Proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus,as demonstrated by the surface enhanced laser desorption/ionization(SELDI)protein chip system

The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorption/ionization （SELDI） protein chip system in order to develop a method of study for the pathogenesis of HCMV infection. In this study, the cultured U251 cells were infected with HCMV in good condition and the supernatants of lysates and the extracellular fluids of the cultivated infected cells were quantitatively defined for the expressed proteins. The proteomics of the differential protein expression in cells before and after infection was analyzed by WCX2 arrays on the protein chip reader. It was demonstrated that the eytopathic effects of infected cells appeared on the 5th day after infection, however, the differential protein expression was evident at 6 h after infection as revealed by RT-PCR and mass spectrometry. The protein peaks captured from different batches of samples, from the same sample detected with different arrays or for the different times were all equivalent. With the molecular weight range from 2000 Da to 3000 Da, chip captured 82 peaks from the intracellular fluids and 11 protein peak from the cellular fluid in which compared with the control group, the protein peaks with molecular weight of 13 536.3 Da, 10 046.1 Da and 17 106.2 Da were close to those of β-amyloid protein, caspase-1 precursor and LPS-induced TNF-α factor respectively, which showed brief up-regulation 4 h after infection, and continued to raise 48 h later. These results infer that these proteins may be related to the apoptosis induced by HCMV infection, thus suggesting that the apoptosis induced by HCMV infection may play a role in the pathogenesis of HCMV infection.

Effect of Human Cytomegalovirus Infection on Nerve Growth Factor Expression in Human Glioma U251 Cells

Objective To explore the change of endogenic nerve growth factor （NGF） expression in human glioma cells infected with human cytomegalovirus （HCMV）. Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD 169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group （P〈0.05）. Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

Apoptosis of Glioblastoma U251 Cells Induced by Carmustine Combined All-trans Retinoic Acid via Regulating Cyclin E and p27kip 1

The effect and mechanism of carmustine（BCNU） combined with all-trans retinoic acid（ATRA） on the apoptosis of human glioblastoma U251 cells were investigated by means of 3-（4,5-dimethylthiazol-2-yl）-2,5-diphe- nyltetrazolium bromide（MTT） assay, flow cytometry, reverse transcription-polymerase chain reaction（RT-PCR） and Western blot analysis. The results show that BCNU or ATRA shows time- and dose-dependent inhibition effects on human glioblastoma U251 cells and the combination of BCNU with ATRA shows an synergistic inhibition effect on human glioblastoma U251 cells, and the combined BCNU and ATRA can significantly inhibit the proliferation of human glioblastoma U251 cells, and induce the apoptosis of them, making the cells arrest in the stage of G1 phase, the stage of S and G2 phases decline, the rate of the apoptosis of human glioblastoma U251 cells increase, the corresponding mRNA expression of cyclin E and cyclin-dependent kinase 2（CDK2） downregulated and the correspon- ding mRNA expression of p27kip 1 unregulated. In addition, the combined BCNU and ATRA reduced the protein expression of nuclear factor kappa B（NF-κB）. Taken together, these results suggest that the treatment of human glioblastoma U251 cells with a combination application of ATRA and BCNU can exert synergistic effect, the course of this kind of combination chemotherapy may likely be associated with multiple molecular mechanisms for apoptosis, furthermore, the cyclin E and p27kip 1 should be considered as novel targets for controlling the growth of glioblastoma cells.

B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium

Previous studies indicated that B7-H4,the youngest B7 family,negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors.Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy.However,the link between B7-H4and tumor stem cells is unclear.In this study,we investigated B7-H4 expression in the medium of human glioma U251 cell cultures.Immunofluorescence results showed that U251 cells cultured in serum-free medium(supplemented with 2%B27,20 ng/mL epidermal growth factor,20 ng/mL basic fibroblast growth factor)maintained stem-like cell characteristics,including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2.In contrast,U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein.Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serumcontaining medium-cultured U251 cells(24%-35%vs.8%-11%,P<0.001).Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of B7-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium.Moreover,conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4expression compared with serum-containing conditioned medium(P<0.01).Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells,and conditioned medium from these cells more effectively induced monocytes to express B7-H4 than conditioned medium from U251 cells cultured in the presence of serum.Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.

