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: The present study was designed to observe the effect of [D- Arg1, D- Trp7,9, Leu11] - substance P (spantide), a non- selective antagonist of NK receptors, on the up- regulation of nitric oxide synthase (NOS) induced by formalin test. METHODS: Formalin (5%, 0.2 mL) was subcutaneously injected into the plantar side of the right hind paw to produce persistent pain and hyperalgesia. The pain response was determined by spontaneous flinch reflex test. NOS expression was examined using NADPH- d histochemical staining. Spantide was intrathecally injected via L5 - L6 intervertebral space 5 min prior to the formalin injection. RESULTS: Injection of formalin resulted in a characteristic behavioral response consisting of vigorous scratching, biting, licking and lifting of the injected hind paw from the box' s bottom. Following these behavioral responses, the NOS expression was up- regulated in the pericentral canal region of the L5 segment of the spinal cord. Pre- treatment with spantide depressed the spontaneous flinches of the injected paw in the second phase of the formalin test. At the same time, the upregulation of NOS was substantially inhibited. CONCLUSION: It might be concluded that substance P played an important role in the up - regulation of NOS in the pericentral canal region of the spinal cord in the formalin test.

AIM: To explore the molecular mechanisms about fibrosis and transforming growth factor (TGF) - β1 as well as inflammation in rats heart after acute myocardial infarction (AMI). METHODS: AMI model in rats was produced by left coronary artery ligation. Samples of rat cardiac tissue were collected at the end of 1 week, 4 weeks and 8 weeks. Hemodynamics had been performed before rats were sacrificed. Reverse transcription polymerase chain reaction (RT- PCR) and immunohistochemical methods were used to analyze mRNA expression and protein production of TGF- β1, respectively. Hydroxyproline was determined by chloramines T method. HE staining was resorted to analyze pathological myocardium after AMI. RESULTS: There were remarkable differences in hemodynamics between AMI groups and sham group (P＜0.01). Compared with sham group, TGF-β1mRNA expression and protein production and hydroxyproline quantification were enhanced greatly. Among them, the levels of TGF -β1 and hydroxyproline at 1 week were higher than those at 4 weeks or 8 weeks. A positive correlation between TGF- β1 protein and hydroxyproline was presented (r=0.75 - 0.99, P＜0.05). In microscope, leucocytes infiltrated significantly in the infarcted and border myocardium at 1 week after AMI, and were rarely seen at 4 weeks and 8 weeks. TGF - β1 protein were detected in cytoplasm of cardiac myocyte and leucocytes at 1 week, and at 4 and 8 weeks in myofibroblast and interstitial. CONCLUSIONS:TGF- β1 is upregulated and found in cytoplasm of cardiac myocyte and leucocytes as well as myofibroblast in heart after AMI,which is associated with dynamic changes of hydroxyproline content and inflammation. TGF - β1 is showed to play an important role in myocardial inflammatory repair and ventricular remodeling after AMI.

AIM: To study the possible role of nitric oxide (NO) in the development of periodontitis and the relationship between the NO concentration and the attachment loss. METHODS: Seventy- two Sprngue- Dawley rats were randomly assigned to two groups, the control group and periodontitis group. Experimental periodontitis in rats was produced by a ligature of braided silk. The nitric oxide concentration was indirectly ascertained by the concentration of nitrite (NO2-) and nitrate (NO3-)in the gingival tissue, which was assayed by spectrophotometry. The attachment loss (AL) was measured by the technology of the cellular graphics engineering research. The histopathologic change in periodontium was observed under a light microscope by using the histotomy. RESULTS: Compared to control group, the NO2-/NO3 - concentration in gingival tissue was significantly higher in periodontitis group at four weeks and eight weeks following ligation (P＜0.01). In periodontitis group, the NO2-/NO3 - concentration in gingival tissue was higher at eight weeks than that at four weeks following ligation (P＜0.01). At four weeks and eight weeks, the AL in experimental periodontitis in rats was significantly increased than that at one week after ligation ( P＜0.01); and the AL was also much higher at eight weeks than that at four weeks (P＜0.01). CONCLUSIONS: The NO2-/NO3- concentration in the gingival tissue in periodontitis group was significantly higher than that in control group. These results demonstrate that the NO2-/NO3- concentration is related to the severity of AL, and NO synthesis is very important to the process of inflammation and lesion in periodontium. Reducing NO production may be of great therapeutic value in the treatment of periodontitis.

