Objective: To investigate the mechanism underlying the promoting effects of epidermal growth factor receptor mutation type III (EGFRv III) on the migration of glioma cells. Methods: Human glioma cell line U87EGFRv III was established by retroviral infection of the parental glioma cells. The effect of EGFRv III on the migration of the cells was detected by gel-drop assay and Transwell migration assay. The phosphorylation of FAK was detected by Western blotting. The FAK Y397F mutant was transfected into U87EGFRv III cells and the change in cell migration was observed. Results: EGFRv III promoted the migration of human glioma U87 cells and increased the FAK phosphorylation level at Tyr397. Transfection with FAK Y397F mutant reduced FAK phosphorylation level in glioma cells U87EGFRvH[ and induced the decrease in the migration abilities of the cells. Conclusion: EGFRv III promoted the migration of glioma cells by up-regulating FAK phosphorylation levels.

Objective To explore the effects of thiamine and riboflavin on H2O2-induced DNA oxidative damage in human umbilical vein endothelial cell line ECV304.Methods ECV304 cells were incubated with 10,100,500,1000 mg/L of thiamine or 20,100,300,500 nmol/L of riboflavin for 24 h,and then oxidative damage of cells were induced by 25 mol/L H2O2 for 30 min.DNA damage was detected with single cell gel electrophoresis(SCGE)assay.ECV304 cells incubated without H2O2,thiamine and riboflavin were served as negative controls,and those incubated with H2O2 and without thiamine and riboflavin were served as positive controls.Results H2O2 induced DNA damage,and the indices of percent of DNA damage cells,percent of tail DNA,tail length and Olive tail moment were increased.The indices of cells pretreated with 10,100,500 mg/L of thiamine or 20,100,300 nmol/L riboflavin were significantly decreased(P0.05).Conclusion Proper supplementation of thiamine and riboflavin may decrease H2O2-induced DNA oxidative damage,while excess thiamine and riboflavin supplementation may be harmful to DNA and enhance the susceptibility to H2O2 potentially.

GJ-4 is crocin enrichments extracted from Gardenia jasminoides J. Ellis, and our previous studies have shown that GJ-4 significantly improved learning and memory impairment induced by Aβ in mice. Herein, a memory deficit model was developed by injecting okadaic acid (OA) into the lateral ventricle of mice, and the neuroprotection and underlying mechanism of GJ-4 on neuronal injury caused by Tau hyperphosphorylation were investigated. The Animal Care & Welfare Committee, Institute of Materia Medica, CAMS & PUMC has approved all procedures (No.00000318). GJ-4 at different doses was intragastric administration to mice for 16 days. Step-down test and Morris water maze test showed that GJ-4 could significantly improve OA-induced memory impairment in mice, and reduced the loss of Nissl bodies in the hippocampus of mice. GJ-4 could also decrease the phosphorylation level of Tau protein at Ser396, Thr231 and Ser404 via increasing protein phosphatase 2A (PP2A) activity and inhibiting glycogen synthase kinase-3β (GSK-3β) activity. Besides, further researches indicated that GJ-4 could inhibit the level of oxidative stress in the brain of OA mice, reduce neuronal apoptosis and inhibit the neuroinflammation mediated by activation of astrocytes in the hippocampus of mice, and eventually achieve its effects in improving learning and memory impairment in mice. According to these findings, we anticipated that GJ-4 might be a potential therapeutic drug for Alzheimer's disease.

The cis and trans isomers separation of 2-butene-1,4-diol and lafutidine were studied by HPLC on two kinds of chiral columns: (S,S)-Whelk-O 1 and ChiraSpher. The isomers of 2-butene-1,4-diol can be separated on both chiral columns while the isomers of lafutidine can only be resolved on ChiraSpher column. The influence of different type and amount of mobile phase modifier on the isomers separation was extensively studied. The resolution of cis and trans isomers of 2-butene-1,4-diol was 2.61 on (S,S)-Whelk-O 1 column with hexane-ethanol (97:3, v/v) as the mobile phase. The resolution of lafutidine was 1.89 on ChiraSpher column with hexane-ethanol-THF-diethylamine (92:3:5:0.1, v/v/v/v) as the mobile phase. LC-MS methods were developed to identify the isomer peaks.
Acetamides ; analysis ; chemistry ; Butylene Glycols ; analysis ; chemistry ; Chemical Fractionation ; methods ; Chromatography, High Pressure Liquid ; methods ; Isomerism ; Mass Spectrometry ; methods ; Piperidines ; analysis ; chemistry ; Pyridines ; analysis ; chemistry