Phosphoinositide 3-Kinase/Akt and Nuclear Factor-κB Are Involved in Staphylococcus Aureus-induced Apoptosis in U937 Cells

Objective To explore the mechanisms involved in Staphylococcus aureus （S. aureus） invading human monocytic U937 cells. Methods S. aureus were added to U937 cells at multiplicity of infections （MOI） of 20：1 for 0, 15, 30, 60, and 90 minutes, respectively. Cell apoptosis was analyzed with Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI） flow cytometry analysis. Akt and nuclear factor-κB （NF-κB） activities were detected by Western blotting. Results Infection of U937 cells with S. aureus induced rapid cell death in a time-dependent manner, and the cells displayed characteristic features of apoptosis. S. aureus-induced apoptosis was associated with a prominent downregulation of activated （phosphorylated） Akt and NF-κB. The inhibition of phosphorylated Akt by LY294002 led to the inhibition of NF-κB in a dose-dependent manner. Inhibition of Akt with LY294002 caused further increase in apoptosis of U937 cells. Conclusions S. aureus can stimulate the apoptosis of U937 ceils. S. aureus induces apoptosis of U937 cells by inhibiting Akt-regulated NF-κB.

Bone marrow mesenchymal stem cells transplantation promotes the release of endogenous erythropoietin after ischemic stroke

This study investigated whether bone marrow mesenchymal stem cell（BMSC） transplantation protected ischemic cerebral injury by stimulating endogenous erythropoietin. The model of ischemic stroke was established in rats through transient middle cerebral artery occlusion. Twenty-four hours later, 1 × 106 human BMSCs（h BMSCs） were injected into the tail vein. Fourteen days later, we found that h BMSCs promoted the release of endogenous erythropoietin in the ischemic region of rats. Simultaneously, 3 μg/d soluble erythropoietin receptor（s EPOR） was injected into the lateral ventricle, and on the next 13 consecutive days. s EPOR blocked the release of endogenous erythropoietin. The neurogenesis in the subventricular zone was less in the h BMSCs ＋ s EPOR group than in the h BMSCs ＋ heat-denatured s EPOR group. The adhesive-removal test result and the modified Neurological Severity Scores（m NSS） were lower in the h BMSCs ＋ s EPOR group than in the heat-denatured s EPOR group. The adhesive-removal test result and m NSS were similar between the h BMSCs ＋ heat-denatured s EPOR group and the h BMSCs ＋ s EPOR group. These findings confirm that BMSCs contribute to neurogenesis and improve neurological function by promoting the release of endogenous erythropoietin following ischemic stroke.

Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor （VEGF） and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concentrations （12.5-200 μmol/L） for different time lengths （12-48 h）. The proliferation of the U937 leukemic cells was determined by MTT assay. Apoptosis was observed by Annexin- V-FIFC/PI double staining and flow cytometry （FCM）. Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose- and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resveratrol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGE induce apoptosis and suppress the proliferation of U937 cells.

MiRNA376a/c靶向调控TGFA对CD133^+ U251胶质瘤细胞增殖的影响

目的:探讨微小核糖核酸(Micro RNA,Mi RNA)376a/c靶向调控转化生长因子α(transforming growth factor alpha,TGFA)在CD133+U251胶质瘤细胞增殖中作用方式及其机制。方法:构建Mi R376a/c靶向调控TGFA慢病毒克隆体,根据Mi R376a/c与TGFA结合位点分为4组,检测各组酶活性。根据转染体外Mi R376a/c和Mi R376a/c模拟物(Mi R-SCR)不同,将CD133+U251细胞分为4组,通过细胞培养、Gimsa染色,检测TGFA蛋白表达量和细胞生长曲线,观察细胞增殖情况,收集资料并进行统计学分析。结果:CD133+组与CD133-组Mi R376a/c表达比较,差异有统计学意义(P<0.001);wt-TGFA组与mut-TGFA组质粒荧光素酶活性表达差异比较,差异有统计学意义(P=0.000);将Mi R376a/c慢病毒导入CD133+U251细胞中,Mi R376a/c组TGFA蛋白表达相对于Mi R-SCR组和空白对照组明显下调,差异有统计学意义(P=0.000);生长曲线检测示:随时间延长,Mi R376a/c组细胞生长速度慢于对照组,差异有统计学意义(P=0.000)。结论:Mi RNA376a/c靶向调控TGFA抑制CD133+U251胶质瘤细胞增殖、克隆。

