To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C termin...To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.展开更多
Objective： To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods： A set of laser captured microdis...Objective： To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods： A set of laser captured microdissected （LCM） specimens from 300 prostate cancer （PCa） patients undergoing radical prostatectomy （RP） were defined. Ten patients representing ＂aggressive＂ PCa, and 10 representing ＂non-aggressive＂ PCa were selected based on prostate-specific antigen （PSA） recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR （TaqMan）, and correlation was analyzed with clinicopathological features. Results： The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas （P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test）. The lower expression of NPY showed association with ＂aggressive＂ PCas based on a larger PCa patient cohort analysis （P=0.0037, univariate generalized linear model （GLM） analysis）. Conclusion： Despite widely noted heterogeneous nature of PCa, gene expression alterations ofAM,4CR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mechanisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.展开更多
AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse t...AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） data from 135 HCC patients undergoing successful hepatectomy. The correlations between PLK1 mRNA expression and clinicopathologic variables were analyzed by Mann-Whitney U test. Prognostic factors were identified by univariate and multivariate analyses. RESULTS： Immunohistochemical results showed overexpression of PLK1 was mainly found in tumor tissues compared with tumor-free tissue. A similar mRNA result was obtained by semi-quantitative RT-PCR. A total of 111 samples were positive for PLK1 mRNA expression. The positive expression was correlated with venous invasion, tumor nodules and Edmondson grade. Furthermore, 1, 3, 5-year survival rates in the positive expression group were significantly lower than the negative control group. Multivariate analysis showed that positive PLK1 expression was an independent risk factor for HCC. CONCLUSION： PLK1 could be a potential biomarker for diagnosis and therapy for HCC.展开更多
Encoded by seven genes, angiopoietin-like （ANGPTL） family members structurally similar to the angiogenic regulating factor angiopoietin are known to possess biological activities in angiogenesis and metabolism. Here...Encoded by seven genes, angiopoietin-like （ANGPTL） family members structurally similar to the angiogenic regulating factor angiopoietin are known to possess biological activities in angiogenesis and metabolism. Here we reports for the first time the identification and expression analysis of all the seven members of bovine ANGPTL gene family, which were designated bANGPTL1 to bANGPTL7 in order. The seven bANGPTL genes consist of 4-9 exons, span 3800M-3000 bp and are located on different chromosomes. The deduced amino acid sequences of the members all possess an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain, both characteristics of angiopoietins. Phylogenetic analysis showed that the 32 identified ANGPTL homologs from 9 species could be classified into two major groups. Real-time quantitative PCR （Q-PCR） analysis revealed that the bANGPTL family members have different expression patterns. This study will be helpful for investigation on the biological role of the bANGPTL family in this economically important species. Furthermore, it provides an insight into the molecular evolution of the emerging ANGPTL family展开更多
Tropomyosin （TM） plays a critical role in skeletal and cardiac muscle development and function. To assess the fimctional significance of a-TM in Japanese flounder （Paralichthys olivaceus） development and metamorph...Tropomyosin （TM） plays a critical role in skeletal and cardiac muscle development and function. To assess the fimctional significance of a-TM in Japanese flounder （Paralichthys olivaceus） development and metamorphosis, cDNA from Japanese flounder was cloned and a-TM mRNA measured during development and metamorphosis. The full-length cDNA is 1 191 bp, including a 5＇- untranslated region of 114 bp, a Y-UTR of 222 bp, and an open reading frame of 855 bp encoding a polypeptide of 284 amino acids. Real-time quantitative PCR revealed that a-TM mRNA is initially expressed in unfertilized ovum, indicating the a-TM gene is maternal. Relatively low mRNA levels were observed in different embryonic stages. A higher level of a-TM mRNA was detected 3 days post hatching （dph）, while the highest level was measured at 29 dph （metamorphic climax） after which it declined towards the end of metamorphosis. The expression of a-TM mRNA was up-regulated in thyroid hormone-treated larvae at 36 dph, but there was no marked difference at other stages when compared to control animals. After thiourea treatment, the expression of a-TM mRNA declined slightly. These data provide basic information that can be utilized in further studies into the role of a-TM in P olivaceus development and metamorphosis.展开更多
