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Identification of Halohydrin Dehalogenase Mutants that Resist COBE Inhibition
Shao-Yun Chen,Xiu-Juan He,Jian-ping Wu,Gang Xu,Li-Rong Yang 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.1
The biocatalytic cascade conversion of ethyl4-chloroacetoacetate (COBE) to ethyl (R)-4-cyano-3-hydroxybutyrate ((R)-HN) for the preparation of atorvastatinrepresents significant economic and environmental benefits,and is catalyzed by alcohol dehydrogenase and halohydrindehalogenase (HHDH). However, as the activity of HHDHis inhibited by COBE, the cascade reaction is an inefficientone-pot reaction. In this study, substrate inhibition kineticsanalysis was performed and the inhibition by COBE wasfound to be competitive reversible inhibition. Molecularsimulation analysis was used to determine the inhibitionmechanism by COBE. The results showed that COBEbound to the active center of HHDH via the formation ofhydrogen bonds with the OH groups of S132 and Y145. Site saturation mutagenesis of residues around the activesite and at the entrance of the access tunnel was performed,and two target mutant residues were identified, F136 andW249. Small focused mutagenesis on these two residueswas performed and the F136V/W249F mutant wassuccessfully found to relieve the activity inhibition ofHHDH by COBE. The half inhibiting concentration ofmutant F136V/W249F was found to be 20-fold higher thanwild-type HHDH. The efficiency of the multi-enzymaticone-pot system for the synthesis of (R)-HN from COBEusing mutant F136V/W249F was improved significantly.
Chen, Shao-Jun Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.10
Tanshinone IIA is a pharmacologically active ingredient extracted from Danshen, a Chinese traditional medicine. Its molecular mechanisms are still unclear. The present study utilized computational approaches to uncover the potential targets of this compound. In this research, PharmMapper server was used as the inverse docking tool andnd the results were verified by Autodock vina in PyRx 0.8, and by DRAR-CPI, a server for drug repositioning via the chemical-protein interactome. Results showed that the retinoic acid receptor alpha ($RAR{\alpha}$), a target protein in acute promyelocytic leukemia (APL), was in the top rank, with a pharmacophore model matching well the molecular features of Tanshinone IIA. Moreover, molecular docking and drug repurposing results showed that the complex was also matched in terms of structure and chemical-protein interactions. These results indicated that $RAR{\alpha}$ may be a potential target of Tanshinone IIA for APL. The study can provide useful information for further biological and biochemical research on natural compounds.
Shao-Chen Sun,Seung-Eun Lee,Yong-Nan Xu,Nam-Hyung Kim 한국발생생물학회 2010 한국발생생물학회 학술발표대회 Vol.29 No.-
Spc25 is a component of the Ndc80 complex which consists of Ndc80, Nuf2, Spc24, and Spc25. Previous work has shown that Spc25 is involved in regulation of kinetochore microtubule attachment, localization of Ndc80, and the spindle assembly checkpoint in mitosis. The role of Spc25 in meiosis remains unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. The Spc25 mRNA level gradually increased from the GV to MI stage, but decreased by MII during mouse oocyte meiotic maturation. Immunofluorescent staining showed that Spc25 was restricted to the germinal vesicle, and associated with chromosomes during all stages after GVBD. Overexpression of Spc25 resulted in oocyte meiotic arrest, chromosome misalignment and spindle disruption. Conversely, Spc25 RNAi resulted in precocious polar body extrusion and caused severe chromosome misalignment and aberrant spindle formation. Spc25 RNAi affected Ndc80 localization, but Ndc80 RNAi did not affect Spc25 localization.Survivin MO caused Ndc80 dispersion but did not affect localization of Spc25. Our data suggest that Spc25 is required for chromosome alignment, spindle formation, and spindle checkpoint activity through the regulation of Ndc80, but that Spc25 function is independent of survivin during meiosis.
