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Expression, Purification and Properties of Shikimate Dehydrogenase from Mycobacterium Tuberculosis
Zhang, Xuelian,Zhang, Shunbao,Hao, Fang,Lai, Xuhui,Yu, Haidong,Huang, Yishu,Wang, Honghai Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.5
Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the main diseases to mankind. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. The shikimate pathway is onsidered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammalian cells. The Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase was cloned, expressed and purified. Sequence alignment analysis shows that shikimate dehydrogenase of Mycobacterium tuberculosis exhibit the pattern of G-X-(N/S)-V-(T/S)-X-PX-K, which is highly conserved within the shikimate dehydrogenase family. The recombinant shikimate dehydrogenase spectrum determined by CD spectroscopy showed that the percentages for $\alpha$-helix, $\beta$-sheet, $\beta$-turn, and random coil were 29.2%, 9.3%, 32.7%, and 28.8%, respectively. The enzymatic characterization demonstrates that it appears to be fully active at pH from 9.0 to 12, and temperature $63^{\circ}C$. The apparent Michaelis constant for shikimic acid and $NADP^+$ were calculated to be about $29.5\;{\mu}M$ and $63\;{\mu}M$. The recombinant shikimate dehydrogenase catalyzes the substrate in the presence of $NADP^+$ with an enzyme turnover number of $399\;s^{-1}$. Zymological studies suggest that the cloned shikimate dehydrogenase from M. tuberculosis has a pretty activity, and the work should help in the discovery of enzyme inhibitors and further of possible antimicrobial agents against Mycobacterium tuberculosis.
An Jin,Xuelian Xiang,Yun-Yun Zhu,Heng-Yi Yu,Hui-Fang Pi,Peng Zhang,Han-Li Ruan 대한약학회 2014 Archives of Pharmacal Research Vol.37 No.3
Three new alkaloids, 2a-hydroxy-6-O-n-butyloduline,O-n-butyllycorenine, (-)-N-(chloromethyl)lycoramine(1–3), and a new phenolic compound, ((7S)-7-(4-hydroxyphenyl)-7-hydroxypropyl)-20-methylbenzene-30,60-diol (14), along with ten known alkaloids (4–13), wereisolated from the bulbs of Lycoris aurea collected fromHuaihua County of Hunan Province, China. Their structureswere elucidated by spectroscopic methods includingHRESIMS, UV, IR, and NMR. All the isolated compoundswere tested for their neuroprotective effects against CoCl2and H2O2-induced SH-SY5Y cell death. Compounds 1–7and 10 exhibited significant neuroprotective effects againstCoCl2-induced SH-SY5Y cell injury, while compounds1–5, 7, 10 and 12 showed obvious neuroprotective effectsagainst H2O2-induced SH-SY5Y cell death.
Wu Weiyu,Zhang Xuelian,He Feng,Wu Longxiang 대한독성 유전단백체 학회 2022 Molecular & cellular toxicology Vol.18 No.3
Background Colorectal cancer (CRC) is one of the most common malignancies worldwide, with the second highest incidence among men and the third incidence among women. Objective To uncover the expression of RNF115 in human colorectal cancer (CRC) tissues and cell lines, investigate its effects on CRC progression, and further explore the mechanisms. Results TCGA database showed the high transcript per million (TPM) in human CRC tissues and its relationship with poor prognosis. RNF115 was highly expressed in CRC tissues and cell lines. RNF115 contributed to the growth of CRC cells via mediating cell cycle and colony formation. RNF115 knockdown also stimulated the apoptosis of CRC cells by increasing Bax and cleaved caspase-3 expression levels. Mechanistically, RNF115 affected the proliferation and apoptosis via targeting WNT/β-catenin pathway in CRC cells. Conclusion RNF115 was highly expressed in human CRC tissues and cell lines and correlated with poor prognosis. RNF115 contributed to the growth of CRC cells via mediating cell cycle and colony formation, and also induced the apoptosis of CRC cells via targeting WNT/β-catenin pathway. RNF115 could serve as a target for CRC treatment.
Shi, Wenjun,Feng, Jianfang,Zhang, Min,Lai, Xuhui,Xu, Shengfeng,Zhang, Xuelian,Wang, Honghai Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.6
Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-Derythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and $Mg^{2+}$. The turnover number of MtIspD is $3.4 s^{-1}$. The Km for MEP and CTP are 43 and $92{\mu}M$, respectively. Furthermore, MtIspD shows thermal instable above $50^{\circ}C$. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.