This paper is aimed to study the effects of nitrogen form on the growth and quality of Chrysanthemums morifolium at the same nitrogen level. In order to provide references for nutrition regulation of Ch. morifolium in field production, pot experiments were carried out in the greenhouse at experimental station of Nanjing Agricultural University. Five proportions of ammonium and nitrate nitrogen were set up and a randomized block design was applied four times repeatedly. The results showed that the growth and quality of Ch. morifolium were significantly influenced by the nitrogen form. The content of chlorophyll and photosynthesis rate were the highest at the NH4(+) -N /NO3(-) -N ratio of 25:75; The activities of NR in different parts of Ch. -morifolium reached the highest at the NH4(+) - N/NO3(-) -N ratio of 0: 100. The contents of nitrate nitrogen in the root and leaves reached the highest at the NH4(+) -N/NO3(-) -N ratio of 50:50. The activities of GS, GOGAT and the content of amylum increased with the ratio of NO3(-) -N decreasing and reached it's maximum at the NH4 + -N/NO3 - -N ratio of 100: 0. The content of ammonium nitrogen were the highest at the NH4 + -N /NO3 --N ratio of 75: 25, while the content of soluble sugar reached the highest at the NH4(+)-N/NO3(-) -N ratio of 25: 75. The content of flavones, chlorogenic acid and 3,5-O-dicoffeoylqunic acid were 57.2 mg x g(-1), 0.673% and 1.838% respectively, reaching the maximum at the NH4(+) -N /NO3(-) -N ratio of 25:75; The content of luteoloside increased with the ratio of NO3(-) -N increasing and reached it's maximum at the NH4(+) -N/NO3(-) -N ratio of 0: 100. The yield of Ch. morifolium reached it's maximum at the NH4(+) -N /NO3(-) -N ratio of 25:75. Nitrogen form has some remarkable influence on the nitrogen metabolism, photosynthesis and growth, Nitrogen form conducive to the growth and quality of Ch. morifolium at the NH4(+) -N /NO3(-) -N ratio of 25: 75.
Ammonium Compounds ; metabolism ; pharmacology ; Chlorophyll ; metabolism ; Chrysanthemum ; drug effects ; growth & development ; metabolism ; Flowers ; drug effects ; growth & development ; metabolism ; Glutamate Synthase ; metabolism ; Glutamate Synthase (NADH) ; metabolism ; Glutamate-Ammonia Ligase ; Nitrates ; metabolism ; pharmacology ; Nitrogen ; metabolism ; pharmacology ; Photosynthesis ; drug effects ; Plant Leaves ; drug effects ; growth & development ; metabolism ; Plant Proteins ; metabolism ; Plant Roots ; drug effects ; growth & development ; metabolism ; Plant Stems ; drug effects ; growth & development ; metabolism

Objective To stratify the risks of patients with acute pulmonary embolism (APE) and pulmonary hypertension (PH) by dynamic pulmonary perfusion imaging (DPPI) and pulmonary perfusion imaging (PPI). Methods From October 2007 to February 2009, 20 healthy volunteers ( 12 males, 8 females; mean age =48.47 ±13.47 years) and 31 APE patients (21 males, 10 females; mean age =47.68 ±18.06 years; from October 2007 to July 2009) were included in the study. DPPI and PPI were performed in all subjects. Percentage of perfusion defect scores ( PPDs% ) were calculated by semi-quantitative analysis of PPI. Risk levels were defined according to PPDs% calculated from PPI: normal (PPDs% =0); very low risk (0 ＜ PPDs% ≤10% ); low risk (10% ＜ PPDs% ≤20% ); moderate risk (20% ＜ PPDs% ≤40% );high risk (40% ＜ PPDs% ≤60% ) and very high risk ( PPDs% ＞ 60% ). Lung equilibrium time (LET)was calculated on region of interest (ROI) drawn over DPPI. Clinical risk was scored by Aujesky method.The t-test, ANOVA and correlation analysis were used with SPSS 13.0 software. Results ( 1 ) LET in healthy volunteers and APE patients was ( 12.18 ± 3.28) and (32.90 ± 14.29) s respectively (t = 6. 81,P ＜ 0. 01 ). (2) The correlation coefficient, coefficient of determination between LET and PPDs% in APE patients were 0.93 and 0. 87, respectively. The correlation coefficient between LET and clinical risk score was 0.86. (3)The mean LET of APE patients in very low risk (n =5), low risk (n = 12), moderate risk (n=9), high risk (n=4) and very high risk groups (n=1) were (19.59 ±0.04), (25.03 ±0.08),(36.07 ±0. 10), (57.15 ±0.06) and (70 ±0.00) s, respectively. There was significant difference among APE patients with different risk levels (F =16. 78, P ＜0.01). Conclusions ( 1 ) DPPI was a reliable, convenient and non-invasive method for the evaluation of PH in APE. (2) Combined LET of DPPI and PPDs% of PPI was valuable for risk stratification and prognosis estimation in APE patients.

