目的 探讨姜黄素(CUR)对纤维蛋白肽A(FPA)诱导血管内皮细胞炎症反应的作用机制.方法 ①体外实验:培养人脐静脉血管内皮细胞(HUVEC),筛选CUR对FPA诱导HUVEC功能障碍的最佳药物浓度,采用CCK-8检测细胞活力,透射电镜观察细胞形态,激光共聚焦显微镜观察活性氧(ROS)水平,ELISA法检测上清中IL-6,IL-1β和CRP炎症因子的含量,免疫荧光检测核因子κB(NF-κB)的活化,Western印迹法检测MAPK信号通路蛋白分子的表达.②体内实验:大鼠体内微渗透泵复制高FPA血症大鼠模型,采用ELISA试剂盒检测血浆中FPA,IL-6,IL-1β和CRP的含量,免疫荧光检测血管内皮脱落情况以及ROS的表达情况,血管肌动扫描实验检测血管内皮功能,Western印迹法检测MAPK,NF-κB信号通路蛋白分子的表达.结果 ①体外实验:姜黄素预处理HUVEC 1 h后,CUR 5,25和50μmol·L-1组细胞活力无影响;与FPA组比较,CUR组细胞较完整,空泡较少,线粒体形态较好,线粒体膜电位有明显升高作用,ROS表达水平显著降低(P<0.05),p-ERK1/2和p-P38蛋白表达量显著下调(P<0.05),NF-κB活化水平明显降低.②体内实验:CUR组干预后能够改善血管内皮功能障碍及内皮脱落情况,血浆中FPA,IL-6,IL-1β和CRP的含量显著降低(P<0.05),血管ROS的表达降低,p-ERK1/2、p-P38和NF-κB蛋白表达量显著下调(P<0.05).结论 姜黄素具有抑制FPA诱导血管内皮细胞炎症反应的作用,其机制与调节ROS-MAPK-NF-κB通路有关.

BACKGROUND:Cervical carcinoma is the second most common cancer and is an important cause of death in women worldwide. CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that regulate cellular differentiation and proliferation in a variety of tissues. However, the role of C/EBPα gene in cervical cancer is still not clear.METHODS:We investigated the expression of C/EBPα gene in cervical squamous cell carcinoma. C/EBPα mRNA level was measured by real-time quantitative RT-PCR in cervical cancer tissues and their adjacent normal tissues. C/EBPα protein level was measured by immunohistochemistry. Methylation in the promoter of C/EBPα gene was detected by MALDI TOF MassARRAY. We transfected HeLa cells with C/EBPα expression vector. C/EBPα expression in HeLa cells was examined and HeLa cell proliferation was measured by MTT assay and HeLa cells migration was analyzed by matrigel-coated transwell migration assays.RESULTS:There were significant difference in C/EBPα protein expression between chronic cervicitis and cervical carcinoma (P < 0.001). CEBPα mRNA level was significantly lower in cervical cancer tissues than in normal cervical tissues (P < 0.01). Methylation of the promoter of CEBPα gene in CpG 5, CpG-14.15, CpG-19.20 were significantly higher in cervical cancer than in normal cervical tissues (P < 0.05, P < 0.01, P < 0.05, respectively). CEBPα pcDNA3.1 construct transfected into HeLa cells inhibited cell proliferation and decreased cell migration.CONCLUSIONS:Our results indicate that reduced C/EBPα gene expression may play a role in the development of cervical squamous cell carcinoma.

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A–B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A–B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.

BACKGROUND: Very low density lipoprotein receptor is regarded as a Swiss Army Knife receptor, and many researchers have studied this receptor. But there are only several kinds of commercial antibodies and the price is on the high side. OBJECTIVE: To get the monoclonal antibody against the c-terminal of very low density lipoprotein receptor for the future study. METHODS: In order to obtain the very low density lipoprotein receptor, hybridoma IgG 6A6 cel s related with very low density lipoprotein receptor intracel ular domain was injected into peritoneal of Balb/c mice. After 2 weeks, ascites was col ected, and the target protein was preliminary purified by salting-out method (n-octanoic acid-saturated ammonium sulfate precipitation method) and further purified by affinity chromatography, and the monoclonal antibody related with very low density lipoprotein receptor intracel ular domain was obtained. The relative molecular mass and purity were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the activity was detected by enzyme-linked immunosorbent assay. RESULTS AND CONCLUSION: Western Blotting results showed that the antibody from the ascites directly can bind the very low density lipoprotein receptor protein, but the background was not clear. The purified antibody from the ascites through salting-out method (n-octanoic acid-saturated ammonium sulfate precipitation method) and then by protein A affinity chromatography can bind specifical y with the very low density lipoprotein receptor. The pure and activity monoclonal antibodies can be obtained through purification by the salting-out method and Protein A Agarose.

