Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic ...Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic research in molecular biology and biotechnological applications.Various chemical inducible systems based on activation and inactivation of the target gene had been described.The transfer of regulatory elements from prokaryotes,insects,and mammals has opened new avenues to construct chemically inducible promoters that differ in their ability to regulate the temporal and spatial expression patterns,and this will dramatically increase the application of transgenic technology.This review provides an overview on regulation of gene expression,promoter activating systems,promoter inactivation systems,inducible gene over expression,and inducible anti suppression.展开更多
Actin is a ubiquitous protein and plays essential roles on cellular structure maintenance and cellular motility in both muscle and non-muscle tissues.Multiple genes encoding muscle actin have been identified from the ...Actin is a ubiquitous protein and plays essential roles on cellular structure maintenance and cellular motility in both muscle and non-muscle tissues.Multiple genes encoding muscle actin have been identified from the ascidians,including those expressed in the larval tail muscle,the adult body-wall muscle,and adult heart muscle.In this study,a novel striated non-tail muscle actin gene was identified from the RNA-seq data of Ciona savignyi embryos.Phylogenetic analysis,alignment of the N-terminal amino acid sequences and comparation of diagnostic residues provided evidence that it had high similarity with vertebrate cardiac and skeletal muscle actin.In situ hybridization and promoter-driven GFP reporter assay revealed that it was specifically expressed in the primordia of the oral and atrial siphon.We hereby defined it as siphon-specific muscle actin coding gene(Cs-SMA).A 201 bp(−1350 bp to−1150 bp)sequence containing T-box and Six1/2 binding motif within the upstream region of Cs-SMA confined the expression of GFP in the siphons of electroporated embryos.Six1/2 binding motif was experimentally confirmed to play indispensable role in controlling the siphon-specific expression of Cs-SMA.The tissue-specific expression of Cs-SMA in the siphon primordia indicated its potential crucial roles in Ciona embryogenesis and organogenesis.展开更多
Objective: In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP ...Objective: In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal ...展开更多
RpoS protein is a σ factor of RNA polymerase that can control the expression of a group-specific gene, thus playing a vital role in bacteria. In bacteria, RpoS expression is under strict control and is mainly regulat...RpoS protein is a σ factor of RNA polymerase that can control the expression of a group-specific gene, thus playing a vital role in bacteria. In bacteria, RpoS expression is under strict control and is mainly regulated at three levels： transcription level, translation level and post-translational level. Environmental stress enters bacterial cells through signal transduction and leads to a series of variations in microenvironment, thereby causing changes of regulator and controlling its levels based on the direct and indirect interaction between regulator and RpoS protein. In addition, RpoS protein has played special roles in bacteria, therefore the changes of RpoS protein levels will lead to variations in expression levels of a large number of genes, thereby causing variations of bacterial response to different environmental stress and changes of certain characteristics of bacteria, which provides a new strategy for the control of bacterial diseases in the future. This paper reviewed the recent progress on the regulation of RpoS protein expression and its function in several common bacteria. Due to the functional complexity of RpoS protein, there are still a lot of unknown functions to be further identified.展开更多
Dendritic spines are small protrusions along dendrites that contain most of the excitatory synapses in principal neurons,playing a crucial role in neuronal function by creating a compartmentalized environment for sign...Dendritic spines are small protrusions along dendrites that contain most of the excitatory synapses in principal neurons,playing a crucial role in neuronal function by creating a compartmentalized environment for signal transduction.The plasticity of spine morphologies provides a tunable handle to regulate calcium signal dynamics,allowing rapid regulation of protein expression necessary to establish and maintain synapses(Cornejo et al.,2022).If excitatory inputs were to be located primarily on dendritic shafts,dendrites would frequently short-circuit,preventing voltage signals from propagating(Cornejo et al.,2022).It is thus not surprising that the structural plasticity of dendritic spines is closely linked to synaptic plasticity and memory formation(Berry and Nedivi,2017).While comprehensive in vitro studies have been conducted,in vivo studies that directly tackle the mechanism of dendritic transport and translation in regulating spine plasticity spatiotemporally are limited.展开更多
