Objective To study the mutations of Env sequence of SIVmac239 after infection of Chinese rhesus monkeys, and compare the differences and characteristics of Gp120 sequences of enterotropic and neurotropic SIV strains. Methods Six strains of simian immunodeficiency virus were analyzed in this study: four separated from peripheral blood mononuclear cells of SIVmac239-infected monkeys and two neurotropic SIVmac251 strains.Isolated and cultured monoclonal virus was obtained by limiting dilution assay.Gp120 sequences were amplified after the RNA extraction and phylogenetic analysis was processed thereafter.So did the Gp120 amino acid sequence and N-glycosylation sites analysis of the enterotropic and neurotropic strains.Results SIVmac239 had different mutations in four rhesus monkeys.The diversity in amino acid sequences of the enterotropic and neurotropic strains concentrated in the V1 and V4 regions of Gp120.The enterotropic strains had an addition of glycosylation site in V4 but the glycosylation site changes of neurotropic strains were located in the conservative regions of C1, C2 and C3.Conclusions The addition of one glycosylation site in V4 region of GP120 and loss of one glycosylation site in C1 region are associated with enhanced enterotropism and neurotropism.The differences between the enterotropic and neurotropic strains are not dipicted in Gp120 V3 region which is closely related with the tropism of strains.

Objective To investigate the mechanisms modulating the functions of dendritic cells (DCs) and suppressing the activation and proliferation of T cells by transforming growth factor-β (TGF-β).Methods Mouse splenic DCs were purified with CD11c+ immunomagnetic beads and the purity of isolated DCs were detected by flow cytometry.Gene chip was used to detect gene expression in DCs after stimulation with TGF-β, and then real-time PCR was performed to analyze the differentially expressed genes in microarray at mRNA level.The activation and proliferation of CD4+ T cells which were co-cultured with DCs after stimulation with TGF-β were detected by flow cytometry.Results The purity of DCs reached over 95% after isolation.TGF-β down-regulated the expression of cell surface markers CD53, CD69, CD33, CD74 and CD93 on DCs;decreased the expression of chemokines Ccl3, Ccl5, Ccl9, Ccl6, Ccl17, Cxcl10, Ccl22, Ccl4, Ccr7, Ccl2, Cxcl9 and Ccl7;inhibited the expression of inflammatory cytokines IL-2ra, IL-12rb2, IL-15ra, IL-1b and IL-15.Moreover, the DCs-mediated activation and proliferation of CD4+ T cells were suppressed by TGF-β.Conclusion TGF-β inhibits the DCs-mediated activation and proliferation of CD4+ T cells by suppressing the expression of surface markers on DCs and down-regulating the expression of chemokines and inflammatory cytokines.

OBJECTIVETo explore the role of phospholipase C-gamma1 (PLC-gamma1) signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells.

METHODSPC12 cells were exposed to 50 micromol/L H(2)O(2) after pretreatment with 10 micromol/L U73122, a specific PLC-gamma1 inhibitor. Hoechst/PI double staining was performed to observe the morphological changes of the cells under light microscope. MTT assay was used to evaluate the cell viability, and the percentage of apoptotic cells was analyzed by flow cytometry. DNA fragmentation assay was carried out to characterize the cell apoptosis.

RESULTSAfter inhibition of the PLC-gamma1 signaling pathway with 10 micromol/L U73122, PC12 cells showed obvious apoptotic morphology, the viable cells decreased significantly, and the percentage of apoptotic cells rose to 35.7%. PC12 cells treated with U73122 presented with a distinct DNA ladder on electrophoresis resulting from DNA cleavage in the apoptotic cells.

CONCLUSIONPLC-gamma1 signaling pathway plays an important protective role in H(2)O(2)-induced PC12 cell apoptosis.

Animals ; Apoptosis ; drug effects ; Estrenes ; pharmacology ; Hydrogen Peroxide ; pharmacology ; PC12 Cells ; Phospholipase C gamma ; antagonists & inhibitors ; metabolism ; Pyrrolidinones ; pharmacology ; Rats ; Signal Transduction

OBJECTIVETo compare the efficacies of portal vein stenting and transcatheter arterial chemoembolization (TACE) combined therapy performed with or without endovascular implantation of iodine-125 (125I) seeds strand in patients with hepatocellular carcinoma (HCC) and main portal vein tumor thrombus (MPVTT).