Bacillus cereus strains containing voitoxin cereulide are pathogenic bacteria. The cereulide is produced by B. cereus strains containing plasmid pCER270. To establish a specific loop-mediated isothermal amplification (LAMP) method detecting pathogenic B. cereus, LAMP primer sets were designed based on 16SrDNA and pCER270 plasmid cesA sequence. The LAMP reaction was carried out under optimal conditions with 2 mM of Mg2+ at 65C for 1 h. Specificity of LAMP primers were validated by assaying 19 B. cereus strains with and without pCER270 plasmid and 42 non-B. cereus species. Detection limits of the LAMP assay were 1 cfu/mL of B. cereus without pCER270 plasmid and 11 cfu/mL of pathogenic B. cereus with pCER270 plasmid. Furthermore, liquid milk samples were detected by the LAMP method. The results showed that LAMP method is a reliable method used for rapid detection of B. cereus and its pathogenic strains of milk and other food stuff.

Failure reasons on the fracture of 40Cr steel earring bar were analyzed through fractography observation,microstructure andchemical composition analysis,mechanical property tests and a follow-up confirmatory experiment.The results show that the failure bar wasnot processed by thermal refining treatment and lots of ferrite networks distributed in the microstructure of the failure part,leading to the lowmechanical properties less than the requirement of GB3077.It is found that the cracks are prone to initiate on the thread roots with stressconcentration,which is the main failure reason.Furthermore,lots of white flakes were found in the failure part with low magnificationinspection.

OBJECTIVE:To study correlation between urinary retinol binding protein (RBP) content and renal tubular damage.METHODS:A total of 1 353 healthy people and 186 patients with renal tubular damage diagnosed by renal biopsy were enrolled. The indicators such as endogenous creatinine clearance rate (Ccr), creatinine(Cr), urinary retinol binding protein(RBP), urinary β(2)-microglobulin(β(2)-MG), urinary N-acety1-beta-D-glucosaminidase (NAG), urine specific gravity(SG), urine osmolality of the 2 groups were examined and compared. Score of tubulointerstitial impairing and all indicators were analyzed by Spearman rank correlation analysis, and the sensitivity and specificity of indicators were calculated.RESULTS:Renal tubular damage was positively correlated with urinary RBP, β2-MG, NAG (r=0.863, P<0.001; r=0.777, P<0.001; r=0.374, P=0.002, respectively), while negatively correlated with urine osmolaling, SG (r=-0.519, P<0.001; r=-0.624, P<0.001, respectively). The specificity and sensitivity for renal tubular damage of RBP were 91.03% and 72.06%.CONCLUSION:RBP is an idea marker for renal tubular damage, and is useful to diagnose renal tubular damage and assess the extent of the damage.

The reported data indicate that oleic acid (OA) decreases cholesterol absorption. To explore the underlying mechanisms, the effects of OA on the expression of cholesterol transport-related proteins (NPC1L1, ABCG5/8, ACAT2, MTP) and the unfolded protein response (UPR) pathway were studied in CaCo-2 enterocytes by incubating CaCo-2 cells with taurocholate micelles or taurocholate micelles containing different concentrations of OA (0.25–1.0 mM). We show that OA effectively induces XBP1 mRNA splicing, a key component of the UPR signaling, and the expression of BiP and mature ATF6 proteins in a concentration-dependent manner, leading to the induction of endoplasmic reticulum (ER) stress and activation of the UPR. Interestingly, OA decreases NPC1L1 expression in a dose-dependent manner while it has no effects on ABCG5 and MTP mRNA level or SREBP-2, ABCG8, and ACAT2 protein level. In CaCo-2 cells treated with 1.0 mM OA, both the NPC1L1 mRNA level and the NPC1L1 protein expression in brush-border membrane fractions were decreased by 39% and 37%, respectively (P < 0.01). A dose of 1 mM dithiothreitol (DTT), a positive control for ER stress induction, also decreases NPC1L1 mRNA and protein expression by 27% and 23%, respectively (P < 0.05). Furthermore, 4-phenyl-butyric acid, an UPR inhibitor, blocks OA- and DTT-induced reduction on NPC1L1 mRNA and protein levels. The results suggest that OA down-regulates NPC1L1 mRNA and protein expression via the induction of the UPR, which may play an important role in reducing intestinal cholesterol absorption.

