Biomedical subjects

Yufang Zheng

Publications and source records attributed to Yufang Zheng.

12 recordsLinked to original sources

PubMed2007

Liquid chromatography-tandem mass spectrometry analysis of the DNA adducts of aristolochic acids.

Electrophilic attack of aristolactam-nitrenium ion by the C7 position to the exocyclic amino group in the DNA bases led to the formation of the major adducts. In this study, liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied to the study of DNA adducts of aristolochic acid (AA). When DNA (bases and CT-DNA) was incubated with AA, dG-AAI, dG-AAII, dA-AAI, dA-AAII, dC-AAI, and dC-AAII were detected and characterized. The dC adducts of AA were identified for the first time. The soft ionization technology allowed detection of the intact DNA adducts. High-resolution MS and MS-MS capabilities of a quadrupole time-of-flight mass spectrometer were shown to be efficient for DNA adducts analysis. DNA-AA adducts showed characteristic fragmentation patterns in MS-MS analysis. The dissociative loss of 116 Da from the DNA-AA adducts, which resulted from internal hydrogen transfer and cleavage at the C-N glycosidic bond, provided a characteristic fragment for the structural elucidation.

Animals↗

PubMed2006

A sensitive method to monitor ectodomain shedding of ligands of the epidermal growth factor receptor.

All ligands of the epidermal growth factor receptor (EGFR) are made as membrane anchored precursors that can be proteolytically processed and released from the plasma membrane. This process, which is referred to as protein ectodomain shedding, is emerging as a key regulator of the function of EGFR ligands. In light of the important roles of EGFR signaling in development and disease, it will be important to understand more about the regulation of proteolytic processing of EGFR ligands. This chapter describes a sensitive and semiquantitative method to measure ectodomain shedding of EGFR ligands that was designed to facilitate studies of this process in cells.

Alkaline Phosphatase↗

PubMed2006

[Metabolite fingerprint and biomarkers identification of rat urine after dosed with ginsenoside Rg3 based on ultra high performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS)].

Porous particles of 1.7 microm was employed for ultra high performance liquid chromatography (UPLC), resulting in higher peak capacity, greater resolution and increased sensitivity in comparison with high performance liquid chromatography (HPLC). Time-of-flight mass spectrometer (TOF-MS) with a lockmass interface was used for the structure identification through exact mass and MS/MS experiment. The hyphenation of these two technologies made it a suitable platform for analysis of complex samples and identification of unknown compounds. Ginsenoside Rg3 has been considered as the major active component of Panax ginseng. Effect of the administration of a single dose of the Ginsenoside Rg3 to male Sprague Dawley rats on the urinary metabolite profiles of a range of endogenous metabolites had been investigated using UPLC/TOF-MS. Urine samples were collected from both dosed and control animals. Analysis of these samples revealed marked changes in the pattern of endogenous metabolites due to the effect of Ginsenoside Rg3. Significant disturbances in the urinary metabolite were observed in the first day after dose. Endogenous metabolites with significant up-regulation identified by accurate mass and MS/MS were xanthurenic acid, and kynurenic acid.

Animals↗

PubMed2005

Strategy for metabonomics research based on high-performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry.

Metabonomics, the study of metabolites and their roles in various disease states, is a novel methodology arising from the post-genomics era. This methodology has been applied in many fields. Current metabonomic practice has relied on mass spectrometry (MS), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) to analyze metabolites. In this study, a strategy was developed for applying high-performance liquid chromatography (HPLC) and LC-MS-MS to metabonomics research. One of the key problems to be solved in this strategy is to match the peaks between the chromatograms. A peak alignment algorithm has been developed to match the chromatograms before the pattern recognition. As an application example, the strategy described above was applied to metabonomics research on liver diseases, and the false-positive result of live cancer diagnosis from the hepatocirrhosis and hepatitis diseases was effectively reduced to 7.40%. Based on the pattern recognition, several potential biomarkers were found and further identified by the following LC-MS-MS experiments. The structures of eight potential biomarkers were given for distinguishing the liver cancer from the hepatocirrhosis and hepatitis diseases.

Adult↗

PubMed2005

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography.

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 mm x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges.

Adult↗

PubMed2005

[Development of high performance liquid chromatographic method using monolithic column for the determination of urinary nucleosides].

Modified nucleosides excreted in urine have been studied as possible bio-markers for malignant tumors. Reversed-phase high performance liquid chromatography (RP-HPLC) is one of the most popular analytical tools. However, its analysis time is longer than desired. The analytical method for urinary nucleosides has been developed using a monolithic column and a linear gradient elution of 25 mmol/L KH2PO4 solution (pH 4.55) and 60% methanol in water with UV detection at 260 nm. Twelve urinary nucleosides were completely separated. The monolithic column presented similar analytical characteristics to reversed-phase column. The time of the analysis is only 23 min, in general much shorter than the methods using a packed RP-HPLC. The method developed is sensitive, reliable and suitable for clinical applications.

Chromatography, High Pressure Liquid↗

PubMed2004

Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases.

Metabonomics, the study of metabolites and their roles in various disease states, is a novel methodology arising from the post-genomics era. This methodology has been applied in many fields. Current metabonomics practice has relied on mass spectrometry (MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) to analyze metabolites. In this study, a novel approach of using high-performance liquid chromatography (HPLC) in conjunction with developed software was employed. Using the principal components analysis method (PCA), all (113) peaks of urinary metabolites with a cis-diol structure from patients with hepatitis and hepatocirrhosis were compared to those from liver cancer patients. The results showed that the metabonomics-PCA method might be useful to differentiate between patients with hepatocirrhosis and hepatitis from patients with liver cancer while lowering false-positive rate. These findings also suggest that a subset of the urinary nucleosides identified with metabonomics correlate better with cancer diagnosis than the traditional single tumor marker alpha-fetoprotein (AFP).