AIM: To study the effect of wild-type p53 gene on the differentiation, apoptosis and expression of scavenger receptor CD36 in U937 cells. METHODS: Recombinant adenovirus vector with wild-type p53 gene was constructed and used to transfect U937 cells. With the expression of wild-type p53 gene following adenoviral infection, transfected U937 cells were largely promoted to differentiate into macrophages. RESUITS: Trypanblue-staining test demonstrated that the percentage of positive cells increased from (14.2±5.5)% to (64.6±9.2)% and nitroblue tetrazolium (NBT) reduction test reached similar results (6.3±1.8)% vs (49.7±12.6)%. Furthermore, CD36 mRNA was up-regulated as confirmed by RT-PCR. The increased expression level of CD 36 was also detected by flow cytometry analysis. CONCLUSION: These results suggest that wild-type p53 gene can affect U937 cells differentiation and apoptosis, up-regulate expression of scavenger receptor CD36. It may have a potential significance on atherogenesis.

AIM: To investigate the role of nuclear factor- κB (NF- κB) in the expression of monocyte chemoatractant protein- 1 (MCP- 1) in human mesangial cells (HMCs) induced by oxidized low- density lipoprotein (Ox- LDL).METHODS: HMCs were used as target cells. Inhibitory κBα (IκBα) and MCP- 1 protein level was measured by cell ELISA.Activities of transcriptional factors NF- κB were determined by electrophoresis mobility shift assay (EMSA). Immunohistochemistry was used to detect the translocation of Rel p65. RESULTS: NF - κB DNA - binding activation in MCs was observed when 10-100 mg/L Ox - LDL was added to the medium, and 50 mg/L Ox - LDL caused the strongest effect (8.50 ± 1.14, P ＜ 0.01vs control; P ＜ 0.05 vs 10, 25 and 100 mg/L Ox - LDL). The most optimal stimulation time was 60 min ( 11.0 ± 2.11, P ＜0.01 vs control; P ＜ 0.05 vs 30 min or 240 min). IκBα protein level in MC dropped down most obviously after 60 min incubation with 50 mg/L Ox - LDL (0.050 ± 0.006, n = 5, P ＜ 0.01 vs control), while MCP- 1 expression level was the highest (0.331± 0.016, n = 5, P ＜ 0.01 vs control). The translocation of Rel p65 from cytoplasm to nucleus was detected too. NF - κB inhibitor pyrroledithiocarbomate (PDTC) could inhibit these effects induced by Ox- DL. CONCLUSION: Activation of NF- κB regulate the expression of MCP- 1 in HMCs induced by Ox - LDL.

AIM: To study the effect and the mechanism of crenulatin, an effective constituent of Chinese traditional medicine, on apoptosis of cerebral microvascular endothelial cells. METHODS: The following terminal concentrations of crenulatin were used in the study: 25 mg/L and 100 mg/L. Apoptosis of mouse cerebral microvascular endothelial cells (bEnd. 3 cell line) was evaluated by flow cytometer, immunocytochemical assay (Fas, Bcl - 2) and Western blotting (caspase - 3) after culture for 24 h. RESULTS: Compared with control group, apoptosis of bEnd. 3 cells in 25 mg/L group was significantly inhibited ( P ＜0.05), but apoptosis in the 100 mg/L group was significantly increased (P ＜ 0.05). In apoptosis inhibited group, the Fas immunocytochemical staining was weaker, the positive cells were significantly decreased ( P ＜ 0.05) and caspase - 3 expression was decreased compared with control group; however, the Bcl - 2 staining was stronger and the positive cells were significantly increased ( P ＜ 0.05). On the other hand, in apoptosis increased group ( 100 mg/L group), the changes were just opposite. CONCLUSIONS: The effect of crenulatin on apoptosis of mouse cerebral microvascular endothelial cells possesses a dual - direction change, inhibitive effect in 25 mg/L and stimulative effect in 100 mg/L group, respectively. The mechanism is related to the alterations of Fas/Bcl - 2 expression and caspase - 3 activity.