METHODSOne-hundred-and-six patients with HCC complicated by MPVTT who were treated with portal vein stents and TACE, either with (Group A, n=56) or without (Group B, n=50) endovascular implantation of 125I seeds strand, between July 2005 and April 2011, were retrospectively analyzed. Overall survival, stent patency, and procedure-related adverse events were compared between the two groups.

RESULTSThe technical success rate was 100% for placement of 125I seeds strands and stents in the obstructed main portal vein. No serious procedure-related adverse events were recorded. Group A had significantly higher median survival (335 days vs. group B: 146 days; P=0.001, hazard ratio (HR)=2.244). Additionally, group A had significantly higher median stent patency (400 days vs. group B: 190 days; P=0.005, HR=2.479).

CONCLUSIONThe combination therapeutic strategy of portal vein stenting and TACE with endovascular implantation of 125I seeds strands improves the survival of HCC patients with MPVTT complication.

Carcinoma, Hepatocellular ; complications ; therapy ; Chemoembolization, Therapeutic ; Combined Modality Therapy ; Female ; Humans ; Iodine Radioisotopes ; administration & dosage ; therapeutic use ; Liver Neoplasms ; therapy ; Male ; Middle Aged ; Neoplastic Cells, Circulating ; Portal Vein ; physiopathology ; surgery ; Retrospective Studies ; Stents ; Treatment Outcome ; Venous Thrombosis ; complications ; therapy

AIM To optimize the preparation of Bishutongqiao Mixture.METHODS With solid total mass,contents of astragaloside A and calycosin-7-glucoside as evaluation indices,water addition,extraction time and extraction times as influencing factors,the aqueous extraction process was optimized by orthogonal test.With the contents and retention rates of astragaloside A and calycosin-7-glucoside as evaluation indices,the effects of normal pressure and decompression on the concentration process were investigated.And the effects of solubilizer (polysorbate 80),taste-masking agent (sucrose) and bacteriostatic agent (sodium benzoate) amounts on the forming process were studied as well.RESULTS The optimal aqueous extraction conditions were determined to be ten times for water addition,1 h for extraction time,and extracting twice,the comprehensive score was 91.67.The contents and retention rates of astragaloside A and calycosin-7-glucoside at normal pressure concentration were higher than those at decompression concentration.The optimal forming process was obtained at polysorbate 80,sucrose and sodium benzoate amounts of 1.43％,15％ and 0.3％,respectively.CONCLUSION This stable and feasible method can be used for the preparation of Bishutongqiao Mixture.

Objective Apoptosis was induced by oxidative stress in nerve cells after cerebral ischemia. It further breaks the dy-namic balance of mitochondrial division of fusion. This study aimed to investigate the effect of butylphthalide combined with edaravone treat-ment on the dynamic change of Drp1 and Mfn2 in rats with focal cere-bral ischemia cells and its protection mechanism. Methods 96 rats were divided into 4 groups according to random number table. The 4 groups were ischemia group,butylphthalide group,edaravone group and butylphthalide combined with edaravone groups(combine group),each group divided into three subgroups(3 d,7 d,14 d). Longa-Zea suppository method is adopted to establish the middle cerebral artery occlusion(MCAO)model.Butylphthalide group,edar-avone group and combine group were injected butylphthalide(0.4 g/kg)and/or edaravone(10 mL/kg)peritoneally 2 hours before surgery and 1 day after surgery. The same volume of isotonic saline was given at the same time of the other 3 groups in ischemia group. The cerebral cortex of the left ischemic region was obtained at the day 3,7,14. The evaluation of the curative effect was evaluated with neurological function score.HE staining was used to observe the cerebral cortex neuron morphological structure,protein and mRNA lev-el of Drp1 and Mfn2 was measured by western blot and RT-PCR. Results At the day 3,7,and 14,the neurological function score was higher in ischemia group than the other 3 groups(P<0.05). Compared with the combine group at day 3,7,and 14[(1.06± 0.18),(0.82±0.13),(0.57±0.10)],the neurological function score was elevated in butylphthalide group[(2.02±0.18),(1.23± 0.13),(0.86±0.10)]and edaravone group[(2.08±0.17),(1.23±0.13),(0.85±0.12)](P<0.05). At the same time point,com-pared with the ischemia group,Drp1 protein and mRNA levels were lower in the other 3 groups(P < 0.05)while Mfn2 protein and mRNA levels were elevated(P<0.05). Compared with the butylphthalide group and edaravone group,Drp1 protein and mRNA levels were lower in combine group(P<0.05)while Mfn2 protein and mRNA levels were elevated(P<0.05). Conclusion The protective effect of edaravone combined with butylphthalide is better than single use. Its mechanism may be related to the removing of oxygen free radicals and reducing oxidative stress by edaravone,and the protection of the mitochondria by butylphthalide.

