The inhibition effects and mechanism of recombinant snake venom metalloproteinase inhibitor on tumor angiogenesis / 中国药理学通报

Aim Toinvestigatetheeffectofrecombi-nant snake venom metalloproteinase inhibitor (rSVM-PI ) on neovascularization and its molecular mecha-nism.Methods Chickenchorioallantoicmembrane (CAM)assay was used to examine the antiangiogenic effect of rSVMPI.Alamar blue analysis was used to de-tect cell proliferation.Annexin V-FITC double labeling flow cytometry was used to assay cell apoptosis. Scratch marker was used to assay cell migration.Boy-den chamber analysis method was used to detect cells chemotaxis in vitro.Tube like structure(TLS)of HU-VECs was used to detect the ability of neovasculariza-tion in vitro.Real-time PCR and Western blot were used to assay the expressions of KDR and FGFR-1 inHUVECs. Results Thevasculardensityindex (VDI)of CAM was drastically decreased after rSVMPI treatment, chemotaxis of HUVECs in response of VEGF was inhibited in the presence of rSVMPI,TLS of HUVECs was less than control group.The expres-sions of KDR and FGFR-1 were down-regulated by re-al-timePCRandWesternblotassay.Conclusion rS-VMPI may inhibit neovascularization by blocking the VEGF-KDR or bFGF-FGFR signal transduction path-way.

Clinical significance of thrombospondin-2 expression in gastric car-cinoma / 中国肿瘤临床

Objective:To investigate the clinical significance of thrombospondin-2 (THBS2) mRNA expression in gastric carcino-ma and its relationships with clinicopathologic features, microvessel density (MVD), and matrix metalloproteinase-2 (MMP-2). Meth-ods:THBS2 mRNA expression was detected in 82 cases of gastric carcinomas and adjacent tissues using real-time quantitative fluores-cence polymerase chain reaction. The correlation of this expression with clinicopathologic features was also analyzed. Cluster of differ-entiation 34 (CD34) and MMP-2 protein expression was examined using an immunohistochemical Elivision method. MVD was deter-mined based on CD34-positive tubular structures. Results:The THBS2 mRNA expression level was significantly higher in the gastric carcinomas than in paraneoplastic tissues (P=0.002). The expression was associated with the depth of tumor invasion, MVD, and MMP-2 (P=0.02, r=0.35, P<0.01, and P=0.004, respectively) but not with patient gender, patient age, tumor size, histological type, and lymph node metastasis (P=0.53, P=0.53, P=0.21, P=0.84, and P=0.96, respectively). Conclusions: THBS2 may be significantly in-volved in the occurrence and progression of gastric carcinoma. The effects of THBS2 on gastric tumor growth and metastasis can be monitored by controlling the MMP-2 expression in the carcinoma. However, the specific functions and underlying mechanisms of TH-BS2 require further investigation.

Beta-amyloid affects retinoid receptor-α exporting into cytoplasm / 中华神经科杂志

Objective To investigate the effect of Alzheimer' s beta-amyloid (Aβ) on the production and the translocation in cytoplasm of retinoid receptor-α (RXRα). MethodsN2awt cells were treated with Aβ peptide or amyloid protein precursor(APP695) transfection. The nucleus were separated from the cytoplasm by kit. The quantity of RXRα in the nucleus and cytoplasm was detected by Westernblot. The translocation of RXRα in the nucleus and cytoplasm of above N2awt cells or of the cortex cells in the brains of Alzheimer' s disease (AD) patients and their normal control groups was observed by immune fluorescence. ResultsIn N2awt cells, the increasing APP or Aβ had no significant effect on the production of RXRα but resulted in RXRα exporting into cytoplasm, the ratio of RXRα in cytoplasm increased from 3.2％ (in control group) to 17.6％ (in APP+ group) and from 3.8％ (in control group) to 14.3％ (in Aβ + group) respectively; compared with normal cortex cells, the translocation of RXRα in the cytoplasm of the cortex cells in the brains of AD increased significantly. ConclusionAβ may affect RXRα exporting into cytoplasm.

