Molecular Biology and Omics : Direct Evaluation of the Effect of Gene Dosage on Secretion of Protein from Yeast Pichia pastoris by Expressing EGFP

( Hai Long Liu ),( Yu Feng Qin ),( Yuan Kai Huang ),( Yao Sheng Chen ),( Pei Qing Cong ),( Zu Yong He ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.2

Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the α-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.

Association of Helicobacter pylori with Elevated Blood Ammonia Levels in Cirrhotic Patients: A Meta-Analysis

Hai-Xing Jiang,Shan-Yu Qin,Zhi-gang Min,Ming-Zhi Xie,Tao Lin,Bang-Li Hu,Xiao-Yun Guo 연세대학교의과대학 2013 Yonsei medical journal Vol.54 No.4

Purpose: The association between Helicobacter pylori (H. pylori) and blood ammonia levels in cirrhotic patients is controversial. We aimed to clarify this controvercy by performing a meta-analysis of published studies. Materials and Methods:We searched PubMed, EMBASE and Cochrane library for studies which explored the association between H. pylori and blood ammonia levels in cirrhotic patients before May 2012. Six cohort studies involved in 632 H. pylori positive and 396 H. pylori negative cirrhotic patients were eligible for our analysis. The summary estimates were presented as standard means differences (SMD) and 95% confidence intervals (CI) from individual studies. Results: Overall, there was significant association between H. pylori infection and the elevated blood ammonia levels in cirrhotic patients (SMD=0.34, 95% CI=0.21-0.47, I2=42.1%). Sensitivity analysis further confirmed this association. Subgroup analysis showed that the association was found only in Asian ethnicity, but not in Caucasian ethnicity. Conclusion:H. pylori infection is associated with elevated blood ammonia levels in cirrhotic patients, and more large scale studies and stratify analysis are warranted in order to further evaluate this association.

HDAC6 siRNA Inhibits Proliferation and Induces Apoptosis of HeLa Cells and its Related Molecular Mechanism

Qin, Hai-Xia,Cui, Hong-Kai,Pan, Ying,Yang, Jun,Ren, Yan-Fang,Hua, Cai-Hong,Hua, Fang-Fang,Qiao, Yu-Huan Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.7

Objective: To investigate the effects of histone deacetylase 6 (HDAC6) siRNA on cell proliferation and cell apoptosis of the HeLa cervical carcinoma cell line and the molecular mechanisms involved. Methods: Division was into three groups: A, the untreated group; B, the control siRNA group; and C, the HDAC6 siRNA group. Lipofectamine 2000 was used for siRNA transfection, and Western blot analysis was used to determine the protein levels. Cell proliferation and apoptosis were characterized using a CCK-8 assay and flow cytometry, respectively. Results: HDAC6 protein expression in the HDAC6 siRNA-transfection group was significantly lower (P < 0.05) than in the untreated and control siRNA groups. The CCK-8 kit results demonstrated that the proliferation of HeLa cells was clearly inhibited in the HDAC6 siRNA transfection group (P < 0.05). In addition, flow cytometry revealed that the early apoptotic rate ($26.0%{\pm}0.87%$) was significantly elevated (P < 0.05) as compared with the untreated group ($10.6%{\pm}1.19%$) and control siRNA group ($8.61%{\pm}0.98%$). Furthermore, Western blot analysis indicated that bcl-2 protein expression in the HDAC6 siRNA-transfection group was down-regulated, whereas the expression of p21 and bax was up-regulated. Conclusion: HDAC6 plays an essential role in the occurrence and development of cervical carcinoma, and the down-regulation of HDAC6 expression may be useful molecular therapeutic method.