Proteomic profile of human monocytic cells infected with dengue virus

Objective: To identify the changes in the proteome of U937 cells infected with dengue virus(DENV).Methods: In this study, differentiated U937 cultures were infected with two DENV-2strains, one of which was associated with dengue(DENV-2/NG) and the other one with severe dengue(DENV-2/16681), with the aim of determining the cellular proteomic profiles under different infection conditions. Cellular proteins were extracted and separated by two-dimensional electrophoresis, and those proteins with differential expression profiles were identified by mass spectrometry. The obtained results were correlated with cellular viability, the number of infectious viral particles, and the viral DNA/protein quantity.Results: In comparison with non-infected cultures, in the cells infected with the DENV-2/NG strain, nine proteins were expressed differentially(five were upregulated and four were downregulated); in those cultures infected with the DENV-2/16681 strain, six proteins were differentially expressed(two were downregulated and four were upregulated). The downregulated proteins included fatty acid-binding protein, heterogeneous nuclear ribonucleoprotein 1, protein disulfide isomerase, enolase 1, heat shock 70 k Da protein 9, phosphotyrosyl phosphatase, and annexin IV. The upregulated proteins included heat shock 90 k Da protein AA1, tubulin beta, enolase 1, pyruvate kinase,transaldolase and phospholipase C-alpha.Conclusions: Because the monocyte/macrophage lineage is critical for disease pathogenicity, additional studies on these proteins could provide a better understanding of the cellular response to DENV infection and could help identify new therapeutic targets against infection.

Effect of Caffeic acid on the Tumor Cells U937 Evaluated by an Electrochemical Voltammetric Method

Electrochemical voltammetric method can;be used to monitor cell health state during its growth. Here we studied the effect of caffeic acid on leukemia cells U937 by the voltammetric behavior of the cells. The result showed that this drug had a negative influence on cell health. which suggests that caffeic acid may be used in inhibition of tumor cells.

The vascular endothelial growth factor genes expression in glioma U87 cells is dependent from ERN1 signaling enzyme function

The expression of different vascular endothelial growth factor (VEGF) genes was studied in glioma U87 cells with endoplasmic reticulum–nuclei-1 (ERN1) loss of function and its regulation by hypoxia and glutamine or glucose deprivation conditions as model of ischemia. The blockade of function of the ERN1 enzyme, which is a major sensor of endoplasmic reticulum stress, leads to a decrease of the VEGFA, VEGFB and VEGFC mRNA expression level. The level of VEGFA proteins also decreases at this experimental condition in the cytosolic fraction, but increases in the nuclear fraction. Hypoxia does not affect VEGFC and increases the expression level of VEGFA and VEGFB mRNA in both used cell types, however, the change was much less profound in cells with suppressed function of ERN1. The expression level of VEGFC mRNA decreases in both used cell types in glutamine deprivation condition, however, the change was more profound in control glioma cells. At the same time, the expression level of VEGFA mRNA increases and VEGFB—decreases in gluta-mine deprivation condition in control glioma cells only. Exposure of glioma cells to glucose deprivation condition increases VEGFB mRNA expression level in both used cell types;however, VEGFA—in control glioma cells only and VEGFC—in cells with ERN1 signaling enzyme loss of function only. Thus, the results of this study clearly demonstrated the down-regulation of the expression of all three VEGF genes in glioma cells with ERN1 loss of function which correlates to the suppressed angiogenesis and proliferation rate of these cells. Moreover, the effect of hy-poxia and glutamine or glucose deprivation condition on the expression level of all VEGF genes is different and mainly depends on ERN1 signaling enzyme function.

Optimal Bandgap of Double Perovskite La-Substituted Bi2FeCrO6 for Solar Cells:an ab initio GGA+U Study

The ab initio generalized gradient approximation (GGA)+U study of multiferroic (La Bi )2FeCrO6 in pnma structure and ferri-magnetic order, including Hubbard corrections ( eV) for transition metal/rare earth d-electrons with 20 atoms cell, shows optimum local magnetic moments of (Cr , Fe equal to (−2.56, 4.14) μB and an ideal spin-down band gap of 1.54 eV. Tuned-band gap La-substituted double oxide perovskites BFCO should exhibit enhanced visible-light absorption and carrier mobility, thus could be convenient light absorbers and then efficient alternatives to wide-gap chalcopyrite absorber-based solar cells failing to achieve highest power conversion efficiencies, and even compete with their metal-organic halide perovskites counterparts.