Yeast two-hybrid system screening liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome / 中华微生物学和免疫学杂志

Objective To screen the liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome. Methods The splicing-specific gene TPss generated by the 2.2 kb singly spliced variant of HBV genome was amplified by PCR and cloned into the bait vector pGBKT7. After exclusion of self-activatian capacities of TPss protein, a two-hybrid library screening was performed using a pre-transformed human liver cDNA library to screen the liver cell proteins interacting with TPss. Mammalian two-hybrid assay was also done to further confirm the interactions between the bait and prey proteins in Huh7 and HepG2 hepatacytes. Results TPss gene with the size of 336 bp was successfully amplified and cloned, and the TPss protein expressed well in AHI09 yeast cells. Four liver cell proteins interacting with TPss, i. e. , cathepsin B, epoxide hydrolase 1, cathepsin D and fibrinogen gamma chain, were screened by yeast two-hybrid assay and further confirmed by mammalian two-hybrid assay. Conclusion TPss could interact with liver cell proteins.

Clinical significance of thrombospondin 4 expression in gastric carcinoma / 解剖学报

Objective The aim of this study was to measure quantitatively the TSP-4 mRNA expression and its significance in gastric carcinoma was evaluated by correlating its expression with clinicopathological features, microvessel density(MVD) and MMP-9.Methods Eighty-two patients with gastric carcinoma were recruited in this study. TSP-4 mRNA expression in gastric carcinoma and adjacent tissue was detected by real-time PCR. CD34 and MMP-9 protein expression were examined by immunohistochemistry. MVD was determined according to CD34-positive tubular structures. Results The expression level of TSP-4mRNA in gastric carcinoma was obviously higher than those of adjacent tissue(P=0.03) and it was associated with tumor size, histological type, lymph node metastasis, MVD and MMP-9(P=0.002, P=0.031, P=0.014,r＝0.67 P<0.01, P=0.008),but not sex, age and depth of invasion. (P=0.53,P=0.57,P=0.15).Conclusion TSP-4 may play an important role in occurrence and progression of gastric carcinoma and that the effect of TSP-4 on tumor growth and metastasis may be exerted through regulation of angiogenesis and MMP-9 expression in gastric carcinoma.

Resistant Mechanism and Strain Relationship of Multi-resistant Pseudomonas aeruginosa / 中华医院感染学杂志

OBJECTIVE To investigate the correlated resistant genes encoding ?-lactamases,aminoglycoside and genetic marker of integron and transposon in multi-resistant Pseudomonas aeruginosa(MRPA) isolated from clinical specimens and study their relationship by phylogenetic analysis.METHODS Twenty-one resistant genes,two integron-Ⅰ genes and one transposon genetic gene were analyzed by PCR and verified by DNA sequencing.Multi-resistant genes cluster analysis was performed by UPGMA.RESULTS The positive rates of CARB,oprD2,aac(3)-Ⅱ,aac(6′)-Ⅱ,ant(2″)-Ⅰ,intⅠ1,qacE△1-sul1 and merA in 20 strains of MRPA were 15%,100%,70%,15%,15%,85%,85% and 85%,respectively,and other genes were negative.It was classfied to three subgroup,by the multi-resistant genes cluster analysis.CONCLUSIONS MRPA isolated from clinical specimens has carried many resistant genes.The deficiency rate of oprD2 gene is very high.The positive rate of genetic mark genes about integron and transposon is very high.It may be the main multi-resistant mechanism of MRPA.Multi-resistant genes cluster analysis shows that there is clone transmission in MRPA and it can induce nosocomial infection prevalence.

Effect of ginsenoside Rb1 on N9 cell activation induced by oxygen deficit / 解剖学报

Objective To activate microglia N9 cell through the oxygen deficit, and to discuss the influence to the N9 cell by ginsenoside Rb1, laying the foundation for the basic study and the clinical medicine development. Methods Through ginsenoside Rb1 intervention, the cell morphology the proliferation ability were observed, ELISA, fluorescent probe DAF-FM DA, Griess the reagent examination, were used to measure TNF-α, the O-2 output, the NO content change, chemiluminescence, the immunofluorescence method, and plastochondria membrane potential, were carried out to detect the cytochrome C content. Results Regardless of being preventive or medical gives, ginsenoside Rb1 can decline the NO,O-2,TNF-α high expression; and reduce the plastochondria membrane potential changing, the cytochrome C redistribution. Conclusion Ginsenoside Rb1 can decline N9 cell activation to a certain extent, reduce expression of the nerve toxic factor, and to stabilize mitochondrial membrane potential and distribution of cytochrome C.

