Protein Kinase C-zeta (PKC-?) is a new member of protein kinase C family and has some specific characteristics as comparing with the classical PKC. It lacks the C 2 domain making its kinase activity Ca 2+ -independent, and it possesses only one zinc-finger region in its regulatory domain. Therefore, PKC-? does not bind Ca 2+ and can not be activated by diacylglycerol or phorbol esters. In addition, many researches showed that PKC-? could induce differentiation, mediate insulin-stimulation protein synthesis, activate immunity of human neutrophils, inhibit proliferation of cancer cells and regulate the function of actin cytoskeleton. What is more, PKC-? plays an important role in signal transduction of cells, such as mediating MAPK and NF-?B activation.

AIM: To investigate the relationship between the prevention of probucol on restenosis and vascular remodeling after percutaneous transluminal angioplasty(PTA) in rabbits. METHODS: New Zealand rabbit thoracic aorta atherosclerosis was induced by 3.5F ballon catheter injury following a 4-weeks feeding of high cholesterol diet, and PTA was performed by using 3.5F balloon catheter. Probucol(1g/d) or vitamin E (400 mg/d) was administrated one week before PTA. Two weeks after PTA, the bore and outside diameter (OD) of arteries, the area circumscribing by intimal elastic lamina (IEL), the area circumscribing by extral elastic lamina (EEL), medial area (MA), neointima area/medial area (NEA/MA) were analyzed by computerized digitizer system. Lipids of serum were measured by means of biochemical assay.RESULTS: After two weeks of PTA, the intima proliferation and lumen restenosis were observed obviously. However, with probucol treatment for 3 weeks, the restenosis of aorta was inhibited significantly by increasing bore, outside diameter, and lumen area of rabbits aortas and decreasing NEA, NEA/MA. Furthermore, probucol regulated vascular remodeling by increasing the area circumscribing by IEL [(3.50?0.20)mm 2 vs (1.59?0.23) mm 2, P

This paper studied the protective effect of gypenosides (GPS) on oxidative damage of myocardium of guinea pig, Using xan-thine-xanthine oxidase (X-XOD) producing free radicals. In the isolated guinea pig papillary muscles, X- XOD produced the quick positive inotropism at first and then the continuous negative one, shortened the functional refractory period (FRP) and elevated the excitability and increased the automaticity induced by adrenaline, inhibited the activity of superoxide dismutase (SOD) and increased the content of malondi-aldehyde (MDA). GPS inhibited the negative inotropism of papillary muscles produced by X-XOD,resisted the changes of FRP, automaticity and excitability induced by X- XOD. Meanwhile, GPS antagonized the effect of X-XOD which decreased activity of SOD and increased the content of MDA. These studies indicate that GPS can protect the myocardium from oxidative damage.

Daxx is found in the nucleus where it localizes to PML oncogenic domains (PODs). Its multiple domains can interact proteins involved in transcriptional regulation and apoptotic signal transduction. In addition, Daxx is associated with viral infection、tumorigenesis and embryonic development.

To produce monoclonal antibody (mAb) specifically against human thrombomodulin (hTM), an immune-tolerizing procedure was employed to generate monoclonal antibodies specific to hTM. Female BALB/c mice were first immunized with CHO cells following at 10 min, 24 h, 48 h by intraperitoneal injection of different doses of cyclophosphamide (CP) 2 times at an interval of 2 weeks, thereby tolerizing the mice to common epitopes shared between CHO and CHO-TM5 cells. Subsequently the selected mice with the lowest titer of serum polyclonal antibody by cellular enzyme-linked immunoabsorbent assay (CELISA) were immunized with CHO-TM5 cells, which have stable high level expression of hTM, to produce antibodies specific to hTM 3 times at an interval of 2 weeks. On the third day after the third immunization, mouse with the highest titer of serum polyclonal antibody was sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with CELISA. To improve probability to obtain specific mAb, CELISA was applied twice. The first CELISA was done with polyethylene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but having CHO cells monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5 +CHO- hybridoma cells. BALB/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded mAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Detection of CELISA showed that 100 mg/kg dose of CP could tolerize the mouse to common epitopes shared between CHO and CHO-TM5 cells. And CELISA also discovered that all hybridomas positive for CHO-TM5 cells were negative for CHO cells. Five lines of positive hybridoma cells had been obtained altogether and 2F7 was selected randomly for next investigation. The Ig subclass of the mAb 2F7 was IgG1 and the titer of ascitic mAb was 1?10-6. Furthermore, the content of ascitic mAb was 19.56 g/L and chromosome numbers is 98. Flow cytometry, CELISA and Western blotting assays demonstrated that mAb 2F7 could specifically recognize hTM expressed on CHO-TM5 and human umbilical vascular endothelial cells (HUVEC). Meanwhile, the tissue specificity of mAb 2F7 was also identified by immunohistochemical ABC staining. On the other hand, Western blotting assays indicated that mAb 2F7 could recognize the antigen protein with 105 ku molecular mass under reduction condition. Moreover, the dissociation constant of mAb 2F7, 1.22? 10-9 mol/L, indicated the affinity higher than some others. The results suggest that the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms. mAb 2F7 can specifically recognize the natural hTM expressed mainly on vascular endothelial cells, which will potentially useful for investigating the functions and clinic values of hTM.

