The Epstein-Barr virus latent membrane protein 1 (LMP1) oncopro tein causes multiple cellular changes, including activation of the NF-κB trans cription factor. To elucidate its possible mechanism, the interaction between LM P1 and the tumor necrosis factor receptor associated factor (TRAF) molecules was detected by the immunoprecipitation-Western blotting assay. Results showed tha t LMP1 was co-precipitated with TRAF1,2,3 in the LMP1-HNE2 cell line. In the m eantime, κB reporter gene analysis revealed that over expression of TRAF1 or TR AF2 augmented LMP1-mediated NF-κB activation from LMP1, suprisingly, overexpr ession of either TRAF3 or an dominant negative TRAF3 inhibited the NF-κB activ ation, indicating that TRAF1 or TRAF2 is a positive modulator of LMP1-mediated NF-κB activation, whereas,TRAF3 is a negative modulator. Rather both CTAR1 (carboxy-terminal activating region 1) and CTAR2 domains of LMP1 can independently activate NF-κB by interacting with TRAF proteins. These data indicate that LMP1 interacts TRAF1,2,3 which are important for LMP1-mediated N F-κB activation, and further suggest that signaling from TRAFs may be involved in the progression to malignancy in cells of epithelial origin such as nasophar yngeal carcinoma (NPC).

BACKGROUND:Previous study reveals that,glycated albumin plays an important role on the apoptosis of endothelial cells and the expression of monocyte chemoattractant protein-1 through increasing the activity of mitogen activated protein kinase and protein tyrosine kinase,besides generating active oxygen products. OBJECTIVE:To investigate the effects of glycated albumin on the expression of monocyte chemoattractant protein-1 in the cultured human umbilical vein endothelial cells(HUVECs) . DESIGN,TIME AND SETTING:A single sample observation was carried out in the Cardiovascular Laboratory of Southeast University(Nanjing,Jiangsu,China) from May to November in 2006. MATERIALS:ECV304 HUVEC strain was provided from Shanghai Institute of Cell Biology,Chinese Academy of Sciences(China) . METHODS:Experimental procedures were assigned to two parts. On one hand,HUVECs were cultured with glycated albumin of the concentration of 400 mg/L for 0,8,16,24,48,or 72 hours. On the other hand,HUVECs were cultured with glycated albumin of the concentrations of 100,200,400,and 800 mg/L for 24 hours. In the control group,HUVECs were incubated with bovine serum albumin of the concentration of 400 mg/L and RPMI 1640 culture medium without addition of the serum. MAIN OUTCOME MEASURES:The proliferation of HUVECs was estimated by MTT colorimetric assay. The content of monocyte chemoattractant protein-1 in the supernatant was measured by enzyme-linked immunosorbent assay. RESULTS:Glycated albumin inhibited the proliferation rate of HUVECs in a time-dependent manner(P

ObjectiveTo examine the protective effects of 17-β estradiol on the experimental model of spinal cord injury (SCI) rats. Methods One hundred and eighty male Sprague Dawley (SD) rats, after Allen' s model, SD rats were divided into three groups: the sham group, the acute spinal cord injury (control groups) and the acute spinal cord injury supplying with 17-β estradiol treatment group. SCI was made by Allen's weight dropping, impacting on the posteriors of spinal cord T10. The content of malonyldialdehyed (MDA), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by chromatometry. The expressions of Caspase-3 and Bcl-2 family in the injured spinal cord were detected by immunohistochemical staining. Results The BBB scores at each time point in 17-β estradiol treatment group were significantly higher than that in SCI group (P<0.05). The contents of GSH, SOD, GSH-Px and the expression of Bcl-2 protein at the majority of time point in 17-β estradiol treatment group were significantly higher than that in SCI group(P<0.05), however, the MDA, Caspase-3 and Bax were markedly decreased (P<0.05). Conclusions This study suggests that 17-β estradiol administration might prevent the cells from SCI-induced apoptosis by triggering to reduce the oxidative stress.