Somatic variants in the cancer genome influence gene expression through diverse mechanisms depending on their specific locations.However,a systematic evaluation of the effects of somatic variants located in 3'untr...Somatic variants in the cancer genome influence gene expression through diverse mechanisms depending on their specific locations.However,a systematic evaluation of the effects of somatic variants located in 3'untranslated regions(3'UTRs)on alternative polyadenylation(APA)of m RNA remains lacking.In this study,we analyze 10,199 tumor samples across 32 cancer types and identify 1333 somatic single nucleotide variants(SNVs)associated with abnormal 3'UTR APA.Mechanistically,these 3'UTR SNVs can alter cisregulatory elements,such as the poly(A)signal and UGUA motif,leading to changes in APA.Minigene assays confirm that 3'UTR SNVs in multiple genes,including RPS23 and CHTOP,induce aberrant APA.Among affected genes,62 exhibit differential stability between tandem 3'UTR isoforms,including HSPA4and UCK2,validated by experimental assays.Finally,we establish that SNV-related abnormal APA usage serves as an additional layer of expression regulation for tumor-suppressor gene HMGN2 in breast cancer.Collectively,this study reveals 3'UTR APA as a critical mechanism mediating the functional impact of somatic noncoding variants in human cancers.展开更多
Selenium （Se） is an essential trace element. Autoimmune thyroid diseases （AITD） are destructive inflammatory or anti-receptor autoimmune diseases characterized by reactivity to self-thyroid antigens. However, the ...Selenium （Se） is an essential trace element. Autoimmune thyroid diseases （AITD） are destructive inflammatory or anti-receptor autoimmune diseases characterized by reactivity to self-thyroid antigens. However, the effects of Se on the cytokines in AITD are still unclear. So we researched the role of Selenium （Se） and Thl/Th2 cytokine productions in the pathogenesis of autoimmune thyroid diseases （AITD）.展开更多
AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor o...AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P【0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P 【 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P 【 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P 【 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P 【 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.展开更多
A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on scre...A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.展开更多
Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role...Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.展开更多
INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer...INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'展开更多
AIM: To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma ...AIM: To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCI-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells. METHODS: The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracycline-responsive element (TRE) to obtain the plasmid pRevTRE-TRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87. The resulting cell line NCI-N87-Tet On TRE-TRAIL and a control cell line, NCI-N87 Tet On-TRE, were established. TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction. RESULTS: The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67-Tet On were obtained,with titers of about 10(8)CFU.L(-1). By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On TRE-TRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCI-N87. When Dox was added, cell death was obvious in the test groups (29%-77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium. CONCLUSION: With the use of the RevTet-On system, a regulated expression system for TRAIL was constructed. Using this system, the selected killing effect of TRAIL on gastric carcinoma cell line NCI-N87 could be observed.展开更多
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 b...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.展开更多
INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat ...INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .展开更多