BACKGROUND:Very low density lipoprotein receptor (VLDLR) has been considered as a multiple function receptor due to binding numerous ligands, causing endocytosis and regulating cellular signaling. Our group previously reported that enhanced activity of type II VLDLR (VLDLR II), one subtype of VLDLR, promotes adenocarcinoma SGC7901 cells proliferation and migration. The aim of this study is to explore the expression levels of VLDLR II in human gastric, breast and lung cancer tissues, and to investigate its relationship with clinical characteristics and β-catenin expression status.METHODS:VLDLR II expression was examined using immunohistochemistry (IHC) and Western blot in tumor tissues from 213 gastric, breast and lung cancer patients, tumor adjacent noncancerous tissues by same methods. Correlations between VLDLR II and clinical features, as well as β-catenin expression status were evaluated by statistical analysis.RESULTS:The immunohistochemical staining of VLDLR II showed statistical difference between tumor tissues and tumor adjacent noncancerous tissues in gastric, breast and lung cancers (P = 0.034, 0.018 and 0.043, respectively). Moreover, using Western, we found higher VLDLR II expression levels were associated with lymph node and distant metastasis in gastric and breast cancer (P < 0.05). Furthermore, highly significant positive correlations were found between VLDLR II and β-catenin in gastric cancer (r = 0.689; P < 0.001)breast cancer (r = 0.594; P < 0.001).CONCLUSIONS:According to the results of the current study, high VLDLR II expression is correlated with lymph node and distant metastasis in gastric and breast cancer patients, the data suggest that VLDLR II may be a clinical marker in cancers, and has a potential link with β-catenin signaling pathway. This is the first to reveal the closer relationship of VLDLR II with clinical information.

PTD4-apoptin protein enters cells and harbors tumor-selective cell death activity. Dacarbazine is the mainstay of treatment for malignant melanoma. In this study, we investigated the cytotoxic effect of PTD4-apoptin protein and/or dacarbazine in mouse B16-F1 and human A875 and SK-MEL-5 melanoma cells in vitro and by means of a mouse B16-F1 melanoma model in vivo. PTD4-apoptin protein inhibits the growth of B16-F1, A875 and SK-MEL-5 melanoma cells in a dose-dependent manner, but not in normal human cell lines WI-38 and L-02. PTD4-apoptin combined with dacarbazine revealed a synergistic cytotoxic effect (coefficient of drug interaction<1) in all three different tumor cell lines. In vivo, PTD4-apoptin protein and dacarbazine alone effectively inhibited the growth of B16-F1 melanoma in C57BL/6 mice. Strikingly, combined PTD4-apoptin/dacarbazine treatment significantly increased the antitumor effect in comparison to the single treatments. As important, a combined PTD4-apoptin/dacarbazine treatment with a 50% reduction of dacarbazine revealed similar antitumor activities, without detectable hematologic side effects. A combined PTD4-apoptin/dacarbazine treatment represents a promising novel efficient and safe anticancer strategy.

This study examined the effect of insulin on the expression of very low density lipoprotein receptor (VLDLR) subtypes of SGC7901 cells and discussed its biological implication. In vitro, moderately or poorly-differentiated human gastric adenocarcinoma cell line SGC7901 was incubated with insulin for different lengths of time, and then the expression of protein and RNA level in VLDLR subtypes were detected by Western blotting and real-time PCR, respectively. The results showed that, at certain time interval, insulin could down-regulate expression of type I VLDLR and up-regulate the expression of type II VLDLR in SGC7901 cells, at both protein and RNA level. We are led to conclude that insulin serves as a regulator in maintaining the balance between glucose and lipid metabolism in vivo, possibly through its effect on the differential expression of VLDLR subtypes.