Prognostic Significance of CYFRA21-1, CEA and Hemoglobin in Patients with Esophageal Squamous Cancer Undergoing Concurrent Chemoradiotherapy

Zhang, Hai-Qin,Wang, Ren-Ben,Yan, Hong-Jiang,Zhao, Wei,Zhu, Kun-Li,Jiang, Shu-Mei,Hu, Xi-Gang,Yu, Jin-Ming Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.1

Purpose: To evaluate the prognostic value of serum CYFRA21-1, CEA and hemoglobin levels regarding long-term survival of patients with esophageal squamous cell carcinoma (ESCC) treated with concurrent chemoradiotherapy (CRT). Methods: Age, gender, Karnofsky Performance Status (KPS), tumor location, tumor length, T stage, N stage and serum hemoglobin, and CYFRA21-1 and CEA levels before concurrent CRT were retrospectively investigated and related to outcome in 113 patients receiving 5-fluorouracil and cisplatin combined with radiotherapy for ESCC. The Kaplan-Meier method was used to analyze prognosis, the log-rank to compare groups, the Cox proportional hazards model for multivariate analysis, and ROC curve analysis for assessment of predictive performance of biologic markers. Results: The median survival time was 20.1 months and the 1-, 2-, 3-, 5- year overall survival rates were 66.4%, 43.4%, 31.9% and 15.0%, respectively. Univariate analysis showed that factors associated with prognosis were KPS, tumor length, T-stage, N-stage, hemoglobin, CYFRA21-1 and CEA level. Multivariate analysis showed T-stage, N-stage, hemoglobin, CYFRA21-1 and CEA level were independent predictors of prognosis. By ROC curve, CYFRA21-1 and hemoglobin showed better predictive performance for OS than CEA (AUC= 0.791, 0.704, 0.545; P=0.000, 0.000, 0.409). Conclusions: Of all clinicopathological and molecular factors, T stage, N stage, hemoglobin, CYFRA21-1 and CEA level were independent predictors of prognosis for patients with ESCC treated with concurrent CRT. Among biomarkers, CYFRA21-1 and hemoglobin may have a better predictive potential than CEA for long-term outcomes.

Inhibitory Effect of Benzyl Isothiocyanate on Proliferation in vitro of Human Glioma Cells

Zhu, Yu,Zhuang, Jun-Xue,Wang, Qin,Zhang, Hai-Yan,Yang, Ping Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.4

Malignant glioma, also known as brain cancer, is the most common intracranial tumor, having an extremely high mortality and recurrence rate. The survival rate of the affected patients is very low and treatment is difficult. Hence, growth inhibition of glioma has become a hot topic in the study of brain cancer treatment. Among the various isothiocyanate compounds, it has been confirmed that benzyl isothiocyanate (BITC) can inhibit the growth of a variety of tumors, including leukemia, glioma and lung cancer, both inside and outside the body. This study explored inhibitory effects of BITC on human glioma U87MG cells, as well as potential mechanisms. It was found that BITC could inhibit proliferation, induce apoptosis and arrest cell cycling of U87MG cells. In addition, it inhibited the expression of SOD and GSH, and caused oxidative stress to tumor cells. Therefore, it is believed that BITC can inhibit the growth of U87MG cells outside the body. Its mechanism may be related to the fact that BITC can cause oxidative stress to tumor cells.

Clinical Study of Thalidomide Combined with Dexamethasone for the Treatment of Elderly Patients with Newly Diagnosed Multiple Myeloma

Chen, Hai-Fei,Li, Zheng-Yang,Tang, Jie-Qing,Shen, Hong-Shi,Cui, Qing-Ya,Ren, Yong-Ya,Qin, Long-Mei,Jin, Ling-Juan,Zhu, Jing-Jing,Wang, Jing,Ding, Jie,Wang, Ke-Yuan,Yu, Zi-Qiang,Wang, Zhao-Yue,Wu, Tian Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.9

