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World Journal of Gastroenterology 2006年第10期12卷 1551-1557页

作者： Ying-Wei Chiang Jaw-Chin Wu Kuei-Chun Wang Chia-Wei Lai Yao-Chi Chung Yu-Chen Hu Department of Chemical Engineering National Tsing Hua University Hsinchu 300 Taiwan China Institute of Clinical Medicine National Yang Ming University and Division of Gastroenterology Department of Medicine Taipei Veterans General Hospital Taipei 112 Taiwan China

AIM： To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen （L-HDAg）. METHODS： A recombinant baculovirus expressing histidine-tagged L-HDAg （L-HDAgH） was constructed to transduce baby hamster kidney （BHK） cells by a simplified transduction protocol. RESULTS： The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly, the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy. Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles. CONCLUSION： The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive posttranslational modifications.

关键词： Baculovirus Hepatitis delta virus L-HDAg Mammalian cell protein expression Transduction

Co-application of ricin A chain and a recombinant adenovirus express-ing ricin B chain as a novel approach for cancer therapy

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Acta Pharmacologica Sinica 2007年第5期28卷 657-662页

作者： Hong-bin WANG~(1,3),Fei XIA~(1,3),Jing GE~1,Juan YIN~1,Li-song TAN~4,Pei-de ZHANG~2,Jiang ZHONG~(1,5)Departments of ~1Mierobiology and Microbial Engineering and ~2Biochemistry and Molecular Biology,School of Life Sciences,FudanUniversity,Shanghai 200433,China ~3Marine College,Huaihai Institute of Technology,Lianyungang 222005,China ~4Shanghai PulmonaryHospital,Shanghai 200433,China Department of Microbiology and Microbial Engineering School of Life Sciences Fudan University Shanghai China Department of Marine College Huaihai Institute of Technology Lianyungang China Department of Shanghai Pulmonary Hospital Shanghai China Department of Biochemistry and Molecular Biology School of Life Sciences Fudan University Shanghai China

Aim:To develop a novel ricin-based approach for the safe and effective therapyof ***:The ricin A chain （RTA） was expressed in Escherichia coli inthe form of a 6×His-tagged fusion protein and purified with Ni2+-NTA affinityresin.A replication-deficient ricin B chain （RTB）-expression adenovirus greenfluorescence protein （AdGFP-RTB） was *** and AdGFP-RTB weretested for cytotoxicity either individually or in combination in human cell linesHEK293,HeLa,SMMC7721,and *** viability was determined with trypanblue staining or MTT ***:The expression and release of RTB,as wellas the entry of RTA into AdGFP-RTB-infected cells were *** RTAand AdGFP-RTB was used individually,neither was toxic to the *** theywere applied together,significant cell death was observed in all of the cell *** cell-killing effect correlated with the amount of RTA protein used,withcell mortality at about 60% at 4.8 μg RTA in combination with AdGFP-RTB at 100pfu/*** major cell killing was seen when RTA was used in combination with acontrol adenovirus *** treatment of healthy HeLa cells with the virus-free supernatant from AdGFP-RTB/RTA-treated HeLa cells resulted in cell death,suggesting the formation of RTA/RTB complex,and a potential by-stander ***:The new approach was successful in *** modifications ofthe adenovirus vector,as well as an in vivo study are needed to confirm its poten-tial in cancer therapy.