ObjectiveTo analyze Echinococcus infection in definitive and intermediate hosts in different zones of Qinghai plateau,Qinghai southern plateau,Qilian mountain-Hehuang valley and Chaidamu basin,and to provideascientificbasisfor developing controlstrategiesagainstEchinococcosisinfection. Methods Echinococcosis infection in definitive hosts,dogs and foxes,was identified by morphological observation; in domesticated and wild intermediate host animals was identified by anatomy and pathology; some of the suspected samples were further identified by molecular biological methods.ResultsStray dogs in different zones of Qinghai plateau were infected with Echinococcus granulosus,the infection rates were 38.71％(300/775),49.60％(124/250),and 9.76％(4/41 ) in Qinghai southem plateau,Qilian mountain-Hehuang valley and Chaidamu basin,respectively,and the difference was statistically significant(x2 =25.72,P ＜ 0.01 ).in addition,only Qinghai southern plateau dogs were infected with Echinococcus multiloularis,and the infection rate was 16.04％(98/611).The infection rates of fox with Echinococcus multilocularis were 22.89％(38/166) and 30.77％(12/39) in Qinghai southern plateau and Qilian mountain-Hehuang valley,respectively,and wolves were also found to be infected with Echinococcus granulosus in the same areas.The infection rates of domesticated sheep,yaks,goats and pigs with Echinococcosis were significantly different statistically in those different areas(x2 =82.70,41.82,212.63,194.58,all P ＜ 0.01 ).The infection rates of sheep and yaks were higher[43.43％(5664/13 042),49.47％(2917/5896),52.99％ (887/1674),42.18％ (779/1847),50.70％ (1049/2069),52.90％ (685/1295) ] in three areas.The infection rates of goats and pigs [3.26％ (7/215),0.00％ (0/108)] in Qinghai southern plateau were lower than that of other two areas[ 19.51％(119/610),26.91％(43/1598),47.91％(343/716),21.91％(71/324)].The infection rates of Ochotona curzoniae with Echinococcosis were 6.21％ (243/3910),1.80％ (3/167) and 0.00％ (0/199) in Qinghai southern plateau,Qilian mountain-Hehuang valley and Chaidamu basin,respectively,and the difference was statistically significant (x2 =18.50,P ＜ 0.01 ).Moreover,wild intermediate hosts of Echinococcosis,such as Microtus fuscus,Lepus oiostolus,Pseudois nayaur,Procapra picticaudata,and Prodorcas gutturosa were found to be infected only in Qinghai southern plateau.ConclusionsHuman is faced with a threat of Echinococcosis infection from various definitive hosts in different zones of Qinghai plateau.And stray dogs are the most crucial factor.The life-cycles of Echinococcus are very complicated in Qinghai plateau.Qinghai plateau is a key area in prevention and control of Echinococcosis infection in China.

Calreticulin (CRT), an important Ca(2+)-binding molecular chaperone in the endoplasmic reticulum (ER), and caspase-12, a pivotal molecule mediating ER-initiated apoptosis, are involved in the ER stress (ERS). Using primary cultured neonatal cardiomyocytes, CRT and caspase-12 expression and activation during hypoxic preconditioning (HPC) and hypoxia/reoxygenation (H/R) were studied to explore the role of ERS in cardioprotection by HPC. And by using SB203580 and SP600125 [the specific inhibitors of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK)] separately, the role of p38 MAPK in HPC-induced ERS was also detected. Neonatal cardiomyocytes were prepared from Sprague-Dawley rats aged 24 h, and cultured in DMEM medium containing 10% fetal bovine serum, and then randomly divided into six groups as follows: H/R, HPC+H/R, SB203580+HPC+H/R, SP600125+HPC+H/R, HPC and control groups. H/R was produced by 2-hour hypoxia/14-hour reoxygenation, and HPC by 20-minute hypoxia/24-hour reoxygenation. Morphological studies, estimation of lactate dehydrogenase (LDH) leakage and flow cytometry were employed to assess cell apoptosis and necrosis. CRT and caspase-12 expression and activation, levels of phospho-p38 MAPK and phospho-JNK were detected by Western blot. All experiments were repeated at least four separate times. The results obtained are as follows: (1) HPC relieved the cell injury caused by H/R. Compared with that in H/R group, cellso survival rate in HPC+H/R group increased by 6.4%, and the apoptosis rate and LDH leakage in the cell culture medium decreased by 6.6% and 70.0%, respectively. (2) H/R induced caspase-12 activation (33.2-fold increase in comparison with control) and CRT expression (8.1-fold increase in comparison with control). HPC itself resulted in mild CRT up-regulation (2.6-fold increase in comparison with control), but the extent of up-regulation was lower than that induced by H/R. HPC before H/R was found to relieve the over-expression of CRT induced by H/R (72.4% decrease), and to inhibit the activation of caspase-12 (59.6% decrease). (3) The protection of HPC and HPC-induced up-expression of CRT and inhibition of caspase-12 activation were almost eliminated when the inhibitor of p38 MAPK, not of JNK, was present before HPC. These results suggest that HPC protects the neonatal cardiomyocytes from severe ERS-induced apoptosis during sustained H/R through pre-invoking proper ERS response. Mild up-expression of CRT and inhibition of caspase-12 activation induced by HPC, which are important protection factors, are mediated by p38 MAPK, not by JNK.
Animals ; Caspase 12 ; physiology ; Cell Hypoxia ; Cytoprotection ; Endoplasmic Reticulum ; metabolism ; Ischemic Preconditioning, Myocardial ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; physiology

