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Yang X.-S.,Liaoning Provincial Core Laboratory of Glycobiology and Glycoengineering | Liu S.-A.,Liaoning Provincial Core Laboratory of Glycobiology and Glycoengineering | Liu J.-W.,Dalian Medical University | Yan Q.,Liaoning Provincial Core Laboratory of Glycobiology and Glycoengineering
Asian Pacific Journal of Cancer Prevention | Year: 2012

Fucosyltransferase IV (FUT4) has been implicated in cell adhesion, motility, and tumor progression in human epidermoid carcinoma A431 cells. We previously reported that it promotes cell proliferation through the ERK/MAPK and PI3K/Akt signaling pathways; however, the molecular mechanisms underlying FUT4- induced cell invasion remain unknown. In this study we determined the effect of FUT4 on expression of matrix metalloproteinase (MMP)-12 induced by EGF in A431 cells. Treatment with EGF resulted in an alteration of cell morphology and induced an increase in the expression of MMP-12. EGF induced nuclear translocation of nuclear factor kB (NF-κB) and resulted in phosphorylation of IkBα in a time-dependent manner. In addition, ERK1/2 and p38 MAPK were shown to play a crucial role in mediating EGF-induced NF-κB translocation and phosphorylation of IκBα when treated with the MAPK inhibitors, PD98059 and SB203580, which resulted in increased MMP-12 expression. Importantly, we showed that FUT4 up-regulated EGF-induced MMP-12 expression by promoting the phosphorylation of ERK1/2 and p38 MAPK, thereby inducing phosphorylation/ degradation of IκBα, NF-κB activation. Base on our data, we propose that FUT4 up-regulates expression of MMP-12 via a MAPK-NF-κB-dependent mechanism. Source


Yang S.,Dalian Medical University | Zhang F.,Dalian Medical University | Kang J.,Dalian Medical University | Zhang W.,Dalian Medical University | And 4 more authors.
BMC Microbiology | Year: 2014

Background: Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results: In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: K m, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S -1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion: We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. © 2014 Yang et al.; licensee BioMed Central Ltd. Source


Li H.,Dalian Medical University | Zhou Y.,Dalian Medical University | Wang N.,Dalian Medical University | Xin Y.,Dalian Medical University | And 3 more authors.
Journal of Hard Tissue Biology | Year: 2012

Streptococcus mutans (S. mutans) is the primary etiological agent of human dental caries. A gene (SMU_1525) encoding MurA was identified in S. mutans UA159 (ATCC700610). The deduced amino-acid sequence has homology (51% identity) with E. coli MurA protein (AAC76221). In this study, we cloned S. mutans murA (SMU_1525) gene, expressed soluble recombinant MurA protein in E. coli BL21(DE3). The recombinant protein was purified by affinity chromatography. The molecular weight of expressed protein was about 45.6 kD. The activity of the protein was identified by high-pressure liquid chromatography (HPLC) and was shown to have UDP-N-acetylglucosamine enolpyruvyl transferase activity. Then one microtiter plate based colourimetric assay for S. mutans MurA activity was developed. The optimal temperature and pH of the purified enzyme were 37°C and 7.5, respectively. The K m for UDP-GlcNAc was 0.120 mM and for PEP was 0.086 mM. The V max for UDP-GlcNAc was 0.048 mM min -1 mg -1 and for PEP was 0.098 mM min -1 mg -1. The activity of S. mutans MurA was inhibited by the fosfomycin, known MurA inhibitor. © 2012 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. Source


Zhou Y.,Dalian Medical University | Xin Y.,Dalian Medical University | Xin Y.,Liaoning Provincial Core Laboratory of Glycobiology and Glycoengineering | Sha S.,Dalian Medical University | Ma Y.,Liaoning Provincial Core Laboratory of Glycobiology and Glycoengineering
Archives of Microbiology | Year: 2011

The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (D-N-GlcNAc-L-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1- phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg2?, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmUenzyme activities and the kinetic properties will facilitate highthroughput screening of GlmU inhibitors. © Springer-Verlag 2011. Source


Sha S.,Dalian Medical University | Zhou Y.,Dalian Medical University | Xin Y.,Dalian Medical University | Xin Y.,Liaoning Provincial Core Laboratory of Glycobiology and Glycoengineering | And 2 more authors.
Journal of Biomolecular Screening | Year: 2012

dTDP-L-rhamnose as a sugar donor provides L-rhamnosyl residue in the synthesis of disaccharide linker (D-N-acetylglucosamine-L-rhamnose), the key structure of the Mycobacterium tuberculosis cell wall. Four enzymes are involved in the formation of dTDP-L-rhamnose and D-glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the first step of D-glucose-1-phosphate and dTTP to dTDP-D-glucose and PPi. The previous studies on RmlA essentiality proved RmlA as a potential target for antituberculosis drugs. However, there has not been a suitable assay for RmlA to screen inhibitors currently. In this study, the authors reported a microtiter plate-based colorimetric assay for RmlA enzyme activity. Using this assay, the kinetic properties of M. tuberculosis RmlA including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and kinetic parameters were determined. The establishment of the accurate and rapid colorimetric assay and kinetic analysis of M. tuberculosis RmlA will facilitate high-throughput screening of RmlA inhibitors. © 2012 Society for Laboratory Automation and Screening. Source

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