关键词： ricin ribosome inactivating protein protein expression recombinant adenovirus cell killing cancer therapy

expression of some tumor associated factors in human carcinogenesis and development of gastric carcinoma

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World Journal of Gastroenterology 2005年第21期11卷 3217-3221页

作者： Ming-DongZhao Xue-MeiHu Dian-JingSun QunZhang Yu-HaoZhang WeiMeng DepartmentofRadiology AffiliatedHospitalofBinzhouMedicalCollegeBinzhou256603ShandongProvinceChina DepartmentofImmunologyofBinzhouMedicalCollege Binzhou256603ShandongProvinceChina MedicalSchoolofShanghaiJiaotongUniversity Shanghai200030China

AIM: To study the effect of IGF-1/IGF-1R and gastrin/CCK-BR on carcinogenesis and development of human gastric carcinoma and to explore its mechanism and provide a credible theoretical foundation for early diagnosis and molecular therapy of gastric ***: mRNA expression levels of IGF-1/IGF-1R and gastrin/CCK-BR were assessed by RT-PCR method in gastric cancer tissues, adjacent mucosa, and tumor-free tissues from 56 patients with gastric carcinoma and normal gastric mucosae from 56 healthy controls. Tissue specimens were obtained by biopsy and confirmed by histological ***: The mRNA levels of IGF-1/IGF-1R were increased in gastric cancer tissues compared with normal tissues from healthy controls and successively increased in tumor-free tissues, adjacent mucosa, and gastric cancer tissues. The mRNA levels of gastrin/CCK-BR were increased in gastric cancer tissues compared with normal tissues from healthy controls. There was a significant difference between gastric cancer tissues and adjacent mucosa and tumor-free tissues, but the mRNA levels of gastrin were not significantly increased in adjacent mucosa and gastric cancer tissues compared with tumorfree tissues. The mRNA levels of CCK-BR were increased in gastric cancer tissues and adjacent mucosa compared with tumor-free tissues, but not significantly increased in adjacent mucosa and gastric cancer tissues compared with gastric cancer ***: Overexpression of IGF-1/IGF-1R and gastrin/CCK-BR promotes the disorderly proliferation of gastric mucosa epithelia and it is of great significance in the carcinogenesis and development of gastric carcinoma.

关键词： Gastric carcinoma mRNA IGF-1 IGF-1R Gastrin CCK-BR protein expression

Biochanin A and CPe-Ⅲ Peptide Improved Hepatic Infl ammation by Regulating the Hepatic Lipid Metabolic Pathways in Diet-Induced Obese Mice

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Transactions of Tianjin University 2018年第3期24卷 234-243页

作者： Zhaohui Xue Ru Wang Haichao Wen Wancong Yu Xiaohong Kou School of Chemical Engineering and Technology Tianjin University Medical Plant Laboratory Tianjin Research Center of Agricultural Biotechnology

Biochanin A(BCA) and CPe-Ⅲ peptide, which both exist in chickpea(Cicer arietinum L.), possess significant antihyperlipidemic properties. However, the actualmechanisms ofthose compounds in inhibiting the dysregulation oflipid metabolism and complicated inflammation have not been wellcharacterized. This study investigated the effects ofBCA, CPe-Ⅲ peptide, and combined BCA and CPe-Ⅲ peptide(BC) on the expression ofgenes involved in hepatic lipid and inflammation metabolism. Results demonstrated that BCA, CPe-Ⅲ peptide, and BC significantly attenuated hepatitis and hyperlipidemia by downregulating those genes involved in pro-inflammatory cytokines(TNF-α), hepatic fatty acid(FA) synthesis(ACC1 and FAS), cholesterolmetabolism(SREBP2, HMGCR, and PCSK9), and upregulating key regulators involved in FA oxidation(PPARα and FABP1), lipolysis(ATGL), LDLR, reverse cholesteroltransport(ABCA1, SR-B1, and LXRα), and cholesterolcatabolism(CYP7 A1). Moreover, they also altered the expression oflipid metabolism-related proteins, including SREBP2, PCSK9, LDLR, ABCA1, and CYP7 A1. Finally, these results revealed that the combination treatment ofBCA and CPe-Ⅲ peptide resulted in greater antihyperlipidemic activity compared with individualcompounds.

