AIM: To investigate the effects of Src family kinases (SFKs) on adenosine 5'-triphosphate (ATP)-induced long-term potentiation (LTP) in the spinal dorsal horn. METHODS: Male Sprague-Dawley rats (250-280 g) were used in the experiments. Western blotting, electrophysiological recording in spinal dorsal horn in vivo and immunohistochemistry were used in the study. The C-fiber-evoked field potentials were recorded at the superficial layers of spinal dorsal horn at the lumbar enlargement and the phosphorylation level and location of SFKs in spinal dorsal horn were examined by Western blotting and immunohistochemistry. RESULTS: Thirty min and 60 min after ATP application, the levels of phosphorylated SFKs (p-SFKs) were significantly increased.The p-SFKs were expressed in microglia, but not in astrocytes or neurons. Spinal application of SFK inhibitors prevented ATP-induced LTP. CONCLUSION: Microglial SFKs may play an important role in ATP-induced LTP of C-fiber evoked field potentials in the spinal dorsal horn.

AIM: To determine the effect of exogenous phosphocreatine (PCr) at different concentrations on transient outward potassium (Ito) current in rat ischemic ventricular mid-myocardial (M) cells and to explore the antiarrhythmia mechanism in the treatment of ischemic heart disease. METHODS: M cells were isolated enzymatically from left ventricular mid-myocardium of rats. Peak Ito current was recorded by patch-clamp technique in the whole-cell configuration when M cells were superfused with normal Tyrode solution,simple ischemic solution,and simulated ischemic solution containing PCr at concentrations of 5,10,20 and 30 mmol/L for 10 min. RESULTS: Peak Ito current density of M cells superfused with simple simulated ischemic solution was significantly reduced by (76.1±6.3)% (P<0.05) compared with M cells superfused with Tyrode solution. Ischemic solution containing 5,10,20 and 30 mmol/L PCr reduced peak Ito current density by (57.1±9.6)% (P<0.05),(40.3±10.3)% (P<0.05),(34.3±9.6)% (P<0.05) and (32.1±10.6)% (P<0.05),respectively. There was statistical difference among ischemic solution without PCr and containing PCr at concentrations of 5 and 10 mmol/L groups (P<0.05). No statistical difference among groups of 10,20 and 30 mmol/L PCr was observed (P>0.05). CONCLUSION: PCr reverses the inhibition of Ito current under ischemic condition in M cells,which may be the mechanism responsible for arrhythmia prevention in ischemic heart disease. PCr at concentrations of 0～10 mmol/L exerts significant dose-effect relationship.

AIM: To investigate the role of N-cadherin in the delamination of neural crest cells. METHODS: The normal expression of N-cadherin in neural tube was identified using in situ hybridization. The cells with N-cadherin over expression were obtained by transfection of wild-type N-cadherin (wt-N-cadherin) ,and the cells with N-cadherin silencing expression were obtained by transfection of dominant-negative N-cadherin (dn-N-cadherin). The migration of cranial neural crest cells was determined by the technique of immunohistochemistry. RESULTS: Either overexpression or down-regulation of N-cadherin significantly affected the migration of cranial neural crest cells. CONCLUSION: Delamination and migration of the cranial neural crest cells rely on the relative N-cadherin expression in the neural tube during neurulation.

AIM:We examined the efficacy ofanti-L3T4 McAb in the T cell signaling pathway in treating ex-Derimental antoimmune cardiomyopathy in BALB/c mice,as a model of the autoimmune mechanism involved in human di-latedardiomyopathy(DCM).METHODS:ADP/ATP carrier peptides were used to induce autoimmune cardiomyopathy in BALB/c mice.Afier 3 months.anti-L3T4 McAb WaS administered to deplete CD4+ T ceHs in the mice.Real-time PCR were used to detect the expression of intracellular signaling molecules(p561ck,p59fyn and Zap-70)and cytokine production(IFN-r,IL-2 and IL-4)in T cells.The expression of CIM5 was determined by immunohistochemistry a-nalysis.RESULTS:Reduced expression of p561ck,p59fyn and Zap-70 and the reduced cytokine production of IFN-r, IL-2 and IL-4 in T cells of anti-IJ3T4-treated DCM mice were found.Also,the expression of CD45 in spleen T cells was significantiv decreased in the anti-L3T4-treated group.In contrast,immunization with irrelevant Ab did not protect the mice.the expression of T cell signaling molecules,CD45,and cytokine were not inhibited.CONCLUSION:Thesestudies provide direct evidenee that anti-L3T4 McAb can be all effective immunomodulator to T cell signal molecules and 8ubsequent cytokine production events in ADP/ATP carrier-induced DCM in BALB/c mice.