AIM:Little has been known about the pathogenesis of non- erosive reflux disease(NERD).Recent studies have implicated interleukin 8(IL-8)in the development and progression of gastroesophgeal reflux disease(GERD).The pu...AIM:Little has been known about the pathogenesis of non- erosive reflux disease(NERD).Recent studies have implicated interleukin 8(IL-8)in the development and progression of gastroesophgeal reflux disease(GERD).The purpose of this study was to determine IL-8 RNA expression levels in NERD patients with or without subtle mucosal changes. METHODS:We studied 26 patients with NERD and 13 asymptomatic controls.Biopsy sample was taken from the esophagus 3 cm above the gastroesophageal junction and snap frozen for measurement of IL-8 mRNA levels by real-time quantitative polymerase chain reaction(PCR).We also examined mRNA expression of IL-8 receptors,CXCR-1 and -2 by reverse transcriptase PCR.The patients were endoscopically classified into grade M(mucosal color changes without visible mucosal break)and N(neither minimal involvement nor mucosal break)of the modified Los Angeles classification. RESULTS:The relative IL-8 mRNA expression levels were significantly higher in esophageal mucosa of NERD patients than those in esophageal mucosa of the controls.There was a significant difference in IL-8 mRNA levels between grades M and N.The CXCR-1 and -2 mRNAs were constitutively expressed in esophageal mucosa.CONCLUSION: Our results suggest that high IL-8 levels in esophageal mucosa may be involved in the pathogenesis of NERD through interaction with its receptors. NERD seems to be composed of a heterogeneous population in terms of not only endoscopically minimal involvement but also immune and inflammatory processes.展开更多
Effects of maternal dietary zinc deficiency on prenatal and postnatal brain development were investigated in ICR strain mice. From d 1 of pregnancy (E0) until postnatal d 20 (P20), maternal mice were fed experimental ...Effects of maternal dietary zinc deficiency on prenatal and postnatal brain development were investigated in ICR strain mice. From d 1 of pregnancy (E0) until postnatal d 20 (P20), maternal mice were fed experimental diets that contained 1 mg Zn/kg/day (severe zinc deficient, SZD), 5 mg Zn/kg/day (marginal zinc deficient, MZD), 30 mg Zn/kg/day (zinc adequately supplied, ZA) or 100 mg Zn/kg/day (zinc supplemented, ZS and pair-fed, PF). Brains of offspring from these dietary groups were examined at various developmental stages for expression of nestin, an intermediate filament protein found in neural stem cells and young neurons. Immunocytochemistry showed nestin expression in neural tube 10.5 d post citrus (dpc) as well as in the cerebral cortex and neural tube from 10.5 dpc to postnatal d 10 (P10). Nestin immunoreactivities in both brain and neural tube of those zinc-supplemented control groups (ZA, ZS, PF) were stronger than those in zinc-deficient groups (SZD and MZD). Western blot analysis confirmed that nestin levels in pooled brain extracts from each of the zinc-supplemented groups (ZA, ZS, PF) were much higher than those from the zinc-deficient groups (SZD and MZD) from 10.5 dpc to P10. Immunostaining and Western blots showed no detectable nestin in any of the experimental and control group brains after P20. These observations of an association between maternal zinc deficiency and decreased nestin protein levels in brains of offspring suggest that zinc deficiency suppresses development of neural stem cells, an effect which may lead to neuroanatomical and behavioral abnormalities in adults.展开更多
Based on bioinformatic analysis, we selected two novel micro RNAs（miRNAs）, bmo-miR-0001 and bmomiR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland（PSG）. Firstly, we examined th...Based on bioinformatic analysis, we selected two novel micro RNAs（miRNAs）, bmo-miR-0001 and bmomiR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland（PSG）. Firstly, we examined the expression of bmo-miR-0001 and bmo-miR-0015 in 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of Bm Fib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pc DNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pc DNA3.0 [ie1-egfp-pribmo-miR-0015-SV40]. Finally, the Bm N cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly（P〈0.05） in Bm N cells co-transfected by pc DNA3.0 [ie1-egfp-pri-bmomiR-0001-SV40] or pc DNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40] with p GL3.0 [A3-luc-Fib-L-3＇UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of Bm Fib-L in vitro.展开更多
AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing rec...AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.展开更多
AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragm...AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.展开更多