Objective: To investigate the relationship between the efficacy and safety of different doses of thalidomide (Thal) plus dexamethasone (Dex) as the initial therapy in elderly patients with newly diagnosed multiple myeloma (MM). Methods: Clinical data of 28 elderly patients with newly diagnosed MM who underwent the TD regimen as the initial therapy were analyzed retrospectively. The patients were divided into two groups according to the maximal sustained dose of Thal: lower dose (group A) and higher dose (group B). The overall response rate (ORR), progression free survival (PFS), overall survival (OS), and adverse events (AES) were compared between the two groups. Results: A total of 28 patients were followed up with a median of 18 months. The ORR was 60.1%. The median response time and PFS were 2.0 and 17.0 months, respectively. The mean sustained dose of Thal in group B was significantly higher than group A (292.9 mg v 180.4 mg, P=0.01). There was no significantly difference in ORR (57.1% v 64.3%, P=1.00) and PFS (9.63months v 17.66 months, P=0.73) between groups A and B. During the follow up, only five patients died (<40%) and, therefore, median OS values were not available. It is estimated, however, that the mean survival time in the two groups was 35.6 and 33.4 months (P>0.05), respectively. All of the patients tolerated the treatment well. The incidence of AES in patients with a grading above 3 in group B was significantly higher than in group A (P=0.033). Conclusions: The TD regimen results in a high response rate and manageable AES as the initial therapy in elderly patients with MM. TD should be considered as the front line regimen for the treatment of elderly patients with MM in areas with financial constraints. The clinical response can be achieved at a low dose Thal with minimal toxicity.

Realgar transforming solution-induced differentiation of NB4 cell by the degradation of PML/RARa partially through the ubiquitin–proteasome pathway

Yang Hai,Xin Wang,Peng Song,Jian-yin Li,Longhe Zhao,Fei Xie,Xiang-min Tan,Qin-Jian Xie,Lan Yu,Yang Li,Zhengrong Wu,Hong Yu Li 대한약학회 2019 Archives of Pharmacal Research Vol.42 No.8

PML/retinoic acid receptor alpha (RARa), as ahallmark of acute promyeloid leukemia (APL), is directlyrelated to the outcome of clinical APL remedy. It isreported that arsenicals can effectively degrade PML/RARa, such as arsenic trioxide and realgar. However, thehigh toxicity or insolubility have hampered their clinicalapplications. Realgar transforming solution (RTS) wasproduced from realgar by bioleaching process in our lab. Previous studies demonstrated that RTS had a significantanti-cancer ability on chronic myeloid leukemia throughoncoprotein degradation. The capacity of RTS on treatingAPL is what is focused on in this study. The results showedthat RTS had a noticeable sensitivity in NB4 cell, and RTSremarkably down-regulated PML/RARa expression andinduced cell differentiation. Further, RTS could accumulatePML/RARa into the nuclear bodies and then executedegradation, which could be reversed by proteasomeinhibitor MG132. The results also exhibited that thereduction of RTS-induced PML/RARa expression accompaniedby the elevation of ubiquitin and SUMO-1 proteinexpression. Finally, PML and SUMO-1 had been demonstratedto be co-localized after RTS treatment byimmunofluorescence co-localization assay and immunoprecipitationassay. In conclusion, these results suggestedthat RTS-induced cell differentiation may attribute to thePML/RARa degradation partially through the ubiquitin–proteasome pathway.

Rapid and Efficient Detection of 16SrI Group Areca Palm Yellow Leaf Phytoplasma in China by Loop-Mediated Isothermal Amplification

Shao-shuai Yu,Hai-yan Che,Sheng-jie Wang,Cai-li Lin,Ming-xing Lin,Wei-wei Song,Qing-hua Tang,Wei Yan,Wei-quan Qin 한국식물병리학회 2020 Plant Pathology Journal Vol.36 No.5