AIM:Panton-Valentine leukocidin(PVL)is a pore-forming toxin secreted by Staphylococcus aureus epidemiologieally associated with the often-lethal necrotizing pneumonia.Until now,the mechanisms of pathogene-sis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult.In the present study，we obtain recombinant staphylococcal F and S components of the Panton-Valentine leukocidin by gene engineering and evaluate its biological activity in vitro,which provides an experimental basis for the further studies of its biological func-tion and its toxicity in pneumonia.METHODS:The full-length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by hiSh-fidelity PCR was cloned into prokaryotic expression vector pET22b(+),and the vector was transformed into BL21(DE3)plysS to construct a prokaryotie expression system.The integrity of the opening-reading frame of each construct was verified by DNA sequencing.The recombinant PVL(rPVL)was induced by1.0 mmol/L IPTG.The expressed products were identified by SDS-PAGE and the fusion proteins(6His-LukS-PV and 6His-LukF-PV)were purified from lysates of transfeeted E.coli cells by affinity chromatography on nitrilotriacetic acid columns.The eytolytie activity was tested by incubation of rPVL with human polymorphonuclear neutrophils(PMNs)in vitro.RESULTS:The nueleotide sequence of the cloned PVL gene was the same as that of reported in GenBank.E coli BL21(DE3)plysS containing recombinant vectors grow at 37℃causes some proteins to accumulate as inclusion bodies.while incubation at 30℃led to a significant amount of soluble active proteins which accounted for about 31.7％ of the total bacterial protein.The relative molecular weight showed on SDS-PAGE profile was consistent with the expected value which the LukS-PV protein was about 34 kD.and the LukF-PV protein was about 35 kD.The purified rPVL was obtained and its cytolytic activity to PMNs was demonstrated.CONCLUSION:The genes of 1ukS-PV and lukF-PV are successfully cloned into plasmid pET22b(+)and expressed in E coli respectively.which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.

AIM,To verify whether p73;a homologue of p53,which supposed]y acts as a tumor suppressor gene in neuroblastoma,might also be a tumor suppressor in non-small cell lung cancer.METHODS:The allelic expres-sion of p73 in the six non-small cell lung cancer cell lines was studied by Sty I polymorphism analysis.The P73 gene ex.pressions in these six cell lines were examined by reverse transcription-PCR,the expressions of P73 protein in the five cell lines inducing tumor8 were also determined by immunohistochemistry.RESULTS:Homozygous allelic expression was dem.onstrated in all six cell lines and the GC/GC genotype Was the predominant type.Complete loss of the p73 expression both at mRNA and the protein level was revealed.CONCLUSION:Taken together,our data suggest that p73 might play a role as a tumor suppressor gene in human non-small cell lung cancer cell lines.

AIM: This study is to determine changes of hippocampal norepinephrine (NE) and serotonin (5 -HT) in long term physical exercise and chronic psychological stress, and to study the roles of the two monoamine transmitters in the effect of exercise counteracting stress - induced hippocampal damages in brain. METHODS : Levels of hippocampal NE and 5 - HT in rats undergoing 4 - week voluntary wheel running exercise (exercise group) or 3 - week restraint stress (stress group) or 4 - week exercise and 3 - week stress (exercise - stress group) were detected by high - performance liquid chromatography using electrochemical detection. RESULTS: It is showed that levels of hippocampal NEand 5 - HT increased significantly (P < 0. 01) in the exercised rats, and in the stressed rats, hippocampal 5 - HT levelssignificantly decreased(P < 0. 05). Additionally, the NE levels maintained significant high (P < 0. 01) in exercise -stressed rats compared to the pure stressed ones. On the other hand, no obvious difference was observed in hippocampal5 - HT levels between stress group and exercise - stress group, which were all significant lower (P < 0. 05) than that in exercise group. CONCLUSION: It is suggested that both the NE and 5 - HT may play important roles in mediating the exercise-induced positive effects and the 5 - HT may play an important role in stress - induced negative effects on the hippocampus. Moreover, NE may take more action in the exercise attenuating stress - induced hippocampal damages. The hippocampal NE may be more susceptible to exercise, and the hippocampal 5 - HT may be more susceptible to stress.`