关键词： Biochanin A CPe-Ⅲ peptide Lipid metabolism Infl ammation Gene expression protein expression

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Hepatoma cells up-regulate expression of programmed cell death-1 on T cells

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World Journal of Gastroenterology 2008年第44期14卷 6853-6857页

作者： Ji Chen Xue-Jie Wu Gui-Qiang Wang Department of Infectious Diseases & Center for Liver Diseases Peking University First Hospital Beijing 10034 China

AIM： To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 （PD-1）, and the function of PD-1 on T cells. METHODS： HepG2 or HepG2.***.1.5 cells were cocultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL：2, INF-γ and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. Cytotoxic action of T cells was determined by MIF reduction assay-direct mononuclear cell cytotoxicity assay. RESULTS： The PD-1 expression on Jurkat cells increased by 16.17% ± 2.5% and 17.43% ± 2.2% after HepG2 or HepG2.***.1.5 cells were co-cultured for 48 h. The levels of IL-2, INF-γ and IL-10 in the culture supernatant were 202.9 ＋ 53.0 pg/mL, 88.6 ± 4.6 pg/mL and 63.7± 13.4 pg/mL respectively, which were significantly higher than those （102.9 ± 53 pg/mL, 39.3 ± 4.2 pg/mL, and 34.6 =E13.7 pg/mL） in the control group （P 〈 0.05）. The OD value for MTT assay in the blocking group （0.29 ± 0.06） was significantly higher than that （0.19 ± 0.09） in the control group （P 〈 0.05）. CONCLUSION： PD-1 expression on Jurkat cells is upregulated by hepatoma cells, cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.

关键词： Hepatoma cell Programmed cell death-l protein expression T cell function Cytokine

Yeast knockout library allows for efficient testing of genomic mutations for cell-free protein synthesis

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Synthetic and Systems Biotechnology 2016年第1期1卷 2-6页

作者： Jennifer *** Lauren G.Clark Alaksh Choudhury C.Eric Hodgman Michael C.Jewett Department of Chemical and Biological Engineering Northwestern University2145 Sheridan RoadEvanstonIL 60208-3120USA Chemistry of Life Processes Institute 2170 Campus DriveEvanstonIL 60208-3120USA Masters in Biotechnology Program Northwestern University2145 Sheridan RoadEvanstonIL 60208-3120USA Robert H.Lurie Comprehensive Cancer Center Northwestern University676 N.St Clair StSuite 1200ChicagoIL 60611-3068USA Simpson Querrey Institute Northwestern University303 E.Superior StSuite 11-131ChicagoIL 60611-2875USA

Cell-free protein synthesis(CFPS)systems from crude lysates have benefitted from modifications to their enzyme *** example,functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis ***,making such modifications can take substantial *** a proof-of-concept to accelerate prototyping capabilities,we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS *** analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors(e.g.,nucleases).We found a statistically significant increase in luciferase yields upon loss of function of GCN3,PEP4,PPT1,NGL3,and XRN1 with a maximum increase of over 6-fold as compared to the wild *** work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.

关键词： Cell-free protein synthesis Saccharomyces cerevisiae Synthetic biology In vitro translation Cell-free biology protein expression

Serial deletion reveals structural basis and stability for the core enzyme activity of human glutaminase 1 isoforms:relevance to excitotoxic neurodegeneration

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Translational Neurodegeneration 2017年第1期6卷 98-107页

作者： Yuju Li Justin Peer Runze Zhao Yinghua Xu Beiqing Wu Yi Wang Changhai Tian Yunlong Huang Jialin Zheng Center for Translational Neurodegeneration and Regenerative Therapy Shanghai Tenth People’s Hospital affiliated to Tongji University School of MedicineShanghaiChina Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical CenterOmahaNEUSA Department of Pathology and Microbiology University of Nebraska Medical CenterOmahaNEUSA Shanghai Tenth People’s Hospital affiliated with Tongji University School of Medicine Shanghai 200072China Laboratory of Neuroimmunology and Regenerative Therapy Departments of Pharmacology and Experimental Neuroscience and Pathology and Microbiology985930 Nebraska Medical CenterOmahaNE 68198-5930USA

