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Detection of anti-preS1 antibodies for recovery of hepatitis B patients by immunoassay

World Journal of Gastroenterology 2002年第2期8卷 276-281页

作者： Jun Wei Guang-Di Li Yuan Wang Zu-Chuan Zhang,Institute of Biochemsitry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031,China Yu-Qin Wang Zhi-Meng Lu,Department of Clinical virology,Rui-Jin Hospital,Shanghai Second Medical University 200025,Shanghai,China Institute of Biochemsitry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China.

AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in *** (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chrom

关键词： Amino Acid sequence Antibodies base sequence Genetic Vectors Hepatitis B Hepatitis B Surface Antigens Humans Immunoassay Molecular sequence Data Peptide Fragments Protein Precursors Recombinant Fusion Proteins Research Support, Non-U.S. Gov't Viral Envelope Proteins

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Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

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World Journal of Gastroenterology 2002年第3期8卷 400-405页

作者： FangJY LuYY RenjiHospital ShanghaiInstituteofDigestiveDiseaseShanghaiSecondMedicalUniversityShanghaiChina BeijingCancerInstitute BeijingUniversityBeijing100000

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, *** inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

关键词： DNA Methylation DNA-Binding Proteins Acetylation Acetyltransferases base sequence Basic Helix-Loop-Helix Transcription Factors Cell Cycle Proteins Cell Transformation, Neoplastic CpG Islands Cyclin-Dependent Kinase Inhibitor p21 Cyclins DNA Histone Acetyltransferases Histones Humans Molecular sequence Data Nuclear Proteins Signal Transduction Sp1 Transcription Factor Trans-Activators Transcription Factors

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Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization

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World Journal of Gastroenterology 2003年第12期9卷 2751-2758页

作者： Atsushi Masamune Masahiro Satoh Kazuhiro Kikuta Noriaki Suzuki Tooru Shimosegawa Division of Gastroenterology Tohoku University Graduate School of MedicineSendaiJapan A.M.and M.S.equally contributed to this work

AIM: Activated pancreatic stellate cells (PSCs) have been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth potential. A continuous cell line may therefore be valuable in studying molecular mechanisms of these pancreatic disorders. The aim of this study was to establish a cell line of rat PSCs by spontaneous ***: PSCs were isolated from the pancreas of male Wistar rats, and conventional subcultivation was performed repeatedly. Telomerase activity was measured using the telomere repeat amplification protocol. Activation of transcription factors was assessed by electrophoretic mobility shift *** of mitogen-activated protein (MAP) kinases was examined by Western blotting using anti-phosphospecific antibodies. Expression of cytokine-induced neutrophil chemoattractant-1 was determined by enzyme ***: Conventional subcultivation yielded actively growing cells. One clone was obtained after limiting dilution,and designated as SIPS. This cell line has been passaged repeatedly more than 2 years, and is thus likely *** cells retained morphological characteristics of primary,culture-activated PSCs. SIPS expressed α-smooth muscle actin, glial acidic fibrillary protein, vimentin, desmin, type Ⅰ collagen, fibronectin, and prolyl hydroxylases. Telomerase activity and p53 expression were negative. Proliferation of SIPS cells was serum-dependent, and stimulated with platelet-derived growth factor-BB through the activation of extracellular signal-regulated kinase. Interleukin-1β activated nuclear factor-κB, activator protein-1, and MAP ***-1β induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-κB and MAP ***: SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.

关键词： Animals base sequence Cell Culture Techniques Cell Line, Transformed Cells, Cultured Cystic Fibrosis Cytoskeletal Proteins Extracellular Matrix Proteins Immunohistochemistry NF-kappa B Oligonucleotide Probes Pancreas Rats Research Support, Non-U.S. Gov't Transcription Factor AP-1

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GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line

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World Journal of Gastroenterology 2006年第5期12卷 723-730页

作者： Gunter Maubach Michelle Chin Chia Lim Institute of Bioengineering and Nanotechnology 31 Biopolis WayThe Nanos#04-01138669Singapore.

AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

关键词： Promoter Regions (Genetics) Animals base sequence Cell Line DNA, Recombinant Gene Expression Glial Fibrillary Acidic Protein Hepatocytes Humans Lac Operon RNA, Messenger Rats Transfection Transforming Growth Factor beta Transforming Growth Factor beta1

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Relationship between genotype and phenotype of flagellin C in Salmonella

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World Journal of Gastroenterology 2001年第6期7卷 864-867页

作者： Wan-Sheng Ji~1 Jia-Lu Hu~1 Jun-Wen Qiu~1 Bo-Rong Pan~1 Dao-Rong Peng~2 Bing-Long Shi~1 Shao-Juan Zhou~1 Kai-Chun Wu~1 Dai-Ming Fan~1 1 Chinese PLA Institute of Digestive Diseases2 Department of Bacteriology,Xijing Hospital,Fourth Military Medical University,Xi’an 710032,Shaanxi Province,China Chinese PLA Institute of Digestive Diseases Xijing Hospital Fourth Military Medical University Xi'an 710032 Shaanxi Province China.

