Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile

Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile（Pt CAT） was cloned using RTPCR and rapid amplification of c DNA ends（RACE）. Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif（^（61）FNRERIPERVVHAKGAG^（77））, a proximal heme-ligand signature sequence（^（352）RLFSYSDP^（359））, and three catalytic amino acid residues（H^（72）, N^（145）, and Y^（356））. Pt CAT also contains two putative N-glycosylation sites（^（34）NKT^（36） and ^（437）NFT^（439）） and a peroxisome-targeting signal（^（511）AQL^（513））. Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA： first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.

Quantitative expression of MMP-2 and FN in high metastatic and low metastatic cell lines of breast cancer

To analyze the relation of matrix metalloproteinase-2(MMP-2) and Fibronection (FN) mRNA expression with metastasis of breast cancer and elucidate the role of MMP-2 and FN in breast cancer metastasis.Methods The expression of MMP-2 and FN mRNA in breast cancer cell lines was detected by fluorescence-quantitative RT-PCR.The expression of MMP-2 and FN protein was detected by Western blots.Results The expression of MMP-2 and FN mRNA was down-regulated in high metastatic cell lines MDA-MB-231,MDA-MB-435,but up-regulated in low metastatic cell lines MDA-453,T47D,SK-BR-3 and non-metastatic cell line MCF-7,ZR-75-30.The protein expression of MMP-2 and FN was up-regulated in high mestastic cell lines,and down-regulated in low metastatic cell lines.Conclusion The mRNA and protein expression of MMP-2 and FN was related with breast cancer metastasis.The mRNA expression of MMP-2 and FN is feed-back regulated with protein expression.6 refs,4 figs,2 tabs.

Regulatory Genes Through Robust-SNR for Binary Classification Within Functional Genomics Experiments

The current study proposes a novel technique for feature selection by inculcating robustness in the conventional Signal to noise Ratio(SNR).The proposed method utilizes the robust measures of location i.e.,the“Median”as well as the measures of variation i.e.,“Median absolute deviation(MAD)and Interquartile range(IQR)”in the SNR.By this way,two independent robust signal-to-noise ratios have been proposed.The proposed method selects the most informative genes/features by combining the minimum subset of genes or features obtained via the greedy search approach with top-ranked genes selected through the robust signal-to-noise ratio(RSNR).The results obtained via the proposed method are compared with wellknown gene/feature selection methods on the basis of performance metric i.e.,classification error rate.A total of 5 gene expression datasets have been used in this study.Different subsets of informative genes are selected by the proposed and all the other methods included in the study,and their efficacy in terms of classification is investigated by using the classifier models such as support vector machine(SVM),Random forest(RF)and k-nearest neighbors(k-NN).The results of the analysis reveal that the proposed method(RSNR)produces minimum error rates than all the other competing feature selection methods in majority of the cases.For further assessment of the method,a detailed simulation study is also conducted.

Feedback regulation between phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 and transforming growth factor β1 and prognostic value in gastric cancer

BACKGROUND Phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1(PREX1)was reported to be overexpressed in some cancers and involved in cancer development,but its expression and significance in gastric cancer remain unclear.AIM To evaluate the expression of PREX1 in gastric cancer and its significance in the development of gastric cancer,especially to evaluate the potential mechanism of PREX1 in gastric cancer.METHODS Bioinformatic analysis was performed in order to examine the expression of PREX1 in gastric cancer.The relationship between the survival rate of gastric cancer patients and PREX1 expression was assessed by Kaplan Meier portal.The Gene Set Enrichment Analysis and the correlation between PREX1 and transforming growth factor(TGF)β1 pathway-related mediators were evaluated by cBioPortal for Cancer Genomics.Western blotting and reverse transcriptase polymerase chain reaction assay were used to test the role of TGFβ1 on the expression of PREX1.Western blotting and dual-luciferase reporter system was used to evaluate the effect of PREX1 on the activation of TGFβ1 pathway.Wound healing and Transwell assay were used to assess the effect of PREX1 on the metastasis activity of gastric cancer cells.RESULTS PREX1 was overexpressed in the gastric tumors,and the expression levels were positively associated with the development of gastric cancer.Also,the high expression of PREX1 revealed poor prognosis,especially for those advanced and specific intestinal gastric cancer patients.PREX1 was closely involved in the positive regulation of cell adhesion and positively correlated with TGFβ1-related mediators.Furthermore,TGFβ1 could induce the expression of PREX1 at both the protein and mRNA level.Also,PREX1 could activate the TGFβ1 pathway.The induced PREX1 could increase the migration and invasion activity of gastric cancer cells.CONCLUSION PREX1 is overexpressed in gastric cancer,and the high level of PREX1 predicts poor prognosis.PREX1 is closely associated with TGFβsignaling and promotes the metastasis of gastric cancer cells.