Yan Gao,Ding Yuan,Liyue Gai,Xuelian Wu,Yue Shi,Yumin He,Chaoqi Liu,Changcheng Zhang,Gang Zhou,Chengfu Yuan 고려인삼학회 2021 Journal of Ginseng Research Vol.45 No.3
Background: The decreased renal function is known to be associated with biological aging, of which the main pathological features are chronic inflammation and renal interstitial fibrosis. In previous studies, we reported that total saponins from Panax japonicus (SPJs) can availably protect acute myocardial ischemia. We proposed that SPJs might have similar protective effects for aging-associated renal interstitial fibrosis. Thus, in the present study, we evaluated the overall effect of SPJs on renal fibrosis. Methods: Sprague-Dawley (SD) aging rats were given SPJs by gavage beginning from 18 months old, at 10 ㎎/㎏/d and 60 ㎎/㎏/d, up to 24 months old. After the experiment, changes in morphology, function and fibrosis of their kidneys were detected. The levels of serum uric acid (UA), β2-microglobulin (β2-MG) and cystatin C (Cys C) were assayed with ELISA kits. The levels of extracellular matrix (ECM), matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), inflammatory factors and changes of oxidative stress parameters were examined. Results: After SPJs treatment, SD rats showed significantly histopathological changes in kidneys accompanied by decreased renal fibrosis and increased renal function; As compared with those in 3-month group, the levels of serum UA, Cys C and β2-MG in 24-month group were significantly increased (p < 0.05). Compared with those in the 24-month group, the levels of serum UA, Cys C and β2-MG in the SPJ-H group were significantly decreased. While ECM was reduced and the levels of MMP-2 and MMP-9 were increased, the levels of TIMP-1, TIMP-2 and transforming growth factor-β1 (TGF-β1)/Smad signaling were decreased; the expression level of phosphorylated nuclear factor kappa-B (NF-κB) was down-regulated with reduced inflammatory factors; meanwhile, the expression of nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) signaling was aggrandized. Conclusion: These results suggest that SPJs treatment can improve age-associated renal fibrosis by inhibiting TGF-β1/Smad, NFκB signaling pathways and activating Nrf2-ARE signaling pathways and that SPJs can be a potentially valuable anti-renal fibrosis drug.
Dongke Wang,Chaofan Duan,Xiaohao Zhang,Junying Xu,Xiaohua Hou,Xuelian Xiang 대한소화기 기능성질환∙운동학회 2022 Journal of Neurogastroenterology and Motility (JNM Vol.28 No.4
Background/Aims Lyon consensus differentiates acid exposure time (AET) as physiological, borderline, and pathological. Mean nocturnal baseline impedance (MNBI) and post-reflux swallow-induced peristaltic wave index (PSPWi) are believed to increase diagnostic yield of gastroesophageal reflux disease (GERD) and correlate with symptom outcome of proton pump inhibitor (PPI) treatment. We aim to explore the clinical characteristics and the correlation of pH-impedance parameters with PPI response in Chinese patients with different AET levels. Methods We retrospectively investigated 177 patients with typical reflux symptoms who received esophageal function tests. The demographics, GERD questionnaire scores, the proportion of esophagitis and PPI responders, and manometric and pH-impedance parameters were compared among patients with AET < 4%, 4-6%, and > 6%. In patients with AET ≥ 4%, manometric and pH-impedance parameters were compared between PPI responders and non-responders. Results Among 177 patients, 69 (39.0%) had AET 4-6%, and 53 (29.9%) had AET > 6%. The demographics, esophagogastric junction type, and occurrence of ineffective esophageal motility were similar between patients with AET 4-6% and > 6%, but different from AET < 4%. MNBI and PSPWi were different among different AET levels, but similar between PPI responders and non-responders in patients with AET ≥ 4%. Conclusions It is reasonable to set 4% as a threshold to define pathological AET in Chinese patients. MNBI and PSPWi could identify GERD patients, but may not correlate with PPI response of Chinese GERD patients.
( Enkhtuya Bayar ),( Yuanyuan Ren ),( Yinghua Chen ),( Yafang Hu ),( Shuncheng Zhang ),( Xuelian Yu ),( Jun Fan ) 한국미생물 · 생명공학회 2021 Journal of microbiology and biotechnology Vol.31 No.12
Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.