Mechanisms for amplified mediator release from colonic mast cells:Implications for intestinal inflammatory diseases

The mast cell is an enigmatic cell type whose physiological function has preoccupied large numbers of investigators for decadest. Some have concluded that the absence of mast cells is incompatible with life, at least in humans, because no human conditions have been documented where these cells are absent from the body. On the other hand, mice harboring specific mutations in certain growth factors, or their receptors, that

Effect of Hypoxia on the Expression of a Subset of Proliferation Related Genes in IRE1 Knockdown U87 Glioma Cells

We have studied the expression of a subset of genes encoding important tumor growth related factors in U87 glioma cells with IRE1 (inositol requiring enzyme-1) knockdown as well as their hypoxic regulation. It was shown that the expression levels of activating transcription factor 6 (ATF6), clusterin (CLU), adhesion G protein-coupled receptor E5 (ADGRE5), transglutaminase?2, C polypeptide (TGM2), leukemia inhibitory factor (LIF), phosphoserine aminotransferase 1 (PSAT1), glyoxalase I (GLO1) and tetraspanin 13 (TSPAN13) are significantly down-regulated in glioma cells with the knockdown of IRE1 signaling enzyme. It was also shown that in glioma cells subjected to hypoxia, the expression levels of PSAT1, TSPAN13, EIF2AK3, and TGM2 genes were up-regulated, whereas the expression of ATF6 gene was down-regulated. At the same time, the expression levels of LIF, CLU, and ADGRE5 genes did not change in response to hypoxic treatment.?Furthermore, inhibition of IRE1, a key effector of an unfolded protein response pathway, modified the effect of hypoxia on the expression of most studied genes. Present study demonstrates that IRE1 knockdown down-regulated the expression of most studied genes and modified their hypoxic regulation and that these changes possibly contributed to the suppression of glioma growth in cells without IRE1 signaling enzyme function.

Suppression mechanisms on proliferation of glioma U251 cells by FePt nanoparticles through intracellular oxidative stress

FePt nanoparticles(NPs)have attracted a great deal of attention in biomedical applications due to their excellent superparamagnetic property and anticancer activity,which display promising potential as the MRI/CT contrast imag-ing agent for cancer detection and therapy.In this study,the suppression mechanism induced by FePt NPs on U251 cells is investigated.Glioma U251 cells are co-cultured with FePt NPs at different concentrations,and the intra-cellular oxidative stress was studied by detecting the activities of various redox enzymes and expression of several apoptosis-related proteins.The cell viability assay shows that the highest suppression on the proliferation of glioma U251 cells is 10.5%±5.6%after treatment with 5μg·ml^(-1)FePt NPs for 24 h.In this case,the LDH release remarkably increases.Flow cytometry and fluorescence assay show that FePt NPs promote the apoptosis and G0/G1 phase arrest of U251 cells.

膜型MHC-Ⅰ类分子高表达的GBM U251细胞疫苗的研制及其体外抗瘤作用

目的探索膜型MHC-Ⅰ类分子高表达人多形性胶质母细胞瘤(GBM)U251细胞疫苗的制备方法及其体外诱导的抗瘤作用机制。方法U251胶质瘤细胞加入0~2000U·mL^(-1)不同浓度梯度的重组人IFN-γ干预48h;流式细胞仪(FCM)检测不同浓度梯度的重组人IFN-γ诱导后的U251细胞胞膜MHC-Ⅰ类分子表达变化情况;取诱导MHC-Ⅰ类分子高表达最高的IFN-γ干预方案作为疫苗的制备方法;体外刺激健康捐献者PBMCs作为效应细胞,MTT比色法检测其对野生型及IFN-γ诱导48h后靶细胞的特异性杀伤活性,同时以抗人HLA-A,B,C行特异性杀伤试验的阻断试验;ELISA法检测效应细胞攻击靶细胞后的IFN-γ、IL-2的分泌情况;FCM检测疫苗刺激前后PBMCs CD4^+、CD8^+T淋巴细胞比例变化。结果 500U·mL^(-1)IFN-γ诱导48h U251细胞胞膜的MHC-Ⅰ类分子表达率最高,达89.9%;高表达膜型MHC-Ⅰ类分子U251细胞疫苗9在体外具有明显特异性的杀瘤活性的诱导能力(P<0.05);体外可分泌大量的IFN-γ和L-2(P<0.05);CD4+、CD8+明显增高(P<0.05)。结论500U·mL^(-1) IFN-γ诱导48h是U251细胞胞膜MHC-Ⅰ类分子体外诱导表达的最佳方案;膜型MHC-Ⅰ类分子高表达的U251细胞疫苗休外具有特异性的抗瘤作用,其杀瘤活性与膜型MHC-Ⅰ类分子表达上调有关。

