CARBOHYDRATE RESEARCH
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2018
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IF 2.074
Ting Wang,Shen-Li Zheng,Li Liu,Josef Voglmeir
PNGases are crucial targets and valuable tools in analyzing asparagine-linked carbohydrate moieties (N-glycans) of glycoproteins . Activity tests of PNGases have been little improved since their discovery four decades ago, and still rely on observing deglycosylation patterns of glycoproteins or glycopeptides using SDS-PAGE or HPLC analysis. These techniques cannot be easily adapted for automated sampling and high-throughput procedures. Herein, we describe a PNGase activity assay which relies on the conversion of WST-1, a yellowish, water-soluble tetrazolium dye (sodium 2-(4-Iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolate), into a blue formazan dye. In this work, we showed that WST-1 could be reduced by N-glycans, which were enzymatically released from glycoprotein substrates. After optimization of the assay conditions, the robustness of the method was challenged by quantifying the activity of various PNGase isoforms at different purification stages using a microwell plate reader. Furthermore, the assay could be used to obtain steady-state kinetics of PNGase H + wild-type and mutant variants, which showed significant differences in their enzymatic reaction rates. The simplicity and robustness of this method might be of benefit for the detection of PNGase activity in routine applications of large amounts of samples.
研究领域:
生物化学
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分子生物学
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DOI
PubMed
高影响因子
Analytical Methods
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2014
·Vol. 7
·期 4
·1535-1542
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IF 1.938
Lu Wang,Shu Liu,Zhong Zheng,Zifeng Pi,Fengrui Song,Zhiqiang Liu
As a result of the donation of one electron, superoxide anion radicals (O2˙−) are produced in vivo, which are closely linked with several diseases in humans. In the past decades, some analytical methods for the determination of O2˙− scavenging capacity have been established. The most common methods in vitro are spectrum-based microplate screening assay using nitroblue tetrazolium (NBT) or cytochrome c as the target/probe for evaluating O2˙− scavenging activity. The target/probe is spectrophotometrically monitored at the ultraviolet-vis (UV-vis) region in the analysis of the samples with restraining UV-vis absorption. Nevertheless, the results of these methods were severely compromised when they were applied to analyze the samples with strong absorption in the visible region, such as natural pigments. To solve these problems, a simple and rapid assay combined with a new probe, a highly water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) and ultra-high performance liquid chromatography-diode-array detection (UPLC-DAD) method was developed. Above all, this method could be adapted to samples of strong absorption in the visible region. In this study, the superoxide anion radical scavenging activities of various natural pigments were evaluated.
研究领域:
分析化学
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生物化学
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药理学
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产品关联
DOI
高影响因子
ANALYTICA CHIMICA ACTA
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2013
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IF 4.387
Chen Xu,Shu Liu,Zhiqiang Liu,Fengrui Song,Shuying Liu
Superoxide anion radical (O 2 − ) plays an important role in several human diseases. The xanthine/xanthine oxidase system is frequently utilized to produce O 2 − . However, false positive results are easily got by using this system. The common spectrophotometric probes for O 2 − are nitroblue tetrazolium (NBT) and cytochrome c. Nevertheless, the application of NBT method is limited because of the water-insolubility of NBT formazan and the assay using cytochrome c lacks sensitivity and is not suitable for microplate measurement. We overcome these problems by using 1,2,3-trihydroxybenzene (pyrogallol) as O 2 − -generating system and a highly water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) which can be reduced by superoxide anion radical to a stable water-soluble formazan with a high absorbance at 450 nm. The method is simple, rapid and sensitive. Moreover, it can be adapted to microplate format. In this study, the O 2 − scavenging activities of superoxide dismutase (SOD), l -ascorbic acid, N-acetyl- l -cysteine (NAC), albumin from human serum, flavonoids and herbal extracts were assessed by using this method. Meanwhile, the activities of tissue homogenates and serum were determined by using this validated method. This method, applicable to tissue homogenates, serum and herbal extracts, proved to be efficient for measuring O 2 − scavenging activities of biological and abiological samples.
研究领域:
生物化学
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分析化学
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药理学
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DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
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2017
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IF 7.504
Xuefang Ding,Yujie Su,Cheng Wang,Fangrong Zhang,Kerong Chen,Yu Wang,Min Li,Wei Wang
Single-walled carbon nanotubes (SWNTs) with unique physicochemical properties have exhibited promising biomedical applications as drug and gene carriers. In this study, polyethylenimine (PEI)-modified SWNT conjugates linked with candesartan (CD) were developed to deliver vascular endothelial growth factor (VEGF)-targeted siRNA (siVEGF) for the synergistic and targeted treatment of tumor angiogenesis. The characterization results revealed that SWNT–PEI–CD conjugates were successfully synthesized and exhibited desirable dispersibility and superior stability. Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) results showed that SWNT–PEI–CD/siVEGF complexes could achieve high cellular uptake and specific intracellular distribution of siRNA in AT1R overexpressed PANC-1 cells. Strong down-regulation of VEGF was also verified by qualitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot in complex-treated PANC-1 cells. The in vitro angiogenesis assay showed that SWNT–PEI–CD/siVEGF complexes highly inhibited tube formation of human umbilical vein endothelial cells. Furthermore, in vivo observation in PANC-1 xenografted nude mice demonstrated that SWNT–PEI–CD/siVEGF complexes exhibited significant distribution at tumor sites and caused obvious inhibition of tumor growth and tumor-associated angiogenesis repression induced by the drug combination of CD and siVEGF. Finally, a WST-1 assay indicated that SWNT–PEI–CD possessed low cytotoxicity, and a hemolysis test showed good biocompatibility of SWNT–PEI–CD. Hematological and histological analyses confirmed that SWNT–PEI–CD/siVEGF complexes did not cause any obvious toxic effects to blood and major organs. These findings suggested that the SWNT–PEI–CD/siVEGF co-delivery system with tumor-targeting specificity, improved endosomal escaping properties, and collaboration of angiogenesis inhibition could be a prospective method for efficient tumor antiangiogenic therapy.
研究领域:
癌症生物学
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纳米医学
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药学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
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