[Influence of hemolytic and non-hemolytic enterococci on TNF-alpha expression in murine macrophage cells].

OBJECTIVE: To study the influence of hemolytic and non-hemolytic enterococci on TNF-alpha expression in Murine macrophage cells. METHODS: The possibility of endotoxin contamination in the cell culture was excluded by polymyxin-B inhibition. RAW264.7 were infected with hemolyticor non-hemolytic enerococci at bacteria: cell ratio of 30:1 for 1 h, followed by washing and re-incubation for 24h in complete medium containing 200 microg/mL Ampicillin. TNF-alpha concentrations in culture supernatants at different time intervals after infection for 3 h, 6 h, 9 h and 24 h were measured by ELISA. TNF-alpha mRNA expression by macrophages infected with hemolytic or non-hemolytic enerococci respectively for 6h was compared by RT-PCR. RESULTS: TNF-alpha was not detected in culture supernatants from uninfected RAW264.7 cells. The concentrations (pg/mL) of TNF-alpha present in culture supernatants from RAW264.7 cells stimulated by hemolytic enterococci were significantly higher than that stimulated by non-hemolytic enterococci at all time intervals tested, p < 0.01, Student t-test. TNF-alpha mRNA expression measured by RT-PCR brought about similar results: more TNF-alpha mRNA was expressed in RAW 264.7 cells stimulated with hemolytic enterococci as compared with non-hemolytic enterococci stimulation (p < 0.05, Student t-test). CONCLUSION: hemolytic enterococci promoted the generation of inflammatory factor TNF-alpha.

Effects of the splicing-specific protein on the regulatory elements of hepatitis B virus per se / 中华微生物学和免疫学杂志

Objective To investigate the effects of the novel protein,TPds encoded by the 2.2 kb doubly spliced variant of hepatitis B virus(HBV)genome on the regulatory elements of HBV.Methods HBV promoters and enhancers were amplified by PCR and respectively cloned into the plasmid pGL3-basic to construct the recombinant firefly luciferase reporter vectors including pGL3-BCP(harboring basal core promoter),pGL3-CP1601(core promoter with enhancer Ⅱ),pGL3-XP(minimal X gene promoter),pGL3 XP1071(X gene promoter with enhancer Ⅰ),pGL3-SP1(promoter of large surface antigen)and pGL3-SP2 (promoter of middle surface antigen).The recombinant reporter plasmids were respectively co-transfected with either the pcDNA3.1/HisC-TPds(TPd8 expression plasmid)or pcDNA3.1/HisC(empty control)into Huh7 hepatocytes by FuGENE6 transfection reagent.Cells were lysed 48 h post transfection,the intracellulax luciferase activities were monitored and calculated by SPSS11.5 software.Results As compared with the empty control of pcDNA3.1/HisC.the intracellular luciferase activities were elevated when the Huh7 hepatocytes were co-transfected by pcDNA3.1/HisC-TPds with one of the recombinant reporter plasmids as pGL3-CP1601,pGL3-XP1071,pGL3-SP1,or pGL3-SP2,while no changes with pGL3-BCP or pGL3-XP.Conduslon TPds could transactivate the HBV regulatory elements a8 promoter SP1,SP2.and enhancer Ⅰ,Ⅱ,while no influences on basal core promoter and minimal X gene promoter.