BACKGROUND Sepsis is a major medical challenge.Magnolol is an active constituent of Houpu that improves tissue function and exerts strong anti-endotoxin and anti-inflammatory effects,but the mechanism by which it redu...BACKGROUND Sepsis is a major medical challenge.Magnolol is an active constituent of Houpu that improves tissue function and exerts strong anti-endotoxin and anti-inflammatory effects,but the mechanism by which it reduces intestinal inflammation in sepsis is yet unclear.AIM To assess the protective effect of magnolol on intestinal mucosal epithelial cells in sepsis and elucidate the underlying mechanisms.METHODS Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,and regulated on activation,normal T-cell expressed and secreted(RANTES)levels in serum and ileal tissue in animal studies.The histopathological changes of the ileal mucosa in different groups were observed under a microscope.Cell Counting Kit-8 and cell permeability assays were used to determine the concentration of drug-containing serum that did not affect the activity of Caco2 cells but inhibited lipopolysaccharide(LPS)-induced decrease in permeability.Immunofluorescence and Western blot assays were used to detect the levels of RANTES,inhibitor of nuclear factor kappa-B kinaseβ(IKKβ),phosphorylated IKKβ(p-IKKβ),inhibitor of nuclear factor kappa-B kinaseα(IκBα),p65,and p-p65 proteins in different groups in vitro.RESULTS In rats treated with LPS by intravenous tail injection in the presence or absence of magnolol,magnolol inhibited the expression of proinflammatory cytokines,IL-1β,IL-6,and TNF-αin a dose-dependent manner.In addition,magnolol suppressed the production of RANTES in LPS-stimulated sepsis rats.Moreover,in vitro studies suggested that magnolol inhibited the increase of p65 nucleation,thereby markedly downregulating the production of the phosphorylated form of IKKβin LPS-treated Caco2 cells.Specifically,magnolol inhibited the translocation of the transcription factor nuclear factor-kappa B(NF-κB)from the cytosol into the nucleus and down-regulated the expression level of the chemokine RANTES in LPS-stimulated Caco2 cells.CONCLUSION Magnolol down-regulates RANTES levels by inhibiting the LPS/NF-κB signaling pathways,thereby suppressing IL-1β,IL-6,and TNF-αexpression to alleviate the mucosal barrier dysfunction in sepsis.展开更多
文摘Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic research in molecular biology and biotechnological applications.Various chemical inducible systems based on activation and inactivation of the target gene had been described.The transfer of regulatory elements from prokaryotes,insects,and mammals has opened new avenues to construct chemically inducible promoters that differ in their ability to regulate the temporal and spatial expression patterns,and this will dramatically increase the application of transgenic technology.This review provides an overview on regulation of gene expression,promoter activating systems,promoter inactivation systems,inducible gene over expression,and inducible anti suppression.
基金funded by the National Key Research and Development Program of China(Nos.2019YFE0190900,2018YFD0900705).
文摘Actin is a ubiquitous protein and plays essential roles on cellular structure maintenance and cellular motility in both muscle and non-muscle tissues.Multiple genes encoding muscle actin have been identified from the ascidians,including those expressed in the larval tail muscle,the adult body-wall muscle,and adult heart muscle.In this study,a novel striated non-tail muscle actin gene was identified from the RNA-seq data of Ciona savignyi embryos.Phylogenetic analysis,alignment of the N-terminal amino acid sequences and comparation of diagnostic residues provided evidence that it had high similarity with vertebrate cardiac and skeletal muscle actin.In situ hybridization and promoter-driven GFP reporter assay revealed that it was specifically expressed in the primordia of the oral and atrial siphon.We hereby defined it as siphon-specific muscle actin coding gene(Cs-SMA).A 201 bp(−1350 bp to−1150 bp)sequence containing T-box and Six1/2 binding motif within the upstream region of Cs-SMA confined the expression of GFP in the siphons of electroporated embryos.Six1/2 binding motif was experimentally confirmed to play indispensable role in controlling the siphon-specific expression of Cs-SMA.The tissue-specific expression of Cs-SMA in the siphon primordia indicated its potential crucial roles in Ciona embryogenesis and organogenesis.
基金National Natural Science Foundation of China(No 39900040)Natiorlal Natural Science Outstanding Youth Foundation of China(No 39825111).
文摘Objective: In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal ...