Areca palm yellow leaf (AYL) disease caused by the 16SrI group phytoplasma is a serious threat to the development of the Areca palm industry in China. The 16S rRNA gene sequence was utilized to establish a rapid and efficient detection system efficient for the 16SrI-B subgroup AYL phytoplasma in China by loopmediated isothermal amplification (LAMP). The results showed that two sets of LAMP detection primers, 16SrDNA-2 and 16SrDNA-3, were efficient for 16SrIB subgroup AYL phytoplasma in China, with positive results appearing under reaction conditions of 64oC for 40 min. The lowest detection limit for the two LAMP detection assays was the same at 200 ag/μl, namely approximately 53 copies/μl of the target fragments. Phytoplasma was detected in all AYL disease samples from Baoting, Tunchang, and Wanning counties in Hainan province using the two sets of LAMP primers 16SrDNA-2 and 16SrDNA-3, whereas no phytoplasma was detected in the negative control. The LAMP method established in this study with comparatively high sensitivity and stability, provides reliable results that could be visually detected, making it suitable for application and research in rapid diagnosis of AYL disease, detection of seedlings with the pathogen and breeding of diseaseresistant Areca palm varieties.

Polysaccharide from Polygonatum Inhibits the Proliferation of Prostate Cancer-Associated Fibroblasts Cells

Han, Shu-Yu,Hu, Ming-Hua,Qi, Guan-Yun,Ma, Chao-Xiong,Wang, Yuan-Yuan,Ma, Fang-Li,Tao, Ning,Qin, Zhi-Hai Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.8

Inhibition of cancer-associated fibroblasts (CAFs) may improve the efficacy of cancer therapy. Polysaccharide extracted from polygonatum can selectively inhibit the growth of prostate-CAFs (p<0.001) without inhibiting the growth of normal fibroblasts (NAFs). Polysaccharides from polygonatum stimulate autophagy of prostate-CAFs. 3-methyl-adenine(3-MA) is an autophagy inhibitor. 3-MA was added to prostate-CAFs with polysaccharide from polygonatum to determine whether autophagy plays an important role in the restrained effect. Finally, polysaccharide from polygonatum treatment significantly increased the activation of Beclin-1 and LC3, key autophagy proteins. Polysaccharide from polygonatum stimulates autophagy of prostate-CAFs and inhibits prostate-CAF growth, indicating that a novel anti-cancer strategy involves inhibiting the growth of prostate-CAFs.

Realgar transforming solution suppresses angiogenesis and tumor growth by inhibiting VEGF receptor 2 signaling in vein endothelial cells

Peng Song,Yang Hai,Xin Wang,Longhe Zhao,Baoqiang Chen,Peng Cui,Qin-Jian Xie,Lan Yu,Yang Li,Zhengrong Wu,Hong Yu Li 대한약학회 2018 Archives of Pharmacal Research Vol.41 No.4

Realgar (As4S4), as an arsenic sulfide mineraldrug, has a good therapeutic reputation for anticancer inTraditional Chinese Medicine, and has recently beenreported to inhibit angiogenesis in tumor growth. However,considering the poor solubility and low bioavailability ofrealgar, large dose of realgar and long period of treatmentare necessary for achieving the effective blood medicineconcentration. In present study, we resolved the crucialproblem of poor solubility of realgar by using intrinsicbiotransformation in microorganism, and investigatedunderlying mechanisms of realgar transforming solution(RTS) for antiangiogenesis. Our results demonstrated thatRTS had a strong activity to inhibit HUVECs proliferation,migration, invasion, and tube formation. Moreover, RTSinhibited VEGF/bFGF-induced phosphorylation ofVEGFR2 and the downstream protein kinases includingERK, FAK, and Src. In vivo zebrafish and chickenchorioallantoic membrane model experiments showed thatRTS remarkably blocked angiogenesis. Finally, comparedwith the control, administration of 2.50 mg/kg RTSreached more than 50% inhibition against H22 tumorallografts in KM mice, but caused few toxic effects in thehost. The antiangiogenic effect was indicated by CD31immunohistochemical staining and alginate-encapsulatedtumor cell assay. In summary, our findings suggest thatRTS inhibits angiogenesis and may be a potential drugcandidate in anticancer therapy.