Objective To study the growth difference and possible mechanism between nasopharyngeal carcinoma (NPC) cell line CNE-2 and its subclone S-18.Methods CNE-2 and S-18 cells were cultured in vitro.6 x 105 cells/mouse were xenografted subcutaneously in the back of nude mice.The volumes of rumors were measured on the 3 rd,7 th,10 th,14 th day after grafting.Mice were sacrificed on the 14 th day and tumors were isolated and weighed.RNA from tumor tissues were extracted and transcriptional levels of HSP27 and NF-K B were detected.Results (1) S-18,instead of CNE-2,grew to form tumor mass 7 days after xenografting subcutaneously;both cell lines formed tumor mass 10 days after xenografting,however,the volumes of S-18 tumors [(223.13 ± 21.32) mm3,10 th day;(420.25 ± 24.52) mm3,14 th day] were significant bigger than CNE-2tumors [(113.70±11.70) mm3,10thday;(279.86±25.78) mm3,14thday];The weights of S-18 umors were significantly higher than CNE-2 tumors on the 14 th day after xenografting;(2) The transcriptional levels of HSP27 and NF-KB in S-18 tumor were significantly higher than in CNE-2 tumor.Conclusion Xenografted S-18 NPC grows faster than Xenografted CNE-2 NPC.HSP27 and NF-κ B are probably involved in the regulation of growth in NPC.

Objective To explore the role of mechanical properties of embryonic neuroepithelial cells in the process of neural tube closure.Methods Neural tube defects (NTDs) mouse model was established by intragastric administration with all-trans retinoic acid at embryo 7.25 day.All the pregnant mice were sacrificed at embryo 9.5 day and 11.5 day,respectively,and the primary neuroepithelial cells were isolated from neural tube tissue of normal and NTDs mice,respectively.The mechanical characteristics of neuroepithelial cells were analyzed using micropipette aspiration technique combined with a standard solid viscoelastic mechanical model.Results The mechanical characteristics of mouse embryonic neuroepithelial cells showed typical viscoelastic solid characteristics.Compared with the control group,the three viscoelastic parameters,i.e.equilibrium modulus,transient modulus and apparent viscosity coefficient,of the neuroepithelial cells in the NTDs group were significantly increased,and the differences were statistically significant (all P＜0.05).However,there was no significant difference in the viscoelastic parameters of the same group between embryo 9.5 d and 11.5 d (all P＞0.05).Conclusion The decrease in the deformability of embryonic neuroepithelial cells may be one of the factors responsible for neural tube closure disorders.