基金Supported by Science and Technology Program of Shandong Province (No. 2010GHY10501)National Department Public Benefit Research Fond of China (No. 200909020)~~
文摘RpoS protein is a σ factor of RNA polymerase that can control the expression of a group-specific gene, thus playing a vital role in bacteria. In bacteria, RpoS expression is under strict control and is mainly regulated at three levels： transcription level, translation level and post-translational level. Environmental stress enters bacterial cells through signal transduction and leads to a series of variations in microenvironment, thereby causing changes of regulator and controlling its levels based on the direct and indirect interaction between regulator and RpoS protein. In addition, RpoS protein has played special roles in bacteria, therefore the changes of RpoS protein levels will lead to variations in expression levels of a large number of genes, thereby causing variations of bacterial response to different environmental stress and changes of certain characteristics of bacteria, which provides a new strategy for the control of bacterial diseases in the future. This paper reviewed the recent progress on the regulation of RpoS protein expression and its function in several common bacteria. Due to the functional complexity of RpoS protein, there are still a lot of unknown functions to be further identified.
基金supported by the National Natural Science Foundation of China(NSFC/RGC/JRF N_HKU735/21)Research Grant Council of Hong Kong,China(17102120,17108821,17103922,C1024-22GF,C7074-21G)+1 种基金Health and Medical Research Fund(HMRF 09200966)(to CSWL)FRQS Postdoctoral Fellowship(to AHKF).
文摘Dendritic spines are small protrusions along dendrites that contain most of the excitatory synapses in principal neurons,playing a crucial role in neuronal function by creating a compartmentalized environment for signal transduction.The plasticity of spine morphologies provides a tunable handle to regulate calcium signal dynamics,allowing rapid regulation of protein expression necessary to establish and maintain synapses(Cornejo et al.,2022).If excitatory inputs were to be located primarily on dendritic shafts,dendrites would frequently short-circuit,preventing voltage signals from propagating(Cornejo et al.,2022).It is thus not surprising that the structural plasticity of dendritic spines is closely linked to synaptic plasticity and memory formation(Berry and Nedivi,2017).While comprehensive in vitro studies have been conducted,in vivo studies that directly tackle the mechanism of dendritic transport and translation in regulating spine plasticity spatiotemporally are limited.
基金financially supported by the National Key R&D Program of China(2023YFC3603300,2021YFA0909300)National Natural Science Foundation of China(92249302,32370592,32400437)China Postdoctoral Science Foundation(BX20230073 and 2023M740709)。
文摘Somatic variants in the cancer genome influence gene expression through diverse mechanisms depending on their specific locations.However,a systematic evaluation of the effects of somatic variants located in 3'untranslated regions(3'UTRs)on alternative polyadenylation(APA)of m RNA remains lacking.In this study,we analyze 10,199 tumor samples across 32 cancer types and identify 1333 somatic single nucleotide variants(SNVs)associated with abnormal 3'UTR APA.Mechanistically,these 3'UTR SNVs can alter cisregulatory elements,such as the poly(A)signal and UGUA motif,leading to changes in APA.Minigene assays confirm that 3'UTR SNVs in multiple genes,including RPS23 and CHTOP,induce aberrant APA.Among affected genes,62 exhibit differential stability between tandem 3'UTR isoforms,including HSPA4and UCK2,validated by experimental assays.Finally,we establish that SNV-related abnormal APA usage serves as an additional layer of expression regulation for tumor-suppressor gene HMGN2 in breast cancer.Collectively,this study reveals 3'UTR APA as a critical mechanism mediating the functional impact of somatic noncoding variants in human cancers.
基金supported by three programs from the National Natural Science Foundation of China (NSFC) (The experimental study on the effect of trace elements iodine and selenium on the autoimmune thyroid disease (No.30571564)The cross-sectional investigation on hypothyroidism induced by excess iodine intake and the experimental research on pathogenesy (No.30972465)The change of thyroid pathology and the levels of T3,T4 in SePP1,GPX3 knock out mice (No.30810103004)
文摘Selenium （Se） is an essential trace element. Autoimmune thyroid diseases （AITD） are destructive inflammatory or anti-receptor autoimmune diseases characterized by reactivity to self-thyroid antigens. However, the effects of Se on the cytokines in AITD are still unclear. So we researched the role of Selenium （Se） and Thl/Th2 cytokine productions in the pathogenesis of autoimmune thyroid diseases （AITD）.
