The retention fractions on the EGFR/CMC model were directly analyzed by combining a 10 port columns switcher with a HPLC/MS system online. As a result, osthole from Radix Angelicae Pubescentis was found to be the active component acting on EGFR like dasatinib as the control drug. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. This new EGFR/CMConline-HPLC/MS method can be applied for screening anti-EGFR antagonists from TCMs, for instance, Radix Angelicae Pubescentis. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource. 相似文献
We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis. 相似文献