Identification of APEX2 as an oncogene in liver cancer

BACKGROUND DNA damage is one of the critical contributors to the occurrence and development of some cancers.APEX1 and APEX2 are the most important molecules in the DNA damage,and APEX1 has been identified as a diagnostic and prognostic biomarker in liver hepatocellular carcinoma(LIHC).However,the expression of APEX2 and its functional mechanisms in LIHC are still unclear.AIM To examine the expression of APEX2 and the potential mechanism network in LIHC.METHODS We conducted a pan-cancer analysis of the expression of APEX1 and APEX2 using the interactive TIMER tool.GEO datasets,including GSE14520,GSE22058,and GSE64041,were used to compare the APEX2 expression level in tumor tissues and adjacent non-tumor tissues.Then,we calculated the 5-year survival rate according to the web-based Kaplan-Meier analysis.We included the TCGA liver cancer database in GSEA analysis based on the high and low APEX2 expression,showing the potential mechanisms of APEX2 in LIHC.After that,we conducted Pearson correlation analysis using GEPIA2.Next,we performed quantitative polymerase chain reaction(qPCR)assay to examine the APEX2 levels in normal liver cell line LO2 and several liver cancer cell lines,including HepG2,Huh7,SMMC7721,and HCCLM3.APEX2 in HCCLM3 cells was knocked down using small interfering RNA.The role of APEX2 in cell viability was confirmed using CCK-8.Dualluciferase reporter assay was performed to examine the promoter activity of CCNB1 and MYC.RESULTS APEX1 and APEX2 are both highly expressed in the tumor tissues of BLCA,BRCA,CHOL,COAD,ESCA,HNSC,LIHC,LUAD,LUSC,READ,and STAD.APEX2 overexpression in LIHC was validated using GSE14520,GSE22058,and GSE64041 datasets.The survival analysis showed that LIHC patients with high expression of APEX2 had a lower overall survival rate,even in the AJCC T1 patients.High level of APEX2 could indicate a lower overall survival rate in patients with or without viral hepatitis.The GSEA analysis identified that kinetochore and spindle microtubules are the two main cellular components of APEX2 in GO Ontology.APEX2 was also positively associated with molecular function regulation of chromosome segregation and DNA replication.The results of KEGG analysis indicated that APEX2 expression was positively correlated with cell cycle pathway and pro-oncogenic MYC signaling.Pearson correlation analysis showed that APEX2 had a significant positive correlation with CCNB1 and MYC.APEX2 level was higher in liver cancer cell lines than in normal liver LO2 cells.Small interfering RNA could knock down the APEX2 expression in HCCLM3 cells.Knockdown of APEX2 resulted in a decrease in the viability of HCCLM3 cells as well as the expression and promoter activity of CCNB1 and MYC.CONCLUSION APEX2 is overexpressed in LIHC,and the higher APEX2 level is associated with a worse prognosis in overall survival.APEX2 is closely involved in the biological processes of chromosome segregation and DNA replication.APEX2 expression is positively correlated with the pro-oncogenic pathways.Knockdown of APEX2 could inhibit the cell viability and CCNB1 and MYC pathways,suggesting that APEX2 is an oncogene in LIHC,which could be a potential pharmaceutic target in the anti-tumor therapy.

High cell density and high expression of recombinant human ApoA-I_(Milano) in Escherichia coli by twice temperature-shifted induction

The effect of temperature on the formation of recombinant protein,apolipoprotein A-IMilano was investigated in the present study.The temperature of the initial growth phase was set at 30ºC,while temperature variation in induction phase was arranged in three modes.High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out.Experimental results showed that ApoA-IMilano reached 4.8 g/L with the final cell density of OD600,150.It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product.The present study provides a basic procedure for the production of recombinant ApoA-IMilano.

High Level Expression of tcs Gene in E.coli

Trichosanthin is a valuable protein in medical applications. An NMR analytical of the trichosanthin solution conformation is made to study the structural-functional relationship of this protein. For preparing a sample labled with 15 N, the tcs gene was cloned into the expression vector pQE 30, and produced a high level expression of trichosanthin in the E coli strain M15. The amount of fusion TCS synthesized in E coli was about 6 4% of the total cellular protein. The fusion protein was purified by using affinity chromatography with Ni NTA resin.

Construction of High Level Expression Cell Lines of Erythropoietin

Human erythropoietin gene was ligated into the mammalian high efficiency expression vector pSV2 dhfr by standard procedures of filling in, trimming, ligation and transformation to construct a high efficiency expression vector. After the expression vector pSEPO25 was transfected into CHO dhfr - cells, the cell line which could express high levels of EPO was successfully selected. The result lays the foundation for production of EPO by genetic biotechnology.

Highly Expressive Hakka Art

SOUTHERN Jiangxi Province was the birthplace of the Hakka ethnic group and has since been the native home and main transfer hub for the spread of the nationality. The highly expressive art of the Hakkas, including folk songs in Xingguo, colored lantern performances in Shicheng,