The anti-IFN-α effects of the novel protein encoded by the 458 nt-1308 nt spliced variant of hepatitis B virus genome / 中华微生物学和免疫学杂志

Objective To investigate the anti-IFN-α effects of the novel protein TSR'r' encodedby the 458 nt-1308 nt spliced variant of hepatitis B virus genome,and to determine its functional domaias.Methods the TSR'r' gene(originated from open reading frame of HBV DNA polymerase,T represents terminal protein region,S represents the Spacer region,R'represents the truncated reverse transcriptase region,and r'represents the truncated RNaseH region)of the 458 nt-1308 nt spliced variant of HBV genome and its deletants were amplified by PCR and were cloned into the pcDNA3.1/HisC vector.The recombinant vector was transfected into Huh7 hepatocytes individually by FuGENE6 transfection reagent,and the expression of the fusion protein was detected by Western blot.Huh7 hepatocytes were co-transfected with p6 16CAT and the recombinant vector harboring either TSR'r'or the related deletant,and treated with IFN-α 2a 48 h post transfection.After 24 h stimulating.the cells were lysed and the intracellular CAT value was calculated.All data were processed with One-way analysis of variance(ANOVA).Resuits Recombinant vectors harboring either the TSR'r'gene or related deletant were constructed successfully,and the fusion proteins were expressed well in Huh7 cells.When Huh7 hepatocytes were co-transfected with p6-16CAT and TSR'r' recombinant.the intracellular CAT values reduced gradually as paralleled with the increasing amount of TSR'r'recombinant.Furthermore,as compared with the empty vector,intracellular CAT values also decreased significantly when the Huh7 cells co-transfected with recombinant harboring TP plus Spacer regions,while any of the other deletants(harboring either TP or Spacer region or neither)showed no significant difference.Conclusion The novel protein encoded by the 458 nt-1308 nt spliced variant of hepatitis B virns genome suppressed the response of Huh7 hepatocytes to IFN-α.and the N-terminal TP plus Spacer region was the functional domain of the protein for anti-IFN-α effects.

The influence of a Grb2 inhibitor on K562 cell growth / 肿瘤研究与临床

Objective To analyze the effects of an inhibitor of the SH3 (Src homology) domains of Grb2 on the growth and proliferation of K562 cells. Methods The peptidimer [(VPPPVPPRRR)2-K], penetratin (RQIKIWFQNRRMKWKK) and peptidimer-c [poptidimer linked to penetratin: (VPPPVPPRRR)2-K-Aha-RQIKIWFQNRRMKWKK] were synthesized by solid-phase synthesis using Fmoc chemistry, and purified by high performance liquid chromatography (HPLC) on a C18 column. Purity was evaluated by HPLC, and the identity of the peptides was checked by electrospray mass spectroscopy (MS). A pull-down assay was used to observe the specific binding of peptidimer-c to the Grb2 of K562 cell lysates. The inhibition of peptidimer-c on K562 cell proliferation was evaluated by trypan blue exclusion assay, the cytostatic effect was tested by clonogenic assay, and the cytotoxicity was examined by WST-1 method. A further experiment was performed with clonogenic assay to analyze the co-effect of peptidimer-c respectively combined with Gleevec, Hydroxyurea and Cytarabine by Jing's method. Results The HPLC analysis showed only a simple peak, which means that the peptide is in high purity. MS analysis showed the peptides were coincided with the design. The molecular weight of peptidimer-c was of 4794.0 and that of the penetratin 2246.7. Pull-down assay demonstrated that the peptidimer-c, not the penetratin, could bind to Grb2 specifically. The trypan blue assay showed that the peptidimer-c could inhibit the proliferation of K562 significantly in a dose-dependent manner, even 3～6 h after the cells were exposed to the drug, and penetratin alone did not influence the cell proliferation. Gleevec inhibited the growth of K562 not only in a dose-dependent manner, but also in a time-dependent manner. WST-1 test showed the cytotoxieity of peptidimer-c or Gleevec on K562 cells, the IC50 of peptidimer-c was (17±2) μmol/L and the IC50 of Gleevec was (0.25±0.05) μmol/L. In the methylcellulose semi-solid medium system, the colony formation of K562 was greatly decreased by peptidimer-c as compared to the penetratin, and the colony number decreased as the dose of peptidimer-c increased. The IC50 value ofpeptidimer-c on K562 colony formation was (3.9±0.9) μmol/L, IC50 of Gleevec was (0.03±0.02) μmol/L, IC50 of Hydroxyurea was (15±7) μmol/L, and that of cytarabine was (0.014±0.012) μmol/L. There were synergistic effects of peptidimer-c with Gleevec, Hydroxyurea or Cytarabine on K562 by colonogenic assay. Combination of 1.5 μmol/L peptidimer-c and 0.05 μmol/L Gleevec showed synergistic effect on K562, as well as the combination of 1.5 μmol/L peptidimer-c and 0.006 μmol/L or 0.01 μmol/L Cytarabine. Conclusion These results suggested that peptidimer-c had an inhibitory effect on K562 cells and combination of peptidimer-c with other drugs would increase the anti-cancer effects.