BACKGROUND:Among many transplanted cells,adult autologous bone barrow-derived mononuclear cells have beenused in clinical practice because they are easy to be obtained,without immunological rejection and ethical disputationand other advantages.How to distinguish donor cells from receptors and observe the survival of donor cells following stem cell transplantation still trouble people.OBJECTIVE: superparamagnetic iron oxide (SPIO)particles-labeled bone marrow-derived mononuclear cells from minipigs were used to observe the feasibility of in vivo tracking with magnetic resonance imaging(MRI).DESIGN:A controlled observation experiment.SETTING:Institute of Cardiovascular Disease,Zhongda Hospital Affiliated to Southeast University.MATERIALS:This experiment was carried out in the Institute of Cardiovascular Disease,Zhongda Hospital Affiliated to Southeast University between April 2006 and August 2006.Healthy Chinese minipigs,aged 3 to 4 months,weighing from 20 to 30 kg,were provided by the Experimental Animal Center of Southeast University[SYXK(Su)2002-0012].METHODS: Autologous bone marrow-derived mononuclear cells of minipigs were isolated and cultured. Bone marrow-derived mononuclear cells in the suspension were traced with SPIO particles.Ferrum in the cells were shown by Prussian blue staining, and cell viability was evaluated by trypan blue exclusion method. Eleven minipigs used for preparation of model of myocardial infarction were divided into experimental group(n=9)and control group(n=2).By means of percutaneous left or right cervical artery or femoral artery puncturation, 1.5 to 2.0 mm balloon was used to occlude 1/3 left anterior descending branch,304 to 405 kPa,60 minutes later,ischemic preconditioning was conducted 3 tO 4 times before operation. When pig models of myocardial infarction were successful that was proved by surface electrocardiogram,bone marrow-derived mononuclear cells were percutaneously injected into coronary artery.Coronary arteriography was performed through femoral artery acupuncture at 24 hours after establishing infarction models.Suspension of bone marrow-derived mononuclear cells was perfused into coronary artery with OTW catheter.Then,the injector and OTW catheter for containing cells were rinsed with normal saline containing heparin and infused with the residual cells within 10 minutes.Non-labeled cells were perfused in 2 minipigs of control group by the same method.Postoperatively, bone marrow-derived mononuclear cells were traced by magnetic resonance and compared with Prussian blue-stained myocardial tissue sections.RESULTS: Seven minipigs of experimental group and one minipig of control group were Involved in the final analysis.One of each group was used for preparation of model of myocardial Infarction.One minipig of experimental group died from anesthetic accident before magnetic resonance.①Bone marrow-derived mononuclear cells all were nearly labeled by SPIO particles. Bone marrow-derived mononuclear cells could further proliferate in culture medium containing Fe2O3-PLL without obvious changes of cellular shape. ②T2+WI showed that 5 of 8 models of myocardial infarction presented fuzzy low-echo signal region in peripheral myocardial infarction after transplantation of labeled cells and the low-echo signal disappeared 4 weeks Iater. Ex vivo T2+WI sequence showed there was a dot-distributed low-echo signal region in the peripheral infarction region.③It was found in histological examination that 5 models(cell number over 106) had Prussian blue-positive cells,which distributed the same as those in magnetic resonance signal reducing region.CONCLUSION:SPIO particles-labeled bone marrow-derived mononuclear cells are safe and effective;T2+ WI is sensitive to tracing SPIO particles-labeled bone marrow-derived mononuclear cells;Magnetic resonance can in vivo trace SPIO particles-labeled stem cells transplanted through coronary artery,magnetic resonance signal change is related with the number of stem cells and division growth.

OBJECTIVETo clarify if Epstein-Barr virus encoded LMP1 induces matrix metalloproteinase 9 expression via NF-kappa B or AP-1 signaling pathway, which gives evidence to the elucidation of the mechanism of LMP1- mediated carcinogenesis.

METHODSTo determine whether LMP1 or its mutants contribute to MMP9 production via NF-kappa B or AP-1 transcription factor, MMP9-chloramphenicol acetyl transferase (CAT), NF-kappa B mut 9-CAT, AP-1 mut MMP9-CAT were transfected into human nasopharyngeal carcinoma cells stably expressing LMP1 (HNE2-LMP1) or its mutants, [HNE2-LMP1 (1-185), HNE2-LMP1 (1-231), HNE2-LMP1 delta 187-351] by electroporation technic. The difference of MMP9 reporter activity among those cell lines was detected by CAT assay and expression of MMP9 was determined in nasopharyngeal carcinoma cells stably expressing LMP1 or its mutants by zymographic analysis. In the meantime, efforts were made to demonstrate if LMP1 regulates NF-kappa B or AP-1 activation using reporter gene analysis.

RESULTSIn contrast with vector-transfected cells, MMP9 CAT activity in HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231), HNE2-LMP1 delta 187-351 increased 7.2, 1.3, 3.3, 4.0 times respectively. Zymographic analysis demonstrated that the 92 kDa MMP9 expression was induced in HNE2-LMP1, HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells, whereas it was negative in HNE2-pSG5 and HNE2-LMP1 (1-185) cells. As compared to the HNE2 cells, NF-kappa B or AP-1 reporter activity in HNE2-LMP1 cells were increased 13.8, 8.4 fold respectively. Moreover, In contrast with MMP9 CAT-transfected cells, MMP9 CAT activity in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells were significantly decreased by 18.1% or 16.3%, 35.0% or 33.3%, 29.1% or 26.1% from the original level. However, there was no difference in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-pSG5, HNE2-LMP1 (1-185) cells.