HCMV Infection Depress NGF Expression in Human Glioma Cells

Human cytomegalovirus （HCMV） is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia ceils by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.

Inhibiting effect of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides on VEGF expression in U937 cells

Objective:To study the effect of VEGF antisense oligodeoxynucleotide(VEGF ASODN)on VEGF expression in acute monocyte leukemic cell line U937 in vitro.Methods:U937 cells were incubated with VEGF ASODN(final concentra-tion as follows:10,20 and 30 μmol/L respectively)or scrambled sequence,compared with negative control.The expression of VEGF mRNA was measured by semi-quantitative RT-PCR,VEGF protein was measured by Western blot.Results:VEGF ASODN obviously inhibited expression of VEGF mRNA in U937 cell,compared with scrambled sequence and negative control(P<0.05).And the inhibition effect was most remarkable after 24 h,which is related with the dose of VEGF ASODN(P<0.05).Scrambled sequence groups had no significant difference compared with negative control groups(P>0.05).VEGF ASODN obvi-ously inhibited expression of VEGF protein,compared with scrambled sequence and negative control(P<0.05).Conclusion:The expressions of VEGF at mRNA and protein levels in leukemic cell line U937 are down-regulated after being treated with VEGF ASODN.

Thermal Diversities of Two Na^+/H^+ Exchanges in Guinea Pig Red Cells

Objective\ To test the effect of hypothermia on Na+/H+ exchange, activated by shrinkage and cytoplasmic acidosis. Method\ Amiloride-sensitive Na+ influx in guinea pig red cells was traced with isotope 22Na and intracellular Na+ concentration was measured by emission flame photometry. Result\ Amiloride-sensitive Na+ influx decreased linearly as a function of temperatures (about 37℃) in shrunken cells, but increased in acidified cells. The up-regulation of acid-induced Na+/H+ exchange by elevated temperature was enhanced by hypo-osmolarity. Less sensitivity of intracellular H+ site at 41℃ may be the mechanism for the inhibition of shrinkage-induced Na+/H+ exchange by elevated temperature. Heating-mediated explosive increase in the activity of acid-induced Na+/H+ exchange may be due to enhanced extracellular Na+ sensitivity and lower intracellular pH caused by acidic metabolites. Acid-induced Na+/H+ ewxchange contributes to cytoplasmic Na+ accumulation. Conclusion\ These two modes of Na+/H+ exchange with different response to elevated temperature may play different roles in the cellular pathogenesis of heatstroke.

Phagocytosis of IgA Immune Complexes by Human U937 Cells

In order to study FcαRⅠ mediated phagocytosis of IgA immune complexes by U937 cells, antigen 8.9NIP/BSA was labeled with FITC and reacted with anti-NIP IgA or anti-NIP IgG antibody to form immune complexes (ICs). They were then incubated with phorbol 12-myristate 13-acetate (PMA) stimulated U937 cells.The phagocytosed ICs were quantified by flow cytometry. The results was that the expression of FcαRⅠ on U937 cells was higher than that of FcγRⅠ, FcγRⅡ and FcγRⅢ. After stimulation by PMA, expression of FcαRⅠ on U937 cells was markedly upregulated and the phagocytosis of IgA ICs was enhanced. FcαRⅠ mediated specific IgA phagocytosis was stronger than FcγRⅠ and FcγRⅡ mediated IgG phagocytosis. Complement receptors, CR1 and CR3, enhanced U937 cell phagocytosis of IgA ICs. It concludes that FcαRⅠ mediated strong phagocytosis of IgA ICs.