BACKGROUND: Focal adhesion kinase (FAK) phosphorylation influences the proliferation, migration and apoptosis of cells. However, there are no reports about the role of FAK phosphorylation in migration and proliferation of vascular smooth muscle cells during the process of vascular restenosis.OBJECTIVE : To analyze the correlation between FAK phosphorylation and vascular remodeling after balloon endothelial denudation in rats.DESIGN: Random controlled experiments in animals.SETTING: Cardiology Institute of Xianning College; Shenzhen Center for Chronic Disease Prevention and Control;Department of Cardiology at the Affiliated Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology.MATERrALS: The experiment was carried out in the Laboratory of Cardiology at the Affiliated Tongji Hospital from March to May in 2005. Forty male Wistar rats were used in the study, and they were managed according to the ethical standard of animals.METHODS: ①Forty rats were randomized into five groups (n =8): non-balloon endothelial denudation control group, and 4-day, 8-day, 16-day and 24-day model groups after balloon endothelial denudation. ②The rat models of restenosis following balloon endothelial denudation were built. At days 4, 8, 16 and 24 postoperatively, the denudated vessels were harvested for the detection of the morphological changes, which were taken as the judgment of successful models;Western blot method was used to detect the expression and activity of FAK. Abdominal aorta of left common carotid in rats was served as controls.MAIN OUTCOME MEASURES: The expressions of FAK and phosphorylation FAK in rat artery.RESULTS: Totally 40 rats were all involved in the result analysis.①A small quantity of FAK without phosphorylation could be detected in the controlled rats. Compared with the control group, the expression and activity of FAK in the model group rapidly increased at 4 days after balloon injury (P ＜ 0.05), and they changed in a similar manner with the increase of injury duration. The expression of FAK reached a peak at 8 days and began to descend at 16 days. The expression of phosphorylation FAK reached a peak at 16 days and then rapidly descended. ②Focal hyperplasia of intima was found at 8 days after balloon endothelial denudation, and diffuse hyperplasia of intima was found at 16-24 days after denudation.CONCLUSION: FAK can take part in vascular remodeling after balloon endothelial denudation in rats. The level of intimal hyperplasia is closely associated with the level of FAK and FAK phosphorylation.

Objective To observe the specific humoral and cellular immune response in BALB/c mice injected with pS and p18S. Methods pS and p18S were constructed separately by inserting HBsAg gene fragment and the fusion gene fragment of HBsAg and mouse interleukin 18(IL 18) into the reading frame of pcDNA3.1+. Mice were injected with either plasmid intramuscularly in a total dose of 300 ?g per mouse. Every serum sample was detected for anti HBs using enzyme linked immunosorbent assay(ELISA). Furthermore, HBsAg specific cytotoxic T lymphocytes activity was measured. Results The expression of HBsAg was demonstrated by ELISA in p815 cells transfected with pS and p18S. pS can stimulate a positive antibody response. The average level was 135 mIU/ml, with the highest level of 530 mIU/ml. p18S could elicit relatively lower antibody response which was 20 mIU/ml. HBsAg specific CTL activities were 37.1% and 34% separately in pS and p18S immunized mice. It was only 13.2% when detected in pcDNA3.1+ immunization. Conclusion pS is effective to stimulate a humoral and cellular response in H 2d mice. IL 18 gene can not enhance the immune response when fused with HBsAg gene. Conversely, it seems to inhibit an immune response.

The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways. Using the yeast-two hybrid method, we have isolated a clone that encodes a novel NS5A--associated binding protein: NS5ABP37. Reverse transcription polymerase chain reaction (RT-PCR) method was employed to amplify the full fragment,and the plasmid pGADT7-NS5ABP37 with the Saccharomyces cerevisiae vector pGADT7 was constructed. To prove the interaction, yeast cell Y187 transformed with pGADT7-NS5ABP37 was mated with yeast cell AH109 containing pGBKT7-NS5A to verify the interaction between the novel protein coded by the new gene NS5ABP37 and NS5A.

Objective HCV NS3 protein plays an important role in disease caused by HCV. We investigate the gene expression of HCV NS3 in yeast for future study of the function of the protein. Methods PCR was performed to amplify the gene of HCV NS3 from the plasmid pBRTM/HCV containing the whole fragment of HCV and the gene was cloned into pGEM T vector. Thereafter, HCV NS3 gene was cut from pGEM T vector and cloned into yeast expression plasmid pGBKT7, and recombinant pGBKT7∶NS3 was transformed into yeast AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. Results HCV NS3 gene was successfully cloned into pGBKT7. The results of SDS PAGE and Western blotting assay showed that the molecular weight of the expressed product was about 22000 Da and HCV NS3 protein was existed within yeast cells.Conclusions HCV NS3 was successfully expressed in yeast expression system.