Background:Glutaminase 1 is a phosphate-activated metabolic enzyme that catalyzes the first step of glutaminolysis,which converts glutamine into *** is the major neurotransmitter of excitatory synapses,executing important physiological functions in the central nervous *** are two isoforms of glutaminase 1,KGA and GAC,both of which are generated through alternative splicing from the same *** and GAC both transcribe 1–14 exons in the N-terminal,but each has its unique C-terminal in the coding *** have previously identified that KGA and GAC are differentially regulated during inflammatory stimulation and HIV ***,glutaminase 1 has been linked to brain diseases such as amyotrophic lateral sclerosis,Alzheimer’s disease,and hepatic *** enzyme structure of KGA and GAC has been published ***,how other coding sequences affect their functional enzyme activity remains ***:We cloned and performed serial deletions of human full-length KGA and GAC from the N-terminal and the C-terminal at an interval of approximately 100 amino acids(AAs).Prokaryotic expressions of the mutant glutaminase 1 protein and a glutaminase enzyme activity assay were used to determine if KGA and GAC have similar efficiency and efficacy to convert glutamine into ***:When 110 AAs or 218 AAs were deleted from the N-terminal or when the unique portions of KGA and GAC that are beyond the 550 AA were deleted from the C-terminal,KGA and GAC retained enzyme activity comparable to the full length *** contrast,deletion of 310 AAs or more from N-terminal or deletion of 450 AAs or more from C-terminal resulted in complete loss of enzyme activity for KGA/***,when both Nand C-terminal of the KGA and GAC were removed,creating a truncated protein that expressed the central 219 AA-550 AA,the protein retained enzyme ***,expression of the core 219 AA-550 AA coding sequence in cel

关键词： Glutaminase 1 protein expression Enzyme activity

expression of Arginyl-tRNA Synthetase in Rats with Focal Cerebral Ischemia

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2014年第2期34卷 172-175页

作者：符荣范云智范宇葱赵洪洋 Department of Neurosurgery Union Hospital Tongji Medical College Huazhong University of Science and Technology

Aminoacyl-tRNA syntheses （AARS） can catalyze the adenosine triphosphate （ATP）-dependent acylation of their cognate tRNA（s） with a specific amino acid. They can be seen as an index to reflect the energy metabolic rate of ischemic brain cells in ischemic penumbra. This study ex- amined the relationship between arginyl-tRNA synthetase （ArgRS）, one of the AARS, and cerebral ischemia in rats. The model of middle cerebral artery occlusion （MCAO） was established in rats. The expression levels of ArgRS protein and mRNA were detected in rat brain tissues at different time points following MCAO by Western blotting and RT-PCR, respectively. The results showed that the MCAO model was successfully established. Western blotting and RT-PCR analysis revealed that the ArgRS protein and mRNA were expressed in brain cells in both ischemic and normal penumbra tissues. The expression levels of ArgRS protein and mRNA peaked at 6 h after MCAO and decreased gradually. At 24 h, the expression levels of ArgRs protein and mRNA in ischemic penumbral tissues were lower than those in normal tissues. The expression levels of ArgRS mRNA and protein in ischemic penumbra var- ied with ischemic time, suggesting that the energy metabolism of brain cells in penumbra changed dy- namically after ischemia to ensure the endogenous self-protection of the body. The brain oxygen supply should be improved as soon as possible, especially within 6-12 h after ischemia, so as to meet the de- mand for energy metabolism in ischemic penumbra and make sure the cell structure remains stable.

关键词： stroke cerebral ischemia arginyl-tRNA synthetase gene expression protein expression

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Physiological Processes Contributing to the Difference in Grain Amino Acid Content Between Two Hybrid Rice(Oryza sativa L.)Cultivars

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Phyton-International Journal of Experimental Botany 2019年第2期88卷 101-107页

作者： Min Huang Hengdong Zhang Chunrong Zhao Guanghui Chen Yingbin Zou Crop and Environment Research Center for Human Health College of AgronomyHunan Agricultural UniversityChangsha410128China.