Very low density lipoprotein receptors (VLDLR) including type I and type II are known to affect cell functions by binding to its extracellular ligands. However, the effect of these ligands on VLDLR expression remains elusive. Tissue factor pathway inhibitor (TFPI) and urokinase plasminogen activator and plasminogen activator inhibitor 1 (uPA-PAI-1) complex, two ligands of VLDLR, were used to examine their effects on VLDLR expression. TFPI treatment decreased type II VLDLR expression, inhibited cell proliferation and migration, and degradated beta-catenin in SGC7901 cells. However, uPA-PAI-1 complex, increased type II VLDLR expression with promoted cell proliferation and migration and stabilization of beta-catenin. These results indicated that extracellular ligands can change the expression of type II VLDLR to affect cell proliferation and migration.

Background and Aim:  Chinese traditional medical science is generally used as a therapeutic method against functional dyspepsia (FD) in China. Although great effort is made to understand the pharmaceutical mechanisms of Chinese traditional medicine, such as typical traditional Chinese medicine, Wei Kangning, there are still many mysteries to be uncovered.

Reported data indicate that cholesterol loading in the liver can cause hepatic injury. To explore the possible mechanisms of cell damage resulting from cholesterol overloading in hepatocytes, cell apoptosis, the unfolded protein response (UPR) and the correlation between them were assessed in the cholesterol-overloaded normal human hepatic cell line L02. L02 cells were incubated with 200 microg/ ml of low density lipoprotein (LDL) for 24 h with or without 20 microg/ml 58035, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). In the LDL+58035 group, the intracellular cholesterol level was dramatically increased, which was measured by an enzymatic combined high performance liquid chromatography assay. Expression of immunoglobulin-binding protein, X-box binding protein 1, activating transcription factor 6, activating transcription factor 4, CCAAT/enhancer-binding protein homologous protein-10, markers of endoplasmic reticulum stress (ERS)/ UPR, were up-regulated as determined using reverse transcription-polymerase chain reaction (RT-PCR) or Western blot analysis. The rate of cell apoptic death increased 21.3+/-2.4%. Meanwhile, the active caspase-3 protein expression was increased 8.4-fold compared to the active caspase-3 protein expression in the controls. Furthermore, 4-phenylbutyric acid, an inhibitor of UPR, partly reduced cell apoptosis and activation of caspase-3. This study suggests that cholesterol overloading in hepatic L02 cells induces ERS and activates the UPR which, in part, leads to the apoptotic damage of cells.

It was to clone and express protein G IgG Fc binding domain(PGFB)for purifying antibody.Four oligonucleotide fragments with the selected E.coli-preferred codon were designed and synthesized according to the amino acid sequence of PGFB.Then the gene of PGFB was synthesized by overlap extension PCR,and cloned into pET-28a-c(+) plasmid.The recombinant protein was purified by a single step affinity chromatography with Ni+-NTA,then it was coupled with Sepharose 6B to purify polyclonal antibody.Results showed PGFB was expressed highly in E.coli BL21(DE3),the purity of which reached 90% with expected molecular weight of 12.25 kD.PGFB coupled with Sepharose 6B which can purify polyclonal antibody.The binding capacity reached 20 mg/mL.Therefore,highly expressed PGFB would provide convenience for the rapid purification of polyclonal antibody.

Background RhoA and RhoC are deregulated by over expression in many human tumors, including colorectal cancer. Some reports show that they play a pivotal role in the carcinogenesis, tumor development and infiltration metastasis. In this study, for the first time we constructed recombinant adenovirus to investigate the inhibitory effects of RhoA and RhoC shRNAs in tandem expression on the cell proliferation and invasion of colorectal cancer HCT116 cells. Methods The recombinant adenovirus carrying RhoA and RhoC shRNAs in tandem expression was transfected into HCT116. The mRNA transcription and protein expressions of RhoA and RhoC were examined by RT-FQPCR and Western blot respectively. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium (MTT) assay and invasive and migrating potential was detected through in vitro Matrigel coated invasion and migration assay. Results Both mRNA and proteins Levels of RhoA and RhoC were significantly reduced in HCT116 cells transfected with Ad-A1+A2+C1+C2 than those in Ad-HK group and control one. The relative RhoA and RhoC mRNA transcriptions were decreased to 40% and 36% (P < 0.05), while proteins expression reducing 42% and 35%, respectively (P < 0.05). Growth curves analysis showed that alive cell number in the Ad-A1+A2+C1+C2 group was lower than others in the third to sixth day and transwell chamber analysis showed that migration/invasion activity was significantly suppressed in Ad-A1+A2+C1+C2 group. Conclusion Our results indicate recombinant adenovirus carrying RhoA and RhoC shRNAs in tandem expression may inhibit the growth and invasion of HCT116 cells. Application of such vector to inhibit one or more genes may be a new method to cancer therapy.

