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Xiaoqing Chang

Publications and source records attributed to Xiaoqing Chang.

8 recordsLinked to original sources
PubMed2006

LTBP-1 blockade in dioxin receptor-null mouse embryo fibroblasts decreases TGF-beta activity: Role of extracellular proteases plasmin and elastase.

In mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR), high levels of latent transforming growth factor-beta (TGF-beta)-binding protein-1 (LTBP-1) correlated with increased TGF-beta1 activity, an observation suggesting that LTBP-1 could contribute to maintain TGF-beta1 levels. Here, using small interfering RNAs (siRNA), we have first analyzed if LTBP-1 expression affected TGF-beta1 activity in MEF cells. We have then determined how LTBP-1 levels could alter the activity of extracellular proteases known to activate TGF-beta1, and finally, whether protease inhibition could reduce TGF-beta1 activation. LTBP-1 inhibition by siRNA in AhR-/- MEF decreased the amount of active TGF-beta1 and reduced plasminogen activators (PA)/plasmin and elastase activities and thrombospondin-1 (TSP-1) expression, without significantly affecting their mRNA levels. On the contrary, LTBP-1 siRNA restored matrix metalloproteinase-2 (MMP-2) activity in AhR-/- MEF. Interestingly, whereas a TGF-beta1 neutralizing antibody mimicked many of the LTBP-1 siRNA effects on extracellular proteases, addition of recombinant TGF-beta1 protein increased proteases activity over basal levels in AhR-/- MEF. These proteases contributed to TGF-beta activation since their specific inhibitors reduced active TGF-beta levels in these cells. These results suggest that LTBP-1 contributes to TGF-beta1 activation in MEF, possibly by influencing the activities of PA/plasmin, elastase, TSP-1, and MMP-2. TGF-beta1, on the other hand, could be also involved in maintaining the activity of these extracellular proteases. Thus, LTBP-1 appears to play a role in TGF-beta1 activation through a process involving extracellular protease activities, which, in turn, could be affected by TGF-beta1 levels.

Animals↗
PubMed2005

Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis.

Testicular necrosis is a sensitive endpoint for cadmium (Cd(2+), Cd) toxicity across all species tested. Resistance to Cd-induced testicular damage is a recessive trait assigned to the Cdm locus on mouse chromosome 3. We first narrowed the Cdm-gene-containing region to 880 kb. SNP analysis of this region from two sensitive and two resistant inbred strains demonstrated a 400-kb haplotype block consistent with the Cd-induced toxicity phenotype; in this region is the Slc39a8 gene encoding a member of the solute-carrier superfamily. Slc39a8 encodes SLC39A8 (ZIP8), whose homologs in plant and yeast are putative zinc transporters. We show here that ZRT-, IRT-like protein (ZIP)8 expression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of intracellular Cd influx and accumulation and 30-fold increase in sensitivity to Cd-induced cell death. The complete ZIP8 mRNA and intron-exon splice junctions have no nucleotide differences between two sensitive and two resistant strains of mice; by using situ hybridization, we found that ZIP8 mRNA is prominent in the vascular endothelial cells of the testis of the sensitive strains of mice but absent in these cells of resistant strains. Slc39a8 is therefore the Cdm gene, defining sensitivity to Cd toxicity specifically in vascular endothelial cells of the testis.

Alternative Splicing↗
PubMed2003

Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity.

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.

Amino Acid Sequence↗
PubMed2002

Arsenic co-exposure potentiates benzo[a]pyrene genotoxicity.