Objective To observe the clinical efficacy and safety of propafenone on paroxysmal supraventricular tachycardia (PSVT).Methods 37 patients with PSVT were injected with 70 mg propafenone intravenously in 5 minutes.The unrecovered patients in 20 minutes were injected with 70 mg propafenone intravenously again,who were given 70 mg propafenone intravenously once again if not controlled in late 30 minuted.Blood pressure,heart beat,12 lead electrocardiogram were recorded before and after use of drug.Results The significant effective rate was 77.5% and effective rate was 21.6% with overall effective rate of 97.3% and effective time of 1～55 (7.1?2.8) minutes.The average accumulated dose of propafenone was 105 mg.Some side effects were observed in part of patients,including slight reduction of blood pressure,P R interval.QRS wave and Q T interval prolonged,temporary type sino atrial block,vertigo and nausea,etc whice could vanish gradually if not treated.Conclusion Propafenone by intravenous injection can quickly contro PSVT with saftey and effectiveness.

BACKGROUND:Adherent migration of vascular smooth muscle cells and proliferated remodeling of vessel walls following vascular endothelial injury play a key role in onset of restenosis after percutaneous transluminal coronary angioplasty,while expression and phosphorylation activation of focal adhesion kinase are attacked during this period.OBJECTIVE:To observe the interventional effect of angiotensin receptor antagonist,telmisartan,on the expression and activation of focal adhesion kinase during vascular-injured remodeling.DESIGN:Randomized controlled animal study.SETTING:Shenzhen Center for Chronic Disease Control and Prevention;Department of Cardiology,Affiliated Hospital of Xianning Medical College;Department of Cardiology,Tongji Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology.MATERIALS:The experiment was carded out in the Cardiovascular Laboratory,Wuhan Tongji Hospital from March to May 2005.A total of 36 male Wistar rats weighing 300-360 g were randomly divided into control group,model group and treatment group with 12 in each group.METHODS:Aortal restenosis models were established after endothelial denudation.Foley's tube technique was used to strip vascular endothetium of rats in the model group and the treatment group;while,rats in the control group and the model group were fed and drunk normally; in addition, rats in the treatment group were also given 5 mg/(kg·d) telmisartan solution.Thirty clays after successive administration,vessels at injured sites were collected to observe the morphological changes and extract RNA and protein.Reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure mRNA expression of focal adhesion kinase and Westem blot technique was USod to measure total protein and phosphorylated protein of focal adhesion kinase.MAIN OUTCOME MEASURES: Proliferation of vessel wall at injured sites; mRNA expression, total protein and phosphorylated protein of focal adhesion kinase at injured sites at 30 days after administration.RESULTS:A total of 36 rats Were involved in the final analysis.Thirty days after operation, aortic tunica intima was thickened in the model group, mRNA expression of focal adhesion kinase was increased, and total protein and phosphorylated protein were higher in the model group than those in the control group;however,proliferation of tunica 5ntima vasorum was lightened in the treatment group, activity of mRNA expression of focal adhesion kinase was decreased, and total protein and phosphorylated protein were lower in the treatment group than those in the model group.CONCLUSION: Telmisartan can remarkably relieve proliferation of tunica intima after denudation and inhibit expression and activation of focal adhesion kinase after vascular injury.Effect of telmisartan on vascular-injured remodeling may be related to inhibiting expression and activation of focal adhesion kinase.

This study investigated the expression of hemeoxygenase-1 (HO-1) in rats with acute lung rejection and its implication. A valid rat orthotopic left lung transplantation model (SD rat-->Wistar rat) was established by using an improved three-cuff anastomosis technique. The rats were divided into control group, CoPP (HO-1 inducer)-treated group and ZnPP (HO-1 inhibitor)- treated group. The severity of acute rejection was graded on the basis of the morphologic changes of the lung samples stained with HE. The expression of HO-1 protein in lung tissue was detected by using immunohistochemistry and Western blot, and HO-1 mRNA activity was assayed by RT-PCR. The results showed that the expression of HO-1 protein was significantly increased with the acute rejection grading in rats (P<0.01). As compared with control and ZnPP-treated groups, the severity of acute rejection was not alleviated and the grade not reduced significantly in CoPP-treated group (P>0.05). It was concluded that HO-1 protein might be involved in the pathological process of post-graft acute rejection. The expression of HO-1 protein was increased gradually with aggravation of acute rejection, and HO-1 protein might be used as an index to monitor acute rejection after lung transplantation.
Graft Rejection/*enzymology ; Heme Oxygenase (Decyclizing)/genetics ; Heme Oxygenase (Decyclizing)/*metabolism ; Lung Transplantation ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Rats, Sprague-Dawley ; Rats, Wistar