基金Supported by the Scientific Research Fund for Doctorate Education,State Educational Commission,No.9837
文摘AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P【0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P 【 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P 【 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P 【 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P 【 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.
基金This work was supported by the National Nat-ural Science Foundation of China(Grant No.30130240),the Chinese Academy of Sciences(GrantNo.KSCX2-SW-303).
文摘A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.
基金Major State BasicResearch (973) Program of China, (G1999053905).
文摘Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.
文摘INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'
基金the National Natural Science Foundation of China,No.39870850
文摘AIM: To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCI-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells. METHODS: The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracycline-responsive element (TRE) to obtain the plasmid pRevTRE-TRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87. The resulting cell line NCI-N87-Tet On TRE-TRAIL and a control cell line, NCI-N87 Tet On-TRE, were established. TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction. RESULTS: The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67-Tet On were obtained,with titers of about 10(8)CFU.L(-1). By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On TRE-TRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCI-N87. When Dox was added, cell death was obvious in the test groups (29%-77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium. CONCLUSION: With the use of the RevTet-On system, a regulated expression system for TRAIL was constructed. Using this system, the selected killing effect of TRAIL on gastric carcinoma cell line NCI-N87 could be observed.
基金the Natural Scientific Foundation of China (NSFC3962526)National High-Technology Project-863 (102-10-01-04)
文摘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.
基金Supported by the National Major Science and Technology Projects,No.96-901-01-54.
文摘INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .
文摘AIM:Little has been known about the pathogenesis of non- erosive reflux disease(NERD).Recent studies have implicated interleukin 8(IL-8)in the development and progression of gastroesophgeal reflux disease(GERD).The purpose of this study was to determine IL-8 RNA expression levels in NERD patients with or without subtle mucosal changes. METHODS:We studied 26 patients with NERD and 13 asymptomatic controls.Biopsy sample was taken from the esophagus 3 cm above the gastroesophageal junction and snap frozen for measurement of IL-8 mRNA levels by real-time quantitative polymerase chain reaction(PCR).We also examined mRNA expression of IL-8 receptors,CXCR-1 and -2 by reverse transcriptase PCR.The patients were endoscopically classified into grade M(mucosal color changes without visible mucosal break)and N(neither minimal involvement nor mucosal break)of the modified Los Angeles classification. RESULTS:The relative IL-8 mRNA expression levels were significantly higher in esophageal mucosa of NERD patients than those in esophageal mucosa of the controls.There was a significant difference in IL-8 mRNA levels between grades M and N.The CXCR-1 and -2 mRNAs were constitutively expressed in esophageal mucosa.CONCLUSION: Our results suggest that high IL-8 levels in esophageal mucosa may be involved in the pathogenesis of NERD through interaction with its receptors. NERD seems to be composed of a heterogeneous population in terms of not only endoscopically minimal involvement but also immune and inflammatory processes.
基金grants from National Basic Research Program (G 1999054000) andNational Natural Science FOundation of China (No.39770643, 398702
文摘Effects of maternal dietary zinc deficiency on prenatal and postnatal brain development were investigated in ICR strain mice. From d 1 of pregnancy (E0) until postnatal d 20 (P20), maternal mice were fed experimental diets that contained 1 mg Zn/kg/day (severe zinc deficient, SZD), 5 mg Zn/kg/day (marginal zinc deficient, MZD), 30 mg Zn/kg/day (zinc adequately supplied, ZA) or 100 mg Zn/kg/day (zinc supplemented, ZS and pair-fed, PF). Brains of offspring from these dietary groups were examined at various developmental stages for expression of nestin, an intermediate filament protein found in neural stem cells and young neurons. Immunocytochemistry showed nestin expression in neural tube 10.5 d post citrus (dpc) as well as in the cerebral cortex and neural tube from 10.5 dpc to postnatal d 10 (P10). Nestin immunoreactivities in both brain and neural tube of those zinc-supplemented control groups (ZA, ZS, PF) were stronger than those in zinc-deficient groups (SZD and MZD). Western blot analysis confirmed that nestin levels in pooled brain extracts from each of the zinc-supplemented groups (ZA, ZS, PF) were much higher than those from the zinc-deficient groups (SZD and MZD) from 10.5 dpc to P10. Immunostaining and Western blots showed no detectable nestin in any of the experimental and control group brains after P20. These observations of an association between maternal zinc deficiency and decreased nestin protein levels in brains of offspring suggest that zinc deficiency suppresses development of neural stem cells, an effect which may lead to neuroanatomical and behavioral abnormalities in adults.