Aim To compare the effects of the non-peptide angiotensin Ⅱ receptor type Ⅰ antagonist,Losartan,and the active vascular peptide,calcitonin gene-related peptide(CGRP),on the proliferation of vascular smooth muscle cells induced by angiotensin Ⅱ,and to explore the mechanism of depressor effect of Losartan and CGRP in vivo.Methods MTT,Thymidine incorporation and flow cytometry,were used to determine the ability of proliferation of VSMC induced by angiotensin Ⅱ in the presence or absence of Losartan or CGRP,Western blotting was used to determine the activity of ERK1/2.Results Losartan or CGRP inhibited the viability,DNA synthesis,cell proliferation index,and the activity of ERK1/2 in a dose-dependent manner.Conclusion Losartan or CGRP significantly inhibits the proliferation of VSMC induced by angiotensin Ⅱ;the inhibitory effect of CGRP is stronger than that of Losartan.The signaling path way is involved in ERK1/2.

In order to study the effects and the possible mechanisms of Daxx overexpressed in HepG2 to hydrogen peroxide treatment, and to search new targets for cancer chemotherapy, HepG2cells were transfected using lipofectamine 2000, and selected by treatment with G418. Stable cell lines were confirmed by reverse transeriptase polymerase chain reaction (RT-PCR) targeting vector gene. Experiments include the following groups: (1) control group (non-transfected cells); (2) transfected with empty vector (HepG2/GFP cells); and (3) transfected with pEGFP-C1-Daxx (HepG2/GFP-Daxx cells). After incubation with hydrogen peroxide (H2O2) for 24 h, cellular viability was analyzed by MTT, and cellular apoptosis was measured by flow cytometric analysis. Gene expression at protein level was detected by Western blot. The RT-PCR results showed that Daxx RNA in cells transfected with pEGFP-C1-Daxx was increased significantly compared with that in the HepG2/GFP cells. Fluorescence microscopy revealed that Daxx protein was localized in the nuclei. Hydrogen peroxide was used to induce apoptosis of HepG2 cells and observed that the hydrogen peroxide decreased the viability of HepG2 cells in concentration-dependent pattern. The IC50 values in three groups (Normal cells, HepG2/GFP cells and HepG2/GFP-Daxx cells) were 0.72, 0.76, and 0.49 mmol/L respectively. The apoptotic ratio was significantly higher in HepG2/GFP-Daxx cells as compared to the other two groups. HepG2/GFP-Daxx cell incubated with hydrogen peroxide, showed a significant increase in the activation of caspase-3 and JNK as compare with the other groups. Over-expression of Daxx facilitated HepG2 cells apoptosis induced by hydrogen peroxide. Furthermore, there may be a synergetic relation with apoptosis and increase of JNK activity.

AIM: To elucidate the effects of losartan on the expression ofmatrix metalloproteinases-2, JNK1/2 and proliferation in cardiac fibroblast. METHODS: Neonatal rat cardiac fibroblasts were cultured. The cells proliferation was determined by MTT. To determine effects of AngⅡ on JNK1/2 activity, cells were incubated (for 0, 2, 5, 10, 30, 60, 120 min) in serum-freemedia with AngⅡ, and the other group fibroblasts were exposed to serum-free media with or without AngⅡ and losartan (AngⅡ 100 nmol/L, AngⅡ 100 nmol/L+losartan 100 nmol/L, losartan100 nmol/L, losartan for 45 min before). Cells protein was collected with MBST buffer. The relative abundance of MMP-2, JNK1/2 and p-JNK1/2 in cells was determined by immunoblotting. The secretion of MMP-2 in media of cell culture was determined by ELISA. RESULTS: AngⅡ increased the proliferation of CFB in a dose-dependent manner, whereas losartan decreased the proliferation of CFB stimulated by AngⅡ in a dose-dependant manner, too (P＜0.05). The relative abundance of JNK1/2 was highest in AngⅡ of the 2-min-stimulated group. AngⅡincreased expression of JNK1/2 and MMP-2 protein (P＜0.05), on the contrary, losartan inhibited JNK1/2 and MMP-2 protein expression.CONCLUSION: AngⅡ induce the increase of proliferation of CFB, expression of JNK1/2 and MMP-2 in CFB, and losartan inhibits these effects of AngⅡ.

To deepen the teaching reform on comprehensive medical function experiment in our school,the mode of opening system of experiment teaching of medical function is proposed.The definition and characteristics of the opening system is discussed in this paper,and its theoretical foundation,guideline,goal location,effect and pending problems are also explored preliminarily.

Objective To investigate the effect of angiotensin Ⅱ-1 receptor antagonist losartan on expression of tumor necrosis factor-alpha (TNF-?) in the ventricular myocardium in the renovascular hypertension rats. Methods Renovascular hypertension model was obtained by clip left renal artery in Sprague-Dawley(SD) rats. After operation the rats were divided into 3 groups: sham group, two-kidney one clip (2K1C) group, and losartan treatment group(2K1C and losartan 20 mg/kg?d by drinking). Tail blood pressure was determined every week. Animals were euthanized after treatment with losartan for four weeks. Cardiac index(CI)was calculated by HW/BW, and TNF-? protein of ventricle myocardium was determined by ELISA and immunohistochemistry. Results Losartan significantly decreased blood pressure(P