Inhibition Effect of a Snake Venom Metalloproteinase Inhibitor on Experimental Pulmonary Metastasis in Mice Inoculated with B16 Melanoma Cells / 中国生物工程杂志

Although there is emerging evidence that BJ46a can function as potent inhibitor of the SVMPs proteolytic activities,its anticancer effect on invasion and metastasis has not yet been evaluated.Inhibition effect of BJ46a on experimental pulmonary metastasis in mice inoculated with B16 melanoma cells at the protein level was investigated. First,BJ46a was produced in baculovirus expression system. SDS-PAGE and Western blot analysis confirmed that BJ46a recombinant protein was produced by Sf9 cells infected with high-titer viral stock. Then,recombinant fusion protein was purified by ProBondTM at the point of maximal expression. B16 cells pre-treated with recombinant BJ46a injected into C57BL/6 mice via the tail lateral vein to form experimental pulmonary metastasis model. The numbers of metastatic lesions in C57BL/6 mice changed dramatically:BJ46a different concentrations of recombinant protein group were 1.1 ? 0.83,0.9 ? 0.7,significantly lower than the control group (6.3?3.00,P

ULTRASTRUCTURE OF NEURAL STEM CELL NEUROSPHERE CULTURED in vitro / 解剖学报

Objective To study the ultrastructure of neural stem cell neurosphere cultured in vitro. Methods Neural stem cell neurospheres from the rat brain were observed under transmission electron microscope or with stain of lanthanum nitrate, ruthenium red and tannic acid. Results The conjunction between the cells within neurosphere was loose and no tight junction was observed. Neural stem cells were proliferating lively. Some neural stem cells differentiated into cells with process and structure of axon, even showed the structures similar to myelin.Conclusion The ultrastructure of neural stem cell neurosphere cultured in vitro was revealed.

Association of ACE and PAI-1 gene polymorphisms with plasma PAI-1 level in type 2 diabetic patients / 中华内分泌代谢杂志

Plasma plasminogen activator inhibitor 1 (PAI-1) antigen level in 204 patients with type 2 diabetes mellitus (DM) was higher than that in 60 healthy subjects (P

Signaling Role of Exogenous Arachidonic Acid in the Invasion of Macrophages by Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To investigate the signaling role of arachidonic acid in the invasion of RAW264.7 macrophage by Toxoplasma gondii . Methods Rate of infection was calculated by both light microscope and flow cytometer. Fluorescent emission spectra were recorded using a microspectrofluometer for the concentration of cytoplasmic free calcium. Results Calcium concentration in macrophages and rate of infection increased with a higher concentration of exogenous arachidonic acid in a dose-dependent manner. The invasion was dependent on the mobilization of calcium from the extracellular medium and from intracellular stores and followed the influx of calcium into the parasitized cell. Conclusion Arachidonic acid may enhance the rate of infection via calcium transduction pathway.