Improving grain amino acid content of rice(Oryza sativa L.)is essential for the health of *** study was conducted to identify the physiological processes that contribute to the higher grain amino acid content in hybrid rice cultivar Lingliangyou 268 compared to Luliangyou *** results showed that total amino acid content in grains was 9%higher in Lingliangyou 268 than in Luliangyou *** was no significant difference in grain nitrogen(N)content between Lingliangyou 268 and Luliangyou 996,while ratio of amino acid to N was 6%higher in Lingliangyou 268 compared to Luliangyou 996.A total of 16 differentially expressed proteins related to amino acid metabolism(e.g.,erythronate-4-phosphate dehydrogenase domain containing protein)were identified in grains between Lingliangyou 268 and Luliangyou *** identified proteins were involved in 10 molecular *** of the 10 defined functions were related to binding(heterocyclic compound binding,nucleoside phosphate binding,nucleotide binding,organic cyclic compound binding,protein binding,and small molecule binding)and the other 4 defined functions were catalytic activity,enzyme regulator activity,hydrolase activity,and transferase *** results indicate that the higher grain amino acid content in Lingliangyou 268 compared to Luliangyou 996 is attributed to increased efficiency of converting N to amino acid that results from altered expression of proteins related to amino acid metabolism.

关键词： Amino acid content grain quality protein expression protein function hybrid rice

Preparation of rabbit anti-rat LRRN3 polyclonal antibody and study of its expression

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Neural Regeneration Research 2010年第13期5卷 1030-1034页

作者： Jing Yang Yanjin Wang Yan Cai Zhiying Yang Ganqiu Wu Jianguo Wen Xuegang Luo He Huang Department of Histology and Embryology Xiangya School of Medicine Central South University Changsha 410013 Hunan Province China Department of Human Anatomy and Neurobiology Xiangya School of Medicine Central South University Changsha 410013 Hunan Province China Department of Neurosurgery Xiangya Hospital Central South University Changsha 410013 Hunan Province China

BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most studies have focused on nucleic acid levels and few have reported on LRRN3 protein levels. OBJECTIVE： To prepare rabbit anti-rat LRRN3 polyclonal antibody and to observe protein tissue expression profiles. DESIGN, TIME AND SEI-rlNG： In vitro, molecular, biological experiments were performed from October 2007 to April 2009 in Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. MATERIALS： Immunization antigen, namely rat MaI-LRRN3C-His recombinant protein, was provided by the Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. METHODS： Rat Mal-LRRN3C-His recombinant protein was used to immunize male, New Zealand rabbits, and rabbit anti-rat LRRN3 polyclonal antibody was prepared. MAIN OUTCOME MEASURES： Antibody purification was conducted using protein A affinity chromatography, and the LRRN3 anti-serum titer was identified using enzyme-linked immunosorbent assay. Immunohistochemical techniques and Western blot preliminary tests were used to determine LRRN3 protein expression profiles in adult rats. RESULTS： A highly purified rabbit anti-rat LRRN3 polyclonal antibody was obtained. Western Blot results from rat brain total protein revealed a band at 79 kD, which was consistent with the size of LRRN3. Immunohistochemistry results showed that protein was mainly expressed in the central nervous system, and no significant positive signals were observed in other tissues. Positive cells included neurons of cerebral cortex and hippocampal dentate gyrus granule cell layer, and cerebellar Purkinje cells. There was no positive expression in glial cells. CONCLUSION： Rabbit anti-rat LRRN3 polyclonal antibody was successfully prepared at a high purity from the prokaryotic-expressed MaI-LRRN3C-His recombinant

关键词： polyclonal antibody preparation protein expression LRRN3 developmental neurobiology neural regeneration

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