Chromatography, High Pressure Liquid↗

PubMed2004

Evaluation of the contribution of different ADAMs to tumor necrosis factor alpha (TNFalpha) shedding and of the function of the TNFalpha ectodomain in ensuring selective stimulated shedding by the TNFalpha convertase (TACE/ADAM17).

Tumor necrosis factor-alpha (TNFalpha), a potent pro-inflammatory cytokine, is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-alpha converting enzyme (TACE; a disintegrin and metalloprotease17; ADAM17) is known to have a key role in the ectodomain shedding of TNFalpha in several cell types. However, because purified ADAMs 9, 10, and 19 can also cleave a peptide corresponding to the TNFalpha cleavage site in vitro, these enzymes are considered to be candidate TNFalpha sheddases as well. In this study we used cells lacking ADAMs 9, 10, 17 (TACE), or 19 to address the relative contribution of these ADAMs to TNFalpha shedding in cell-based assays. Our results corroborate that ADAM17, but not ADAM9, -10, or -19, is critical for phorbol ester- and pervanadate-stimulated release of TNFalpha in mouse embryonic fibroblasts. However, overexpression of ADAM19 increased the constitutive release of TNFalpha, whereas overexpression of ADAM9 or ADAM10 did not. This suggests that ADAM19 may contribute to TNFalpha shedding, especially in cells or tissues where it is highly expressed. Furthermore, we used mutagenesis of TNFalpha to explore which domains are important for its stimulated processing by ADAM17. We found that the cleavage site of TNFalpha is necessary and sufficient for cleavage by ADAM17. In addition, the ectodomain of TNFalpha makes an unexpected contribution to the selective cleavage of TNFalpha by ADAM17: it prevents one or more other enzymes from cleaving TNFalpha following PMA stimulation. Thus, selective stimulated processing of TNFalpha by ADAM17 in cells depends on the presence of an appropriate cleavage site as well as the inhibitory role of the TNF ectodomain toward other enzymes that can process this site.

ADAM Proteins↗

PubMed2004

Analysis of coenzyme Q(10) in human plasma by column-switching liquid chromatography.

A new method of determining coenzyme Q10 in human plasma was developed based on column-switching high performance liquid chromatography (HPLC). CoQ10 was quantitatively extracted into 1-propanol with a fast one-step extraction procedure, after centrifugation, the supernatant was cleaned on an octadecyl-bonded silica column and then transferred to reversed-phase column by a column-switching valve. Determination of CoQ10 was performed on a reversed-phase analytical column with ultraviolet detection at 275 nm and the mobile phase containing 10% (v/v) isopropanol in methanol at a flow-rate of 1.5 ml/min. The sensitivity of this method allows the detection of 0.1 microg/ml CoQ10 in plasma (S/N=3). The linearity between the concentration and peak height is from 0.05 to 20 mg/l. The reproducibility (R.S.D.%) of the method is less than 2% (within day) and less than 3% (between day), the average recovery is 100.9 + 2.1%, it takes only 30 min to complete an analysis procedure, suitable for the determination of CoQ10 in human plasma especially for batch analysis in clinical laboratories. Finally, the method was applied to determine the plasma CoQ10 levels in healthy subjects, hyperthyroid and hypothyroid patients.

Chromatography, High Pressure Liquid↗

PubMed2003

Catalytic properties of ADAM19.

ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.

ADAM Proteins↗

PubMed2002

Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer.

Nucleosides in human urine and serum have frequently been studied as a possible biomedical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. Fifteen normal and modified nucleosides were determined in 69 urine and 42 serum samples using high-performance liquid chromatography (HPLC). Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached 100%. In the validating set, 95.8 and 92.9% of people were correctly classified into cancer patients and healthy persons when urine and serum were used as the sample for measuring the nucleosides. The results show that the artificial neural network technique is better than principal component analysis for the classification of healthy persons and cancer patients based on nucleoside data.

Biomarkers, Tumor↗

PubMed2002

Evidence for regulation of the tumor necrosis factor alpha-convertase (TACE) by protein-tyrosine phosphatase PTPH1.

Tumor necrosis factor alpha-convertase (TACE) is a metalloprotease-disintegrin involved in the ectodomain shedding of several proteins and is critical for proper murine development. TACE-mediated ectodomain shedding is regulated, and the cytoplasmic domain of TACE contains several potential signaling motifs, suggesting that this domain may play a role in regulating the metalloprotease activity. Here we report that the protein-tyrosine phosphatase PTPH1, which contains both a band 4.1 domain and a single PDZ domain, can interact with the cytoplasmic domain of TACE. The interaction was initially observed in a yeast two-hybrid screen and was confirmed using an in vitro binding assay and co-immunoprecipitations from eukaryotic cell extracts. The interaction is mediated via binding of the PDZ domain of PTPH1 to the COOH terminus of TACE. The latter represents a novel group I PDZ binding sequence characterized by a terminal cysteine residue. In co-expression experiments, significantly lower levels of TACE were observed in the presence of catalytically active forms of PTPH1 compared with catalytically inactive forms of PTPH1. Furthermore, phorbol ester-stimulated shedding of the TACE substrate tumor necrosis factor-alpha was decreased in cells expressing catalytically active PTPH1 compared with inactive PTPH1. Taken together, these results suggest that PTPH1 may be a negative regulator of TACE levels and function, and thus provide the first evidence for the regulation of TACE through a cytoplasmic protein.

ADAM Proteins↗