OBJECTIVEHuman herpesvirus 6 (HHV-6) isolates are classified into two variants, HHV-6A and HHV-6B, based on distinct genetic, antigenic and biological characteristics. HHV-6 has been associated with encephalitis in children recently. This study aimed to establish a real time PCR assay for simultaneous detection of the two subtypes of HHV-6, and apply this new assay to children with suspected encephalitis, then analyze the relationship between the infection with HHV-6 and encephalitis in children.

METHODThe universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene (U38) of HHV-6. The 5' end of the probes for HHV-6A and HHV-6B was labeled with the fluorescein reporter tetrachloro-6-carboxyfluorescein and 6-carboxyfluorescein (6-FAM), separately, while the 3' end were quenched with 6-carboxy-tetramethylrhodamine. The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established. Then, the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 10(9) to 10(0) copies/microl, together with controls were used for testing both sensitivity and specificity of the real time PCR assay. The cerebrospinal fluid (CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced.

RESULTBoth HHV-6A (strain ZJ-159) and HHV-6B (strain GS) were positive on the real time PCR assay. There were no cross-reaction with herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus, Staphylococcus aureus, Mycoplasma pneumoniae and human DNA. A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6. The sensitivity threshold was 10 copies/microl for the real time PCR. HHV-6 positive rate by the real time PCR assay was 4.72% (21/445), including 4 cases with HHV-6A infection, 16 cases of HHV-6B infection and 1 case with mixed HHV-6A and HHV-6B infection. The new PCR assay usually took 2 to 3 hours to provide results.

CONCLUSIONThis new real time PCR assay can simultaneously detect both subtypes of HHV-6, and have high specificity and sensitivity. It will provide an early and sensitive diagnosis of HHV-6 encephalitis in children.

Adolescent ; Child ; Child, Preschool ; DNA Fingerprinting ; DNA Primers ; DNA, Viral ; Encephalitis, Viral ; cerebrospinal fluid ; diagnosis ; virology ; Female ; Fluorometry ; Genotype ; Herpesvirus 6, Human ; genetics ; isolation & purification ; Humans ; Infant ; Infant, Newborn ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

This study was aimed to explore the role of human fetal bone marrow stromal cells (FBMSC) in combination with exogenous cytokines in supporting the in vitro expansion of cord blood mononuclear cells and the expression of CXCR4(+) and CD49d(+) in CD34(+) cells. Mononuclear cells (MNC) separated from cord blood (CB) were cultured in a serum-free support culture system with FBMSC or exogenous cytokines or both of them. On day 0, 6, 10 and 14, total cells were counted, CD34(+), CD34(+)CXCR4(+) and CD34(+)CD49d(+) cells were quantitated by FACS, and hematopoietic progenitor cells were assessed by semisolid culture assay. The results showed that after culturing for 14 days, CD34(+) cells, CD34(+)CXCR4(+) cells, CD34(+) CD49d(+) cells and colony forming unit (CFU) were significantly increased (P < 0.05). Compared with other groups, expansion multiple of CD34(+), CD34(+)CXCR4(+), CD34(+)CD49d(+) cells and CFU were higher than that in FBMSC and cytokine group (P < 0.05). It is concluded that the culture system used in this study can not only support the expansion of CB MNCs but also increase the number of hematopoietic stem and progenitor cells which has chemokine and adhesion capacity. This culture system may be a feasible way for in vitro culture of cord blood cells.
Antigens, CD34 ; blood ; Bone Marrow Cells ; cytology ; immunology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Cytokines ; pharmacology ; Fetal Blood ; cytology ; Fetus ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Integrin alpha4 ; blood ; Interleukin-3 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; immunology ; Receptors, CXCR4 ; blood ; Stromal Cells ; cytology ; immunology ; Time Factors