AIM: To discover the relationship between the genotype and antigen serotype of flagellin C among Salmonella strains. METHODS: Fragment of Salmonella flagellin C in plasmid pLS408 was cloned, sequenced and compared with the corresponding sequence in other strains. Salmonella strains including two typhi strains, one paratyphoid strain, one enteritidis and one typhimurium strain were isolated from outpatients. Genome DNA was purified respectively from these clinical isolates, then the corresponding flagellin C fragment was amplified by polymerase chain reaction,and the amplification products were analyzed by agarose gel electrophoresis. RESULTS: The cloned fragment includes 582 nucleotides encoding the variable region and partial conservative region of Salmonella flagellin C in plasmid pLS408. With comparison to the corresponding sequences reported previously, there is only a little difference from other strains with the same flagellar serotype in both nucleotide and amino acid level. Specific PCR products were amplified in Salmonella strains with flagellar serotype H-1-d including S. muenchen, typhi and typhimurium, but not in S. paratyphoid C or S. enteritidis strains. CONCLUSION: In this experiment, the specificity of nucleotide sequence could be found in flagellin C central variable regions as it exists in flagellar serotypes in Salmonella. It may be helpful to developing a rapid, sensitive, accurate and PCR-based method to detect Salmonella strains with serotype H-1-d.

关键词： Amino Acid sequence base sequence Flagellin Genotype Molecular sequence Data Phenotype Protein Isoforms Salmonella Species Specificity

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hOGG1 Ser326Cys polymorphism modifies the significance of the environmental risk factor for colon cancer

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World Journal of Gastroenterology 2003年第5期9卷 956-960页

作者： Jae-Il Kim Young-Jin Park Ki-Hong Kim Ji-Il Kim Byung-Joo Song Meung-Soo Lee Chul-Num Kim Seok-Hyo Chang Department of Surgery Ilsan Paik HospitalInje University College of Medicine.2240Daehwa-dongIlsan-guKoyang shiKyunggido 412-270Korea

AIM:To determine the association of hOGG1 (8-oxoguanine glycosylase I,OGG1) polymorphism of Ser326Cys substitution with colon cancer risk and possible interaction with known environmental risk factors. METHODS:A case-control study with 125 colon cancer cases and 247 controls was conducted, RESULTS:There was no major difference in Ser326Cys genotype distribution between cases and *** meat intake tended to increase the odds ratio for colon cancer with an OR of 1.72 (95 % confidence interval;CI=1.12-2.76). Such tendency was more prominent in Cys/Cys carriers (OR=4.31,95 % CI=1.64-11.48),but meat intake was not a significant risk factor for colon cancer in Ser/Ser or Ser/ Cys *** OR for colon cancer was elevated with marginal significance in smokers who were Cys/Cys carriers (OR=2.75,95 % CI=1.07-7.53) but not in Ser/Ser or Ser/ Cys carriers. CONCLUSION:These results suggest that the hOGG1 Ser326Cys polymorphism is probably not a major contributor to individual colon cancer susceptibility overall,but the Cys/ Cys genotype may alter the impact of some environmental factors on colon cancer development.

关键词： base sequence Case-Control Studies Colonic Neoplasms DNA DNA-Formamidopyrimidine Glycosylase Diet Female Genotype Humans Male Meat Middle Aged N-Glycosyl Hydrolases Odds Ratio Polymorphism, Genetic Risk Factors Smoking

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Expression of mucosal addressin cell adhesion molecule 1 on vascular endothelium of gastric mucosa in patients with nodular gastritis

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World Journal of Gastroenterology 2003年第12期9卷 2701-2705页

作者： HiroshiOhara HajimeIsomoto Chun-YangWen ChiekoEjima MasahiroMurata MasanobuMiyazaki FuminaoTakeshima YoheiMizuta IkuoMurata TakehikoKoji HiroshiNagura ShigeruKohno SecondDepartmentofInternalMedicine 1-7-1SakamotoNagasakiJapan DepartmentofMolecularPathology AtomicBombDiseaseInstitute1-12-4SakamotoNagasakiJapan DepartmentofPathology TohokuUniversitySchoolofMedicineAobakuSendalJapan DepartmentofHistologyandCellBiology NagasakiUniversitySchoolofMedicineNagasaki1-12-4SakamotoNagasakiJapan

