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.     

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.     

Comparison of antioxidant capacity and phenolic composition of peel and flesh of some apple varieties

BACKGROUND: Apples contain a large concentration of phenolic compounds, dependent on factors such as cultivar, harvest, storage conditions, and processing. This study aims to identify the essential phenolic compounds present in various apple varieties, to measure their total antioxidant capacity (TAC) with the CUPRAC (cupric ion reducing antioxidant capacity) and ABTS (2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonate)) methods, and to correlate their TAC values with HPLC findings. RESULTS: The order of TAC (mmol Trolox g^?1 fresh weight) of apple peels determined with the CUPRAC method was: Granny Smith > Amasya > Sky Spur > Ervin Spur > King Luscious ≥ Arap Kizi ≥ Lutz Golden. The theoretically calculated TAC values of HPLC‐quantified compounds, with the aid of the combined HPLC‐CUPRAC method, accounted for 18.4–33.5% of the experimentally observed CUPRAC capacity of peel extracts and 19.5–56.3% of flesh extracts, depending on apple variety. CONCLUSION: In synthetic samples of apple antioxidants, the CUPRAC‐TAC values of constituents, identified and analyzed by HPLC, proved to be additive, enabling measurement of the cooperative action of antioxidants using the proposed methodology. Apple peel showed higher contents of phenolics and therefore higher TAC than apple flesh, confirming the health benefit of the consumption of apples together with peel. © 2012 Society of Chemical Industry     

Comparison of total antioxidant capacity and phenolic composition of some apple juices with combined HPLC–CUPRAC assay

It was aimed in this study to identify and quantify various constituents (particularly phenolics) of apple juice and to quantitatively compare the total antioxidant capacities of juices obtained from apple varieties grown in Turkey.     

Evaluation of radiosensitivity of human tumor cells after irradiation of γ-rays based on G2-chromosome aberrations

The aim of the present investigation is to determine initial G2-chromosome aberrations and to validate whether the G2-chromosome aberrations can predict the cellular clonogenic survival in human tumor cell lines. Cell lines of human ovary carcinoma cells (HO8910) and human hepatoma cells (HepG2) were irradiated with a range of doses and assessed both for initial G2-chromosome aberrations and for cell survival after γ-irradiation. The initial G2-chromosome aberrations were measured by counting the number of G2-chromatid breaks after irradiation, detected by the premature chromosome condensation technique, and the G2-assay method. Cell survival was documented by a colony formation assay. A linear-quadratic survival curve was observed in both cell lines. The dose-response results show that the numbers of G2-chromatid breaks increase with the increase in dose in the two cell lines. At higher doses (higher than 4 Gy) of irradiation, the number of G2-chromatid breaks for the G2-assay method cannot be determined because too few cells reach mitosis, and hence their detection is difficult. A good correlation is found between the clonogenic survival and the radiation-induced initial G2-chromatid breaks per cell (r=0.9616). The present results suggest that the premature chromosome condensation technique may be useful for determining chromatid breaks in G2 cells, and the number of initial G2-chromatid breaks holds promise for predicting the radiosensitivity of tumor cells.     

Evaluation of topical application of clobetasol 17-propionate from various cream bases

The effect of clobetasol 17-propionate (CP), a potent corticosteroid, in various cream bases on the permeation through artificial membrane was sought. Four formulations were then chosen for a further in vivo skin blanching assay. After calculation of the relationship between in vivo flux_0-8hr determined from a surface recovery technique and in vitro release rate_0-8hr of CP from various formulations, a high correlation coefficient of 0.9996 was achieved. Therefore, the in vitro release study could be used as an index to predict and evaluate the in vivo penetration capacity of CP cream to screen the effective formulation preclinically. After a series of in vivo investigations in this study, it was concluded that myristic acid-added formulations may show a bioequivalence with commercial Dermovate®. Furthermore, the flux calculated from the surface recovery technique and ΔE^* detected from the skin blanching assay may be useful as parameters evaluating the quality and effectiveness of CP cream.     

可移动式高分辨率分段γ扫描现场测量装置的研制

描述了１台用于核设施、核材料现场测量的可移动式高分辨分段γ扫描装置。本装置用７５Ｓｅ和１６９Ｙｂ作为透射源测量样品对γ射线的透射率，采用近立体角三维自吸收校正模型计算样品自吸收校正系数ＣＦ（ＡＴ），较准确计算出样品对γ射线的自吸收校正量。本装置适合于准确测定中低密度非均匀核返料和核废物中核材料含量或裂变产物的含量，对２３５Ｕ硝酸铀酰均匀介质的盲样，测量结果与控制电位库仑测定的标准值之偏差小于１．４％。