CONCLUSIONIn nasophargyngeal carcinoma, Epstein-Barr virus-encoded LMP1 induces MMP9 transcription and enzymatic activity via an NF-kappa B or AP-1 signaling pathway, which may contribute to invasiveness and metastasis.

Gene Expression ; drug effects ; Herpesvirus 4, Human ; chemistry ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; NF-kappa B ; metabolism ; Nasopharyngeal Neoplasms ; pathology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; pharmacology

OBJECTIVESTo identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-kappaB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).

METHODSA stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.

RESULTSLMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMP1. Moreover, LMP1-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the kappaB site kappaB1 or kappaB5 was disrupted, whereas mutation of kappaB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.

CONCLUSIONLMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-kappaB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.

Apoptosis ; physiology ; Humans ; NF-kappa B ; physiology ; Nasopharyngeal Neoplasms ; physiopathology ; Protein Biosynthesis ; Signal Transduction ; physiology ; TNF Receptor-Associated Factor 1 ; Tumor Cells, Cultured ; Viral Matrix Proteins ; physiology

OBJECTIVE: To study the prevalence and clinical characteristics of decreased myocardial blood flow (MBF) quantified by dynamic computed tomography (CT) myocardial perfusion imaging (MPI) in symptomatic patients without in-stent restenosis. MATERIALS AND METHODS: Thirty-seven (mean age, 71.3 ± 10 years; age range, 48–88 years; 31 males, 6 females) consecutive symptomatic patients with patent coronary stents and without obstructive de novo lesions were prospectively enrolled to undergo dynamic CT-MPI using a third-generation dual-source CT scanner. The shuttle-mode acquisition technique was used to image the complete left ventricle. A bolus of contrast media (50 mL; iopromide, 370 mg iodine/mL) was injected into the antecubital vein at a rate of 6 mL/s, followed by a 40-mL saline flush. The mean MBF value and other quantitative parameters were measured for each segment of both stented-vessel territories and reference territories. The MBFratio was defined as the ratio of the mean MBF value of the whole stent-vessel territory to that of the whole reference territory. An MBFratio of 0.85 was used as the cut-off value to distinguish hypoperfused from non-hypoperfused segments. RESULTS: A total of 629 segments of 37 patients were ultimately included for analysis. The mean effective dose of dynamic CT-MPI was 3.1 ± 1.2 mSv (range, 1.7–6.3 mSv). The mean MBF of stent-vessel territories was decreased in 19 lesions and 81 segments. Compared to stent-vessel territories without hypoperfusion, the mean MBF and myocardial blood volume were markedly lower in hypoperfused stent-vessel territories (77.5 ± 16.6 mL/100 mL/min vs. 140.4 ± 24.1 mL/100 mL/min [p < 0.001] and 6.4 ± 3.7 mL/100 mL vs. 11.5 ± 4 mL/100 mL [p < 0.001, respectively]). Myocardial hypoperfusion in stent-vessel territories was present in 48.6% (18/37) of patients. None of clinical parameters differed statistically significantly between hypoperfusion and non-hypoperfusion subgroups. CONCLUSION: Decreased MBF is commonly present in patients who are symptomatic after percutaneous coronary intervention, despite patent stents and can be detected by dynamic CT-MPI using a low radiation dose.
Angiography ; Blood Volume ; Contrast Media ; Coronary Artery Disease ; Heart Ventricles ; Humans ; Male ; Multidetector Computed Tomography ; Myocardial Perfusion Imaging ; Percutaneous Coronary Intervention ; Prevalence ; Prospective Studies ; Stents ; Veins