In hypertriglyceridaemic individuals, atherosclerogenesis is associated with the increased concentrations of very low density lipoprotein (VLDL) and VLDL-associated remnant particles. In vitro studies have suggested that VLDL induces foam cells formation. To reveal the changes of the proteins expression in the process of foam cells formation induced by VLDL, we performed a proteomic analysis of the foam cells based on the stimulation of differentiated THP-1 cells with VLDL. Using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, 14 differentially expressed proteins, containing 8 up-regulated proteins and 6 down-regulated proteins were identified. The proteins are involved in energy metabolism, oxidative stress, cell growth, differentiation and apoptosis, such as adipose differentiation-related protein (ADRP), enolase, S100A11, heat shock protein 27 and so on. In addition, the expression of some selected proteins was confirmed by Western blot and RT-PCR analysis. The results suggest that VLDL not only induces lipid accumulation, but also brings about foam cells diverse characteristics by altering the expression of various proteins.

Apoptin protein harbors tumor‐selective cell death activity, which makes it a potential anticancer therapy candidate. This study reports an apoptin therapy approach based on protein transduction domain 4 (PTD4)‐mediated transduction of recombinant apoptin protein. In vitro , the PTD4‐apoptin fusion protein is located in the nucleus and induces cell death in, e.g ., human hepatocarcinoma HepG2 cells. In normal human L‐02 hepatocytes, PTD4‐apoptin protein retained mainly cytoplasmic and did not induce detectable levels of cell death, illustrating that the PTD4 domain does not affect apoptin's tumor‐selective characteristics. In vivo , liver, cervix and gastric carcinoma xenografts treated with PTD4‐apoptin protein for 6 days via the tumor epidermis exhibited a significant tumor growth inhibition because of apoptin‐mediated cell death. In addition, treatment of human hepatocarcinoma xenografts during 3 weeks showed that PTD4‐apoptin protein has significant anticancer activity, whereas control treatment with PTD4‐enhanced green fluorescence protein or saline did not. Cell death and disruption of the tumor integrity were apparent in the PTD4‐apoptin transduced xenografted tumors. As important, although PTD4‐apoptin protein could be detected in the epidermal tissue covering the subcutaneous tumor tissue and in several organs, such as liver and brain, of the treated mice, no tissue disruption or signs of cell death could be detected. Our in vivo data reveal that apoptin protein delivery constitutes a novel powerful and safe anticancer therapy. © 2009 UICC

Very low density lipoprotein receptor （VLDLR） is thought to participate in the patho- genesis of atherosclerosis induced by VLDL and β-VLDL. The present study was undertaken to elu- cidate the effects of VLDL and β-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and β-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway in- volving VLDLR expression regulation. The results showed that VLDL and β-VLDL stimulated ERK1/2 activity in a PKC-dependent manner. VLDL or β-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERK1/2 or PKC. Our findings revealed that VLDL or β-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.

Objective To construct the expression vector of VLDL-R's ligand binding domain fusion protein and study its expression and purification in E.coli.Methods The DNA fragment of LBR1-8 was amplified from VLDL-R cDNA recombinant plasmid by PCR and was inserted into plasmid pET28a(+)to construct the prokaryotic expression plasmid pET28a(+)-LBR1-8.The positive recombinant plasmid was transformed into E.coli strain Rosetta and then was induced by IPTG,with the expression conditions optimized.The fusion protein was purified by Ni+-NTA chromatography column,analyzed and identified by SDS-PAGE and Western blotting.Result The recombinant expression plasmid was identified by PCR,digested with restricted endoenzymes and subjected to DNA sequencing.It was found to be consistent with the predicted result.The host cells containing the recombinant plasmid that successfully expressed LBR1-8 after being induced by IPTG and the optimal inducing conditions were determined.Conclusion The prokaryotic expression and plasmid pET28a(+)LBR1-8 was successfully constructed and the target recombinant protein LBR1-8 was expressed and purified,which lays a foundation for the study on the functions of VLDL-R function and its application,especially on its ligand recognition and binding characteristics.