Objective To investigate the effects of tetramethylprazine (TMP) on left ventricular hypertrophy and the regulation of nicotinamide-adenine dinucleotide(NADPH) oxidases in renovascular hypertension of rat.Methods Hypertensive rats were obtained by using two-kidney-two-clip (2K2C) method.Rats were classified into sham-operated group,model group,low,high dose experimental groups (TMP,30,60 mg · kg-1 · d-1) and control group (candesartan,10 mg · kg-1 · d-1).The drugs were continuously administered for 2 weeks.Left ventricular (LV) hemodynamic parameters,serum superoxide ismutase (SOD),dmalondialdehyde (MDA) and hydrogen peroxide(H2O2)levels were measured.The ratio of LV weight to body weight (LVW/BW) was calculated.Hematoxylin-eosin(HE) staining was used for morphological analysis.RT-polymerase chain reaction and Western-blot were performed to investigate the protein expressions of brain natriuretic peptide (BNP),β-myosin heavy chain(β-MHC) and NADPH oxidases(Nox2,Nox4 and P47phox).Results Compared with model group,aortic systolic pressure (AoSP),aortic diastolic pressure (AoDP),LV end-systolic pressure (LVESP),LV end-diastolic pressure(LVEDP) significantly decreased in low,high dose experimental groups with siginificanly (all P ＜ 0.05) while (+ dp/dtmax) and (-dp/dtmax) increased in low,high dose experimental groups with siginificanly (P ＜ 0.05).Compared with model group on LVW/BW (2.8 ± 0.3) mg · g-1,LVW/BW of (1.9 ± 0.2),(1.7 ± 0.4) mg · g-1 in low,high dose experimental groups significantly decreased (P ＜ 0.05).Compared with model group SOD significantly increased while MDA and H2O2 decreased in low,high dose experimental groups with significandy (all P ＜ 0.05).The protein expressions of Nox2 in low,high experimental groups and model group were 0.82 ± 0.08,0.57 ± 0.05,0.30 ± 0.13;the protein expressions of Nox4 in above three groups were 1.00±0.10,0.79 ± 0.07,1.32 ± 0.15 and the protein expressions of P47phox in above three groups were 0.88 ± 0.08,0.65 ± 0.06,1.23 ± 0.15,compared with model group,the protein expression significantly decreased in low,high dose experimental groups with significantly (all P ＜ 0.05).Conclusion TMP attenuated cardiac hypertrophy possibly through decreasing NADPH oxidases (Nox2,Nox4,P47phox) expressions and oxidative stress in renovascular hypertensive rats.

Objective To comparatively analyze the changes of environmental risk factors in 9 years in an area polluted by arsenic coal-burning in Xingren County of Guizhou Province,in order to provide evidence for reasoning the occurrence and development as well as its effective prevention and control.Methods Epidemiological sampling methods was used to conduct follow-up investigation on 181 arsenism patients who were diagnosed in 1998 in arsenic polluted area.Control group included 65 residents who lived far from polluted area of 12 km.The follow up investigation included age,sexuality,family economic situation,time of use or stop use of arsenic coal, ventilation of the room,desiccation of food etc.Diethyl dithiocarbamate silver(Ag-DDC)method was used to detect arsenic content of coal,soil,air,water and rice,corn,chili;Single factor and multivariate factors non-conditional Logistic regression models were used to analyze exposure factors of patients and related environmental risk factom, and the differences of those in 1998 and 2006 were compared. Results The arsenic content in indoor and outdoor air,coal,chili and corn went down from 0.0880 and 0.0220 mg/m3,397.20,45.07 and 2.64 mg/kg in 1998 to 0.0790 and 0.0070 mg/m3,93.01,3.46 and 1.50 mg/kg in 2006. Arsenic contents of other samples were less than national standard. The analysis of single factor and multivariate factors non-conditional Logistic regression showed that time of using high arsenic coal,age,fluorosis and smoking(x2 = 50.159,12.195,37.69,6.358,P ＜ 0.05 or ＜ 0.01 ) were still the main risk factors for arsenism,while family economic situation was still influential factors (X2 = 4.614,P ＜ 0.05);Ventilation of the room changed from a risk factors at 1998 to an influencing factors at 2006(X2 = 38.093,P ＜ 0.01 ). Single factor non-conditional Logistic regression model analysis showed that food desiccation by arsenic coal-burning and educational level were no longer risk and influencing factors,while food preservation and gender had become influencing factors(x2 = 17.463,11.004,all P ＜ 0.01 ) nine years after. Conclusions Environmental arsenic pollution in arsenism area in Guizhou Province has been obviously improved after nine years. However,the continued existence of low doses of arsenic pollution is still a major cause of failure of controlling arsenism. Time of using high arsenic coal,age,smoking,fluorosis,family economic situation and ventilating room are closely related to the occurrence and the development of arsenism. Prohibition of use of high arsenic coal,furnace improvement,health education and economic development are effective measures