AIM:To investigate the activation and inactivation of nuclear factor kappa B(NF-κB)when tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is applied to induce the apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS:After the treatment of TRAIL or LPS at different doses,we tested the nuclear translocation of NF-κB by cell immunohistochemical staining and electrophoretie mobility shift assay(EMSA),and evaluated the level of IκB by RT-PCR under pyrrolidine dithiocarbamate(PDTC)treatment.RESULTS:EMSA and cell immunohistochemical analysis showed that the translocation of NF-κB was significantly activated when PC-3M cells were treated with TRAIL or LPS(P＜0.05).The pretreatment of PDTC upregulated the expression of IκB and blocked the nuclear translocation of NF-κB.CONCLUSION:TRAIL remarkably stimulates the activation of nuclear NF-κB in androgen-independent prostate cancer cells.On the other hand,the translocation of NF-κB can be significantly and efficiently inhibited in PC-3M cells by pretreatment with PDTC.The increased expression of IκB might be a clue for this inhibition,which means the possible way to enhance the effect of TRAIL in the apoptosis of prostate cancer cells.

AIM:To evaluate the ability of dendritic cells (DCs) loaded with tumor lysate to initiate cell mediated immune responses by stimulating naive T cells, and the efficiency of activated T cells to kill autologous tumor cells in vitro. METHODS: The peripheral blood lymphocytes and monocytes were obtained from the advanced renal cell carcinoma patient by eonglutination method. The immature dendritic cells were generated in the presence of interleukin -4(IL-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF) from monocytes of healthy individuals.These cells were pulsed with tumor lysate or not. Induction of tumor-specific cytotoxic T lymphocytes(CTLs) response by mature dendritic cells (mDCs) was evaluated by the CD95(Fas) expression assay through FCM and the cytotoxic assay a gninst autolognns human tumor cells. RESULTS: Human immature dendritic cells and T cells obtained from healthy donors were stimulated with tumor- pulsed dendritic cells. The immature dendritic cells were applied to the cytotoxicity assay a gainst target autologons tumor cells. The CD95 (Fas) expression, IFN-γ, and TNF -α secreted by the CTLs in tumor lysate-plused DC group were higher than those of other groups. The capacity of the CTLs to kill autolognns tumor cells was significantly different(P<0. 05). Antigen-specific DCs vaccine can induce T cells activation and proliferation, thus we can obtain higher proportion of tumor specific cytotoxic T cells(CTLs), and enhance the CTLs to secret IFN-γ and TNF-α. CONCLUSION: Our results indicate that monocyte-derived human dendritic cells pulsed with tumor lysate could in duce the specific antitumor effect against autologons tumors. This in vitro model offers a new and simple approach to the development of DC + CTL - based immunotherapy.

AIM: To verify the protection of astragalus polysaecharide (APS) on H2O2 - stressed skin fibro-blasts. METHODS: A model of acute H2O2 stress in primary skin fibroblast was used at concentration of 0.5 mmol/L by 30 rain incubation. Dose responses of APS on cell survival was measured by MTI, cell death was evaluated by DAPI, and effect of APS on mitochondria, mitochondrial membrane potential and lysesome stabilization were measured by confocal microscopy. RESULTS: APS improved cell survival in a dose -dependent manner, starting at 0. 5 mg/L and with a maximum at 1 mg/L. Moreover, APS inhibited mitochondrial membrane potential collapse, protected mitechondrial morphology and stablized lysosomal membrane. CONCLUSION: The results suggest the existence, at the mitochondria-lysosome level, of a new pathway of apoptotic regulation by APS. This might constitute a new therapeutic target where oxidative stress and lysesomal impairment are involved.