Mycotoxins Containing Diet Affects Oocyte Quality in Mouse
Shao-Chen Sun,Yan-Jun Hou,Xiang-Shun Cui,Nam-Hyung Kim 한국동물생명공학회(구 한국동물번식학회) 2013 Reproductive & Developmental Biology(Supplement) Vol.37 No.2s
Background: Mycotoxins which mainly consist of Aflatoxin (AF), Zearalenone (ZEN) and Deoxynivalenol (DON) are commonly found in many food commodities, each component has been shown to cause organ toxicity and oxidative stress in several species. Our previous study showed that mycotoxin-contaminated diet could cause oxidative stress in liver, kidney, spleen. Recently we examined its effects on oocyte quality. Materials and Methods: Mycotoxins-contaminated maize (AF 597μg/kg, ZEN 729μg/kg, DON 3.1mg/kg maize) was incorporated into the diet at three different doses (0, 5 and 20%) to feed the mice for 4 weeks. Results: Our results showed that the both the index of ovary and the number of good GV oocytes decreased in the mycotoxin-treated mice. The oocytes from mycotoxin- treated mouse displayed low developmental competence showing with lower GVBD and polar body extrusion rate; the embryo developmental competence also showed the similar pattern, most embryos could not develop to blastocyst stage. The cytoskeleton component actin expression in both oocyte cortex and cytoplasm decreased, and the expression of actin nucleation factor Profilin and mDia1 also decreased, indicating that mycotoxin may affect oocyte quality through the effects on actin. Moreover, a big proportion of oocytes with mycotoxin contaminated diet treatment showed disrupted cortical granule free domain, spindle morphology and mitochondria distribution, further confirmed the oocyte quality declination. We also used the in vitro model to confirm this, we cultured the oocytes in the medium with Zearalenone, a key component of mycotoxins, and the results were similar with the in vivo model. Conclusion: Our data indicated that the mycotoxins were toxic to mouse reproductive system and induced the oocyte quality declination.
Significance of Human Telomerase RNA Gene Amplification Detection for Cervical Cancer Screening
Chen, Shao-Min,Lin, Wei,Liu, Xin,Zhang, You-Zhong Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.5
Aim: Liquid-based cytology is the most often used method for cervical cancer screening, but it is relatively insensitive and frequently gives equivocal results. Used as a complementary procedure, the high-risk human papillomavirus (HPV) DNA test is highly sensitive but not very specific. The human telomerase RNA gene (TERC) is the most often amplified oncogene that is observed in cervical precancerous lesions. We assessed genomic amplification of TERC in liquid-based cytological specimens to explore the optimal strategy of using this for cervical cancer screening. Methods: Six hundred and seventy-one residual cytological specimens were obtained from outpatients aged 25 to 64 years. The specimens were evaluated by the Digene Hybrid Capture 2 (HC2) HPV DNA test and fluorescence in situ hybridization (FISH) with a chromosome probe to TERC (3q26). Colposcopic examination and histological evaluation were performed where indicated. Results: The TERC positive rate was higher in the CIN2+ (CIN2, CIN3 and SCC) group than in the normal and CIN 1 groups (90.0% vs. 10.4%, p < 0.01). In comparison with the HC2 HPV DNA test, the TERC amplification test had lower sensitivity but higher specificity (90.0% vs. 100.0%, 89.6% vs. 44.0%, respectively). TERC amplification test used in conjunction with the HC2 HPV DNA test showed a combination of 90.0% sensitivity and 92.2% specificity. Conclusion: The TERC amplification test can be used to diagnose cervical precancerous lesions. TERC and HPV DNA co-testing shows an optimal combination of sensitivity and specificity for cervical cancer screening.
Chen, Feiyan,Zhu, Kexuan,Chen, Lin,Ouyang, Liufeng,Chen, Cuihua,Gu, Ling,Jiang, Yucui,Wang, Zhongli,Lin, Zixuan,Zhang, Qiang,Shao, Xiao,Dai, Jianguo,Zhao, Yunan The Korean Society of Ginseng 2020 Journal of Ginseng Research Vol.44 No.3
Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, the mechanism of action is not completely understood, and its molecular targets remain largely unknown. Methods: By screening for proteins that interact with the primary components of ginseng (ginsenosides) in an affinity chromatography assay, we have identified muscle-type creatine kinase (CK-MM) as a potential target in skeletal muscle tissues. Results: Biolayer interferometry analysis showed that ginsenoside metabolites, instead of parent ginsenosides, had direct interaction with recombinant human CK-MM. Subsequently, 20(S)-protopanaxadiol (PPD), which is a ginsenoside metabolite and displayed the strongest interaction with CK-MM in the study, was selected as a representative to confirm direct binding and its biological importance. Biolayer interferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPD specifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by molecular docking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activity in vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the function of the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delaying exercise-induced lactate accumulation, and improving exercise performance in mice. Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginseng reduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can help in further developing better CK-MM activators based on the dammarane-type triterpenoid structure.