OBJECTIVETo investigate the relationship between the pathologic responses and histologic type, grade, the expression of ER, PR and HER2 and their changes in breast carcinoma before and after neoadjuvant chemotherapy (NAC).

METHODSTwo-hundred and nine cases of breast cancer with NAC were analyzed and clinical, pathologic data were evaluated based on the Miller and Payne ( MP) grading system. The expression of ER, PR and HER2 in the cancers before and after NAC were detected by immunohistochemistry (MaxVision method). SPSS 15.0 software was used to conduct statistical analysis.

RESULTS(1) Pathologic responses to the NAC were graded as MP1 (14 cases), MP2 (35 cases), MP3 (106 cases), MP4 (36 cases) and MP5 (18 cases); (2) The expression of ER in core needle biopsy had related negatively to the pathologic response (chi2 = 33.083, P = 0.001). However, the histologic type, grade, ER and PR status, and HER2 expression in surgically-removed specimens had not related to the pathologic response (P>0.05); (3) After NAC, the pathologic type and grade changed in 6. 8% (9/132) and 34.9% (30/86) of the cases, and the rates of changes in the expression of ER, PR and HER2 were 42.4% (75/177), 55.4% (98/177) and 26.6% (46/173) , respectively. Only the expression of HER2 had significant difference between before and after neoadjuvant chemotherapy (P = 0.049). The changes in other data had no relationship with the pathologic response (P>0.05).

CONCLUSIONSAnalysis of core needle biopsy can provide important information to predict the pathologic responses to the NAC. The pathologic appearance, grade, ER, PR and HER2 in breast carcinoma may change after NAC. It is necessary to examine the histologic type, grade and the expression of ER, PR and HER2 after NAC once more.

Adenocarcinoma, Mucinous ; drug therapy ; metabolism ; pathology ; surgery ; Adult ; Aged ; Biomarkers, Tumor ; metabolism ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Carcinoma, Ductal, Breast ; drug therapy ; metabolism ; pathology ; surgery ; Carcinoma, Lobular ; drug therapy ; metabolism ; pathology ; surgery ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Neoadjuvant Therapy ; Neoplasm Grading ; Neoplasm Staging ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

OBJECTIVETo clone the recombinant human islet neogenesis-associated protein (rhINGAP) gene for its secretory expression in Pichia pastoris.

METHODSINGAP gene was amplified with PCR and inserted between Xho I and EcoR I downstream sites of the alpha factor of the recombinant plasmid alpha/pUC18. The fusion gene of alpha factor and INGAP was subsequently inserted between BamH I and EcoR I sites of the plasmid pPIC9K of P. pastoris. After confirmation with restriction enzyme digestion and sequencing, the positive recombinant plasmid that integrated INGAP gene was linearized with Sal I digestion and transformed into the yeast host strain GS115 through electroporation. The yeast transformants that harbored the INGAP gene with high copies were selected with the auxotroph medium and G418, followed then by PCR verification of the positive transformants, from which the expression of recombinant human INGAP was induced with methanol as the only carbone source. The antigenic activity of the desired protein was then detected using Western blotting and enzyme-linked immunosorbent assay (ELISA).

RESULTS AND CONCLUSIONThe recombinant expression plasmid INGAP/pPIC9K was successfully constructed, and 3 positive transformants were obtained. The expressed protein showed good antigenic activity as confirmed by Western blotting and ELISA.

Antigens, Neoplasm ; genetics ; metabolism ; Biomarkers, Tumor ; genetics ; metabolism ; Blotting, Western ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Islets of Langerhans ; metabolism ; Lectins, C-Type ; genetics ; metabolism ; Pancreatitis-Associated Proteins ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; metabolism