Objective To evaluate the changes in the expression of CXCR3 in regulatory T cells (Tregs) in the renal tissues of mice with renal ischemia-reperfusion (I/R) injury .Methods Forty-eight SPF male C57BL/6J mice ,aged 8-12 yr ,weighing 20-25 g ,were randomly divided into 3 groups ( n=16 each ) using a random number table:sham operation group (group S) ,group I/R and CD25 monoclonal antibody PC61 group (group P) . Bilateral kidneys were exposed and their pedicles were occluded for 45 min with atraumatic mini-clamp followed by 72 h reperfusion .PC61 250 μg was injected intraperitoneally at 24 h before the model was established .Blood samples were collected from the inferior vena cava at 24 and 72 h of reperfusion (T1 ,2 ) for determination of serum blood urea nitrogen (BUN) and creatinine (Cr) concentrations .Bilateral kidneys were obtained for determination of CD4+ CD25+ Foxp3+ Treg count and CXCR3+ CD4+ CD25+ Foxp3+ Treg count in renal tissues and the pathological changes of the kidney were scored .Results Compared with group S , the serum BUN and Cr concentrations and pathological scores were significantly increased at T1 ,2 in I/R and P groups ,and the number of CD4+ CD25+ Foxp3+ Treg and CXCR3+ CD4+ CD25+ Foxp3+ Treg was increased at T2 in I/R group ( P<0.05) .Compared with group I/R ,the serum BUN and Cr concentrations and pathological scores were significantly increased at T2 ,and the number of CD4+ CD25+ Foxp3+ Treg and CXCR3+ CD4+ CD25+ Foxp3+ Treg was decreased at T2 in P group ( P<0.05 ) .Conclusion Up-regulation of CXCR3 is helpful in migration of Tregs into the renal tissues of mice with renal I/R injury .

Polymerase chain reaction was employed to amplify the whole HBV CP region from the serum of patients with chronic hepatitis B virus(HBV) infection, and then the PCR products were subcloned into pGEM Teasy vectors. Clones were randomly selected to be sequenced and the selected clones were compared to look for the difference.The sequencing results suggested that each sequence of selected clones was different and there were HBV quasispecies groups in patients. There were hot deletion region and point mutation near the TATA like box of CP gene. To address whether the mutations were responsible for the transcriptional activity, the wild type(wt) and the mutants of HBV CP genes were subcloned into pcDNA3 1( ) vectors, respectively. The reverse oriented clones were digested with KpnI and XhoI, and cloned into the KpnI and XhoI sites of the chloramphenicol acetyltransferase (CAT) expressing vector (pCAT3 basic).The recombinant CAT plasmids were transfected into HepG2 cells using lipofectamine PLUS reagent, and the CAT expression which indirectly represented the transcriptional activity of HBV CP lying upstream of CAT gene was detected with a CAT ELISA kit. The restriction enzyme digesting results indicated that the recombinant CAT plasmids were successfully constructed, and the transfection tests indicated that the transcriptional activity of the mutants with deletion or substitute point mutation of TATA like box were reduced in comparison with that of CPwt. The HBV CP gene heterogeneity downregulated the transcriptional activity to some extent.

mRNAs of alpha-adrenoceptor (α-AR) subtypes are found in neurons in dorsal root ganglion (DRG) and change after peripheral nerve injury. In this study, the distribution of α-AR subtype proteins was studied in L5 DRG of normal rats and rats with chronic constriction injury of sciatic nerve (CCI). Using immunofluorescence technique, it was found that α1A-, α1B-, and α2A-AR proteins were expressed in large, medium, and small size neurons in normal DRG, and significantly increased in all size neurons 14 days after CCI. α1D- and α2C-AR was also expressed in all size neurons in normal DRG. However, α1D-AR was significantly increased and α2C-AR was decreased in small size neurons 14 days post CCI. α2B-AR neurons were not detectable in normal and CCI DRG. Co-expression of α1A- and α2A-AR in the same neuron was observed in normal DRG and increased post CCI. Collectively, these results indicated that there is distinct distribution of α-AR subtypes in DRG neurons, and the distribution and levels of expression of α-AR subtypes change differently after CCI. The up-regulation of α-AR subtypes in DRG neurons may play an important role in the process of generating and transmitting neuropathic pain.