The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative...The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus （rt306P） and the polymerase gene from a mutant, with rt306P substituted by serine （rtP306S） were separately fused to the maltose binding protein （MBP） gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Westem blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.展开更多
Strawberry is an economically valuable crop all over the word. Its fruits contain large amounts of polyphenol compounds, anthocyanins for instance. Anthocyanins play important roles in attracting pollinators and prote...Strawberry is an economically valuable crop all over the word. Its fruits contain large amounts of polyphenol compounds, anthocyanins for instance. Anthocyanins play important roles in attracting pollinators and protecting plants from various stresses. ANS （Anthocyanidin synthase） catalyzes the conversion of leucoanthocyanidins to colored anthocyanidins. In this study, the total anthocyanins concentration at seven developmental stages of the strawberry fruit were investigated by UV spectrophotometry at 496 nm and 700 rim; and the expression levels of the Fa-ANS gene were studied by qRT-PCR. The results showed that the anthocyanins contents gradually increased along with the fruit maturation; while the expression patterns of Fa-ANS were consistent with the anthocyanins.展开更多
RNA-Seq technology is becoming widely used in various transcriptomics studies;however,analyzing and interpreting the RNA-Seq data face serious challenges.With the development of high-throughput sequencing technologies...RNA-Seq technology is becoming widely used in various transcriptomics studies;however,analyzing and interpreting the RNA-Seq data face serious challenges.With the development of high-throughput sequencing technologies,the sequencing cost is dropping dramatically with the sequencing output increasing sharply.However,the sequencing reads are still short in length and contain various sequencing errors.Moreover,the intricate transcriptome is always more complicated than we expect.These challenges proffer the urgent need of efficient bioinformatics algorithms to effectively handle the large amount of transcriptome sequencing data and carry out diverse related studies.This review summarizes a number of frequently-used applications of transcriptome sequencing and their related analyzing strategies,including short read mapping,exon-exon splice junction detection,gene or isoform expression quantification,differential expression analysis and transcriptome reconstruction.展开更多
基金Supported by the National Natural Science Foundation of China (No.20336010 and No.20176025).
文摘To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.
基金Project supported by the Center for Prostate Disease Researchthe Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville, MD, USA
文摘Objective： To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods： A set of laser captured microdissected （LCM） specimens from 300 prostate cancer （PCa） patients undergoing radical prostatectomy （RP） were defined. Ten patients representing ＂aggressive＂ PCa, and 10 representing ＂non-aggressive＂ PCa were selected based on prostate-specific antigen （PSA） recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR （TaqMan）, and correlation was analyzed with clinicopathological features. Results： The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas （P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test）. The lower expression of NPY showed association with ＂aggressive＂ PCas based on a larger PCa patient cohort analysis （P=0.0037, univariate generalized linear model （GLM） analysis）. Conclusion： Despite widely noted heterogeneous nature of PCa, gene expression alterations ofAM,4CR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mechanisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.
文摘AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） data from 135 HCC patients undergoing successful hepatectomy. The correlations between PLK1 mRNA expression and clinicopathologic variables were analyzed by Mann-Whitney U test. Prognostic factors were identified by univariate and multivariate analyses. RESULTS： Immunohistochemical results showed overexpression of PLK1 was mainly found in tumor tissues compared with tumor-free tissue. A similar mRNA result was obtained by semi-quantitative RT-PCR. A total of 111 samples were positive for PLK1 mRNA expression. The positive expression was correlated with venous invasion, tumor nodules and Edmondson grade. Furthermore, 1, 3, 5-year survival rates in the positive expression group were significantly lower than the negative control group. Multivariate analysis showed that positive PLK1 expression was an independent risk factor for HCC. CONCLUSION： PLK1 could be a potential biomarker for diagnosis and therapy for HCC.
