On-line HPLC–MS–DPPH assay for the analysis of phenolic antioxidant compounds in fruit wine: Antidesma thwaitesianum Muell.

A reversed phase high performance liquid chromatography (RP-HPLC) separation coupled with an electrospray ionisation mass spectrometry detection (ESI-MS) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used for the screening of multiple antioxidant compounds in Antidesma thwaitesianum Muell. fruit wine. The active compounds were identified by comparison with authentic standards and published mass spectra. With the help of the multidimensional information of LC–ESI-MS/MS and DPPH assay, the compounds with different chemical structures could be determined in one run successfully. The antioxidant compounds were separated and identified as gallic acid, cyanidin-3-sophoroside, monogalloyl glucoside, delphinidin-3-sambubioside, catechin, caffeic acid, and pelargonidin-3-malonyl glucoside. This result shows that an on-line HPLC–MS–DPPH assay can be a powerful technique for the rapid characterisation of antioxidant compounds in plant extracts.     

肽类抑菌物质效价的测定

采用琼脂扩散法以Nisin为标准样品,对副干酪乳杆菌(LactobacillusparacaseiHD1.7)产生的肽类抑菌物质效价测定条件进行了研究,结果表明在2号检测培养基中添加0.75%琼脂、以浓度为106cfu/mL枯草芽孢杆菌为指示菌是最佳检测效价的条件,在此条件下测得的肽类抑菌物质的效价为1.6×104IU/g。     

Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.     

Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them,PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts.Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting antiserum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.     

电痛阈测定法对光敏剂亚甲兰镇痛效果的评定

目的:利用电痛阈测定法精确评定光敏剂亚甲兰的镇痛效应.方法:50只SD大鼠随机分为5组:生理盐水组、0.25%亚甲兰组、0.5%亚甲兰组、0.75%亚甲兰组、1%亚甲兰组.将清醒大鼠的右下肢给予刺激,左肢引导,给予以0.28mA为起始强度、0.02mA为阶梯值的5个串长的连续递增的电刺激,观察给予一定的刺激强度后经过一定的潜伏期后出现第一个复合波形后的相对应的电流值,作为动物给药前之基础痛阈值.选择靠近刺激电极中枢段的右侧坐骨神经为阻断点,神经刺激器定位,注射药物,同样的方法观察记录给药后的1h、2h、3h、4h、6h、8h、12h、24h的痛阈的变化,来确定约物的镇痛效果及药物的起效时间和作用高峰时间.结果:0.25%组亚甲兰无效应,0.5%组在4小时起效,与对照组比较有显著差异,但是很快又恢复至对照水平.0.75%组滓射后3d小时起效果,与对照组比较有显著差异,且这种效果可以持续12小时:1%组注射后2小时起效果,与对照组比较有显著差异,且这种效果可以持续24小时之久.结论:亚甲兰可以提高大鼠的痛阈值,且有浓度递增效应.0.75%组和1%组阻滞效果显著,2小时至3小时起效,4小时达高峰.     

Indian Hedgehog in Synovial Fluid Is a Novel Marker for Early Cartilage Lesions in Human Knee Joint

To determine whether there is a correlation between the concentration of Indian hedgehog (Ihh) in synovial fluid (SF) and the severity of cartilage damage in the human knee joints, the knee cartilages from patients were classified using the Outer-bridge scoring system and graded using the Modified Mankin score. Expression of Ihh in cartilage and SF samples were analyzed with immunohistochemistry (IHC), western blot, and enzyme-linked immunosorbent assay (ELISA). Furthermore, we detected and compared Ihh protein levels in rat and mice cartilages between normal control and surgery-induced osteoarthritis (OA) group by IHC and fluorescence molecular tomography in vivo respectively. Ihh expression was increased 5.2-fold in OA cartilage, 3.1-fold in relative normal OA cartilage, and 1.71-fold in OA SF compared to normal control samples. The concentrations of Ihh in cartilage and SF samples was significantly increased in early-stage OA samples when compared to normal samples (r = 0.556; p < 0.001); however, there were no significant differences between normal samples and late-stage OA samples. Up-regulation of Ihh protein was also an early event in the surgery-induced OA models. Increased Ihh is associated with the severity of OA cartilage damage. Elevated Ihh content in human knee joint synovial fluid correlates with early cartilage lesions.     

Simplified method for determining cadmium concentrations in rice foliage and soil by using a biosensor kit with immunochromatography

BACKGROUND: The behavior of cadmium in ecosystems needs to be monitored because of the human toxicity of this heavy metal. The need recently arose for a simple and quick on‐site test for trace levels of Cd in food and environmental samples. In response, an immunochromatographic assay kit for detecting Cd was manufactured by Kansai Electric Power Co. of Japan. This kit uses the antigen–antibody complex reaction between the Cd–EDTA complex and an anti‐Cd–EDTA antibody and shows the results in terms of the degree of color developed on a test paper. We previously reported the successful use of this kit to determine Cd concentrations in brown rice. Here, we applied the kit to the determination of Cd concentrations in rice foliage and soil. RESULTS: Cadmium in rice foliage was not extracted successfully by the method used for brown rice. However, it was successfully extracted by 0.1 mol L^?1 HCl solution at a rice foliage:HCl ratio of 1:20, and coexisting metals were removed sufficiently by the column treatment. The Cd concentrations determined by immunochromatographic assay were well correlated with the values obtained by acid decomposition and inductively coupled plasma mass spectrometry. The 0.1 mol L^?1 HCl‐extractable Cd concentration in soil was also determined successfully with the kit. CONCLUSION: Approximate Cd concentrations in rice plants and 0.1 mol L^?1 HCl‐extractable Cd concentrations in soil can be monitored easily and quickly by this method at locations where facilities for acid digestion and precision analysis are not available. Copyright © 2009 Society of Chemical Industry     

Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles(GNPs)based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotiC., tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay.In the presence of tunicamycin, the dose-and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy(CLSM)and classic flow cytometry(FCM) assay, and satisfactory results were obtained.