Biological Features of Mouse Macrophage Transfected with Toxoplasma gondii GRA-1 Gene / 中国寄生虫学与寄生虫病杂志

Objective To investigate the change of biological features of macrophages after transfected by Toxoplasma gondii GRA 1 genes (P24). Methods The transfected cells Cyto P24 RAW264.7, Nuc P24 RAW264.7, Mito P24 RAW264.7 and ER P24 RAW264.7 were studied by RT PCR to determine the P24 mRNA expression. Growth features of the cells were examined with microscopy and the cell growth curve was developed. Results In four cell lines, expression of ER P24 RAW 264.7 was found to be higher than the other three, and there was no P24 mRNA expression in either of the cells without P24 insert. The attachment and the proliferation of ER-P24-RAW264.7 were more rapid than normal RAW264.7. Conclusion Transfection of mouse macrophages ER RAW264.7 strain with T. gondii P24 gene leads to a prominent change of biological features in the studied cell line.

OBSERVATION OF TEGUMENT AND CAECUM OF ADULT SCHISTOSOMA JAPONICUM BY TRANSMISSION ELECTRON MICROSCOPY / 中国血吸虫病防治杂志

The ultrastructrual features of tegument and caecum of Chinese mainland strain S. japonicum abult were studied by transmission electron microscopy.l.The tegumental surface folds and pits of male worms are more obvious than those of female worms, and ultrastructural difference was also detected in various regions of the tegumental surface.2.The gut of male and female worms consists of a single-layered columnar epithelial cell surrounded by a thin muscle layer, but the ultrastructural differences are found between different regions of the gut.3.The comparative study of the tegument and the caecal epithelium has shown that in function the caecal syncytium appears to be responsible for the digestion and absorption, while the tegument is responsible for both absorption and defence.

Intra-cellular Signal Pathway and Synthesis of Prostaglandin E_2 During Invasion of Macrophage by Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To investigate the signal transduction pathway of arachidonic acid(AA) and prostaglandin E-2(PGE-2) synthesis in macrophage invaded by Toxoplasma gondii. Methods Synthesis of AA and PGE-2, expression of COX_2 mRNA and protein following stimulation infection by Toxoplasma gondii were evaluated in RAW264

Cloning, Expression and Purification of the Major Surface Antigen P30 of Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To obtain protein of the major surface antigen P30 of Toxoplasma gondii by molecularcloning. Methods The gene of P30 (containing the whole P30 gene sequence, without the gene encoding signalpeptide) was obtained by polymerase chain reaction (PCR) using the primer designed according to the DNA sequence ofP30. The recombinant plasmid was constructed using EcoR Ⅰ, Xho Ⅰ and was then transformed into E. coli Top10. Thepositive clones were identified by restriction enzymes and DNA sequence analysis. The fusion protein was induced by IPTGand purified by affinity chromatography using ProBond~(TM) Resin (a kind of nickel-charged sepharose resin) and was identi-fied by SDS-PAGE and Western blotting. Results The products of PCR, cleavage and link reaction were same as ex-pected and the sequence of inserted fragment in the recombinant plasmid was same as reported except one synonymy muta-tion. A 58 kDa fusion protein was induced by IPTG and was purified by chromatography. Conclusion Fusion proteincontaining Toxoplasma gondii P30 was achieved and was provided as experiment material for further research.

Exogenous Nitric Oxide Induces Apoptosis in Tachyzoites of Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To explore whether NO is able to induce apoptosis in Toxoplasma gondii tachyzoites.Methods Apoptosis induced by NO in T^gondii tachyzoites was investigated by TUNEL (terminal\|deoxynucleotidyl transferase mediated d\|UTP nick end labeling ) method, electron microscopy and agarose gel electrophoresis. Results NO donor, sodium nitroprusside (SNP),was found to induce apoptosis in Toxoplasma gondii tachyzoites in a time\| and dose\|dependent manner by TUNEL detection. N\|acetylcysteine, a NO scavenger, could inhibit SNP\|induced apoptosis in the tachyzoites while potassium ferricyanide could not induce apoptosis in the tachyzoite. Electron macroscopy showed that SNP\|treated tachyzoites possessed typical morphological features of apoptosis, including chromatin condensation below the nuclear membrane, nuclear pyknosis, and formation of apoptotic body.Agarose gel electrophoresis revealed that SNP\|treated tachyzoite DNA fragment exhibited characteristic "DNA ladder" after 15 to 20 h. Conclusion SNP, NO donor, might induce apoptosis in T^gondii tachyzoites in terms of characteristic morphological and biochemical features.