基金supported by the Fundamental Research Funds for the Central Universities (Grant No. JUSRP10916)the Program of 'Qinglan Project' of Jiangsu Province
文摘Encoded by seven genes, angiopoietin-like （ANGPTL） family members structurally similar to the angiogenic regulating factor angiopoietin are known to possess biological activities in angiogenesis and metabolism. Here we reports for the first time the identification and expression analysis of all the seven members of bovine ANGPTL gene family, which were designated bANGPTL1 to bANGPTL7 in order. The seven bANGPTL genes consist of 4-9 exons, span 3800M-3000 bp and are located on different chromosomes. The deduced amino acid sequences of the members all possess an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain, both characteristics of angiopoietins. Phylogenetic analysis showed that the 32 identified ANGPTL homologs from 9 species could be classified into two major groups. Real-time quantitative PCR （Q-PCR） analysis revealed that the bANGPTL family members have different expression patterns. This study will be helpful for investigation on the biological role of the bANGPTL family in this economically important species. Furthermore, it provides an insight into the molecular evolution of the emerging ANGPTL family
基金This work was supported by the National Science Foundation of China （31172392） and the Foundation for Graduate Excellent Paper Breeding Program by Shanghai Ocean University （B-5201-11-000101）
文摘Tropomyosin （TM） plays a critical role in skeletal and cardiac muscle development and function. To assess the fimctional significance of a-TM in Japanese flounder （Paralichthys olivaceus） development and metamorphosis, cDNA from Japanese flounder was cloned and a-TM mRNA measured during development and metamorphosis. The full-length cDNA is 1 191 bp, including a 5＇- untranslated region of 114 bp, a Y-UTR of 222 bp, and an open reading frame of 855 bp encoding a polypeptide of 284 amino acids. Real-time quantitative PCR revealed that a-TM mRNA is initially expressed in unfertilized ovum, indicating the a-TM gene is maternal. Relatively low mRNA levels were observed in different embryonic stages. A higher level of a-TM mRNA was detected 3 days post hatching （dph）, while the highest level was measured at 29 dph （metamorphic climax） after which it declined towards the end of metamorphosis. The expression of a-TM mRNA was up-regulated in thyroid hormone-treated larvae at 36 dph, but there was no marked difference at other stages when compared to control animals. After thiourea treatment, the expression of a-TM mRNA declined slightly. These data provide basic information that can be utilized in further studies into the role of a-TM in P olivaceus development and metamorphosis.
基金grants from China National 973 Project （Grant G 1999054105） National Natural Science Foundation of China （No. 30530040）.
文摘The reverse transcriptase （RT） protein of hepatitis B virus （HBV） has been successfully expressed by recombinant technology in Eschericahia coli （ E. coli ）. In this study we aimed to develop a semi-quantitative assay for the study of HBV RT protein using this system. Complete HBV polymerase gene from a wild type virus （rt306P） and the polymerase gene from a mutant, with rt306P substituted by serine （rtP306S） were separately fused to the maltose binding protein （MBP） gene and expressed in E. coli respectively. The expression levels of HBV polymerase genes from the wild type virus and its counterpart mutant at rt306 were compared. When these proteins were semi-quantified by Westem blotting using rabbit anti-TP serum, the rtP306S mutant showed decreased expression of MBP-HBV polymerase. By this method, we have shown that the expression level of HBV RT could be affected by substitutions in its amino acid sequences, and this method could be used to study the characteristics of HBV RT protein.
文摘Strawberry is an economically valuable crop all over the word. Its fruits contain large amounts of polyphenol compounds, anthocyanins for instance. Anthocyanins play important roles in attracting pollinators and protecting plants from various stresses. ANS （Anthocyanidin synthase） catalyzes the conversion of leucoanthocyanidins to colored anthocyanidins. In this study, the total anthocyanins concentration at seven developmental stages of the strawberry fruit were investigated by UV spectrophotometry at 496 nm and 700 rim; and the expression levels of the Fa-ANS gene were studied by qRT-PCR. The results showed that the anthocyanins contents gradually increased along with the fruit maturation; while the expression patterns of Fa-ANS were consistent with the anthocyanins.
基金supported by the National Basic Research Program of China (Grant Nos. 2010CB945401, 2007CB108800)National Natural Science Foundation of China (Grant Nos. 30870575,31071162,31000590)Science and Technology Commission of Shanghai Municipality (Grant No. 11DZ2260300)
文摘RNA-Seq technology is becoming widely used in various transcriptomics studies;however,analyzing and interpreting the RNA-Seq data face serious challenges.With the development of high-throughput sequencing technologies,the sequencing cost is dropping dramatically with the sequencing output increasing sharply.However,the sequencing reads are still short in length and contain various sequencing errors.Moreover,the intricate transcriptome is always more complicated than we expect.These challenges proffer the urgent need of efficient bioinformatics algorithms to effectively handle the large amount of transcriptome sequencing data and carry out diverse related studies.This review summarizes a number of frequently-used applications of transcriptome sequencing and their related analyzing strategies,including short read mapping,exon-exon splice junction detection,gene or isoform expression quantification,differential expression analysis and transcriptome reconstruction.