基金supported by the National Natural Science Foundation of China(Nos.31172266/C1703 and 31402143/C1703)
文摘Based on bioinformatic analysis, we selected two novel micro RNAs（miRNAs）, bmo-miR-0001 and bmomiR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland（PSG）. Firstly, we examined the expression of bmo-miR-0001 and bmo-miR-0015 in 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of Bm Fib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pc DNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pc DNA3.0 [ie1-egfp-pribmo-miR-0015-SV40]. Finally, the Bm N cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly（P〈0.05） in Bm N cells co-transfected by pc DNA3.0 [ie1-egfp-pri-bmomiR-0001-SV40] or pc DNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40] with p GL3.0 [A3-luc-Fib-L-3＇UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of Bm Fib-L in vitro.
文摘AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.
基金Supported by National 863 Project,No.102-07-02-079th Five-Year Sci-Tech Plan,No.96-906A-03-08
文摘AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.
基金Basic Public Welfare Research Foundation of Zhejiang Province,China,No.GD21H290001and Traditional Chinese Medicine Science and Technology Project Foundation of Zhejiang Province,China,No.2020ZB072.
文摘BACKGROUND Sepsis is a major medical challenge.Magnolol is an active constituent of Houpu that improves tissue function and exerts strong anti-endotoxin and anti-inflammatory effects,but the mechanism by which it reduces intestinal inflammation in sepsis is yet unclear.AIM To assess the protective effect of magnolol on intestinal mucosal epithelial cells in sepsis and elucidate the underlying mechanisms.METHODS Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,and regulated on activation,normal T-cell expressed and secreted(RANTES)levels in serum and ileal tissue in animal studies.The histopathological changes of the ileal mucosa in different groups were observed under a microscope.Cell Counting Kit-8 and cell permeability assays were used to determine the concentration of drug-containing serum that did not affect the activity of Caco2 cells but inhibited lipopolysaccharide(LPS)-induced decrease in permeability.Immunofluorescence and Western blot assays were used to detect the levels of RANTES,inhibitor of nuclear factor kappa-B kinaseβ(IKKβ),phosphorylated IKKβ(p-IKKβ),inhibitor of nuclear factor kappa-B kinaseα(IκBα),p65,and p-p65 proteins in different groups in vitro.RESULTS In rats treated with LPS by intravenous tail injection in the presence or absence of magnolol,magnolol inhibited the expression of proinflammatory cytokines,IL-1β,IL-6,and TNF-αin a dose-dependent manner.In addition,magnolol suppressed the production of RANTES in LPS-stimulated sepsis rats.Moreover,in vitro studies suggested that magnolol inhibited the increase of p65 nucleation,thereby markedly downregulating the production of the phosphorylated form of IKKβin LPS-treated Caco2 cells.Specifically,magnolol inhibited the translocation of the transcription factor nuclear factor-kappa B(NF-κB)from the cytosol into the nucleus and down-regulated the expression level of the chemokine RANTES in LPS-stimulated Caco2 cells.CONCLUSION Magnolol down-regulates RANTES levels by inhibiting the LPS/NF-κB signaling pathways,thereby suppressing IL-1β,IL-6,and TNF-αexpression to alleviate the mucosal barrier dysfunction in sepsis.