Co-exposures to complex mixtures of arsenic and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) are common in the environment. These two environmental pollutants are carcinogenic, but the nature of their molecular interactions in the induction of cancer is not well understood. Additive or synergistic interactions have been proposed to explain why arsenic, which is not a potent mutagen itself, is comutagenic with a variety of DNA-damaging agents. We have examined the genotoxicity of BaP-arsenic mixtures. We find that exposure of mouse hepatoma Hepa-1 cells to low concentrations of arsenite increases BaP-DNA adduct levels by as much as 18-fold. This effect requires the activation of BaP by cytochrome p450 1A1 (CYP1A1), although arsenite does not alter BaP-inducible CYP1A1 enzymatic activity, suggesting that arsenite acts downstream of metabolic BaP activation. Glutathione homeostasis was important in modulating the potency of arsenite. In cells depleted of reduced glutathione, arsenite increased BaP-DNA adduct formation by an even greater degree than in cells co-treated with BaP and arsenite in control medium. Although arsenic comutagenicity has been attributed to inhibition of DNA repair, arsenite treatment did not alter adduct removal kinetics in BaP-treated cells, suggesting that mechanisms upstream of DNA repair are responsible for increased adduct levels. Concentrations of arsenite and BaP that had no measurable mutagenic effect alone, increased mutation frequency at the Hprt locus by eight-fold when given in combination, demonstrating a comutagenic response between BaP and arsenite. These results provide strong support for the positive interaction between arsenic and PAH-induced cancer observed in epidemiology studies, and help to identify additional mechanistic steps likely to be involved in arsenic comutagenesis.

Animals↗
PubMed2002

NMR studies of the aggregation of glucagon-like peptide-1: formation of a symmetric helical dimer.

Nuclear magnetic resonance (NMR) spectroscopy reveals that higher-order aggregates of glucagon-like peptide-1-(7-36)-amide (GLP-1) in pure water at pH 2.5 are disrupted by 35% 2,2,2-trifluoroethanol (TFE), and form a stable and highly symmetric helical self-aggregate. NMR spectra show that the helical structure is identical to that formed by monomeric GLP-1 under the same experimental conditions [Chang et al., Magn. Reson. Chem. 37 (2001) 477-483; Protein Data Bank at RCSB code: 1D0R], while amide proton exchange rates reveal a dramatic increase of the stability of the helices of the self-aggregate. Pulsed-field gradient NMR diffusion experiments show that the TFE-induced helical self-aggregate is a dimer. The experimental data and model calculations indicate that the dimer is a parallel coiled coil, with a few hydrophobic residues on the surface that may cause aggregation in pure water. The results suggest that the coiled coil dimer is an intermediate state towards the formation of higher aggregates, e.g. fibrils.

Amino Acid Sequence↗
PubMed2002

An NMR approach to structural proteomics.

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins.

Bacterial Proteins↗
PubMed2002

Activation of mitogen-activated protein kinases (MAPKs) by aromatic hydrocarbons: role in the regulation of aryl hydrocarbon receptor (AHR) function.

The aromatic hydrocarbon (Ah) receptor (AHR) is the only known cellular receptor of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of many other widespread environmental contaminants that cause diverse toxic effects in animals and humans. Most, if not all, the biological effects of TCDD are mediated by the activation of AHR, which is a ligand-activated transcription factor required for ligand-induced expression of several detoxification genes, including those encoding for cytochrome P450 enzymes CYP1A1, CYP1A2, and CYP1B1. Environmental agents also activate several mitogen-activated protein kinase (MAPK) pathways, believed to modulate transcription factor function and to regulate gene expression. However, the contribution to TCDD toxicity resulting from cross-talk between AHR and MAPK pathways has yet to be determined. In this study, we show that TCDD and other AHR ligands induced the immediate activation of the extracellular signal-regulated kinases and the Jun N-terminal kinases, but not the p38 MAPKs. MAPK activation by TCDD did not require the AHR, since it occurred equally well in AHR-negative CV-1 cells and in Ahr (-/-) mouse embryonic fibroblasts as in AHR-positive cells. Distinct from serum factors and the tumor promoter TPA-induced MAPKs, which resulted in transcriptional activation of ELK or c-JUN, TCDD-stimulated MAPKs were critical for the induction of AHR-dependent gene transcription and CYP1A1 expression. These data indicate that AHR ligands elicit AHR-independent non-genomic events that are essential for AHR activation and function.

Animals↗