AIM:The interaction of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) with integrin α4β7 mediates lymphocyte recruitment into mucosa-associated lymphoid tissue (MALT).Nodular gastritis is characterized by a unique military pattern on endoscopy representing increased numbers of lymphoid follicles with germinal center,strongly associated with H pylori *** purpose of this study was to address the implication of the MAdCAM-1/integrin β7 pathway in NG. METHODS:We studied 17 patients with NG and H pylori infection and 19 H pylori-positive and 14 H pylori-negative controls.A biopsy sample was taken from the antrum and snap-frozen for immunohistochemical analysis of MAdCAM- 1 and integrin β*** simultaneous viewing of serial sections, the percentage of MAdCAM-1-positive to von Willebrand factor-positive vessels was *** also performed immunostaining with anti-CD20,CD4,CD8 and CD68 antibodies to determine the lymphocyte subsets co- expressing integrin β7. RESULTS:Vascular endothelial MAdCAM-1 expression was more enhanced in gastric mucosa with than without H pylori *** note,the percentages of MAdCAM-1-positive vessels were significantly higher in the lamina propria of NG patients than in H pylori-positive *** expression of MAdCAM-1 was identified adjacent to lymphoid follicles and dense lymphoid *** β7-expressing mononuclear cells,mainly composed of CD20 and CD4 lymphocytes,were associated with vessels lined with MAdCAM-1-expressing ***: Our results suggest that the MAdCAM一1/ integrin a4p7 homing system may participate in gastric inflammation in response to H py/o}i-infection and contributes to MALT formation, typically leading to the development of NG.

关键词： base sequence Comparative Study DNA Primers Endothelium, Vascular Gastric Mucosa Gastritis Helicobacter Infections Helicobacter pylori Humans Immunoglobulins Immunohistochemistry Lymphoma, Mucosa-Associated Lymphoid Tissue Mucoproteins Reverse Transcriptase Polymerase Chain Reaction

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Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

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World Journal of Gastroenterology 2002年第2期8卷 258-262页

作者： Feng-Tian He Rong-Fen Li Yun-Sheng Kang Yan Zhang,Department of Biochemistry & Molecular Biology,Third Military Medical University,Chongqing 400038,China Yong-Zhan Nie Bao-Jun Chen Tai-Dong Qiao Dai-Ming Fan,Institute of Digestive Disease,Xijing Hospital,Fourth Military Medical University,Xi’an 710032,Shaanxi Province,China Department of Biochemistry & Molecular Biology Third Military Medical University Chongqing 400038 China. hefengtian@***

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into *** *** transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect *** HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

关键词： Animals Antibodies, Monoclonal base sequence Carcinoma Colorectal Neoplasms Enzyme-Linked Immunosorbent Assay Humans Immunoglobulin Fragments Immunoglobulin Variable Region Mice Molecular sequence Data Recombinant Proteins Stomach Neoplasms Tumor Cells, Cultured

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Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

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World Journal of Gastroenterology 2002年第2期8卷 213-216页

作者： Yong Li You-Yong Lu,Beijing Institute for Cancer Research,Beijing Laboratory of Molecular Oncology,School of Oncology,Peking University,Beijing 100034,China Beijing Institute for Cancer Research Beijing Laboratory of Molecular Oncology School of Oncology Peking University 1 Da-Hong-Luo-Chang Street Western District Beijing 100034 China.

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

关键词： Gene Expression Regulation, Neoplastic sequence Analysis, DNA Allyl Compounds Amyloid beta-Protein Precursor base sequence Carrier Proteins Cloning, Molecular Expressed sequence Tags Garlic Gene Library Humans Molecular sequence Data Plasminogen Inactivators Platelet Aggregation Inhibitors Receptors, Cell Surface Research Support, Non-U.S. Gov't Stomach Neoplasms Sulfides Tumor Cells, Cultured Viral Nonstructural Proteins

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Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

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World Journal of Gastroenterology 2004年第22期10卷 3356-3360页

作者： JianZhuge Ying-NianYu DepartmentofPathophysiology EnvironmentalGenomicsCenterSchoolofMedicineZhejiangUniversityHangzhou310031ZhejiangProvinceChina

AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

关键词： Alternative Splicing base sequence Carcinoma, Hepatocellular Cytochrome P-450 CYP2D6 DNA, Complementary Exons Humans Liver Liver Neoplasms Molecular sequence Data Mutation RNA, Messenger Research Support, Non-U.S. Gov't Reverse Transcriptase Polymerase Chain Reaction

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