Aim To evaluate the effects of fibrinolytic enzyme FⅡ from agkistrodon acutus venom on an experimental model of kidney thrombus induced by lipopolysaccharide(LPS). Methods The model of microvascular thrombosis in the rabbits kidney was performed by the method of Hermida, which was induced by infusing LPS. Treatments were begun simultaneously with LPS infusion, through the contralateral marginal ear vein. Six different groups were established: NS 10 ml?h~-1 was infused as the negative control group, urokinase ~20 000 IU?kg~-1 ?h~-1 as positive control group, FⅡwas infused with the dosage of 0.1(Low-dose), 0.3 (medium-dose),0.6 (high-dose) mg?kg~-1 ?h~-1 . The further rabbits, which were given neither LPS nor FⅡ, were infused with saline solution through both marginal ear veins. Kinney sections were examined for the presence of fibrin microthrombi. The measurement of FDP concentrations was used to assess the degradation of microvascular thrombosis. Results Intense fibrin deposition was also detected and FDP concentrations were (78.21?4.79)% and (84.27?6.21)% at 2 and 6 hours after LPS administration in LPS-control group. Little fibrin deposition was detected and FDP concentration also increased in urokinase control group. A lot of fibrin deposition was detected in Low-dose FⅡ group,little fibrin deposition was detected in medium-dose FⅡ group, and no fibrin deposition was detected in high-dose FⅡ group. Additional all doses of FⅡ led to a significant increase in FDP concentration as compared with LPS-control group (P

Aim To find out the relationship between muscarinic receptor and reactive oxygen species (ROS) and the probable differences between the four muscarinic receptor subtypes. Methods We transfected the plasmid encoding muscarinic receptor (including subtypes: M_1, M_2, M_3 and M_4) into PC12 cells. Then PC12 cells were exposed to hydrogen peroxide (H_2O_2), carbachol and other inhibitors such as atropine, LY294002 and PD98059. Results The results showed that activation of muscarinic receptor by carbachol protected PC12-M_1, PC12-M_2,PC12-M_3 and PC12-M_4 cells from apoptosis induced by H_2O_2. There was no statistical difference in the protective effect between these four muscarinic receptor subtypes. By using the inhibitors, we found that atropine and LY294002 blocked the protective effect of activation of muscarinic receptor on apoptosis induced by H_2O_2. Conclusion Activation of muscarinic receptor retarded the apoptosis induced by H_2O_2. There was no difference between the four muscarinic receptor subtypes. The protective effect was mainly mediated by the activation of muscarinic receptor and phosphatidylinositol-3 kinase (PI3K).

Objective To investigate the biological effects of bcl-2 gene on neurons. Methods The recombinant expression plasmid pc DNA3-bcl-2 was constructed from pSFFV-bcl-2,then it was introduced into PC12 cell line by liposome method.Western blotting and immunohistochemistry in situ were applied to exam the exogenous gene expression.The two groups of cells(Group A,PC12 transfected by pcDNA3-bcl-2 and Group B,PC12 transfected by pcDNA3) were exposed to cisplatin with the concentration of 10*!?mol/L,50*!?mol/L, and 100*!?mol/L 72 hours later,the survival cells were estimated.Cell cycle indexes between these two groups were also studied by FCM. Results The recombinant expression plasmid pcDNA 3-bcl-2 was constructed successfully and PC12 cell line transfected by the plasmid could express Bcl-2 protein effectively.Compared with the control(25 79%),there was a significant decrease of cells from the S phase in PC12 with bcl-2 gene(8.81%).After exposed with 10*!?mol/L,50*!?mol/L,and 100*!?mol/L cisplatin,the surviving cells in group A were 276?13,185?11 and 108?10 respectively,which increased much more than in group B while they were 100?9,12?3 and 2?2 accordingly.Conclusions bcl-2 can protect PC12 cells against cytotoxic insults of cisplatin,and it suggested that it might act via cell cycle controlling.

AIM To investigate the protection of plicamycin on apoptosis in cerebellar granule neurons (CGN) of rat. METHODS TUNEL, Hoechst 33258 staining, agarose gel electrophoresis and fluorescein diacetate staining were used to detect morphological and biochemical characteristics of apoptosis in primary rat CGN. RESULTS Being pre-incubated with plicamycin for 1 h and lasting for 24 h, rat CGN apoptosis induced by low potassium basal modified Eagle′s medium for 24 h was inhibited in a plicamycin concentration-dependent manner. This effective concentrations of plicamycin were from 50 to 200 nmol·L-1, and the maximum inhibitory rate of plicamycin on CGN apoptosis was near 80% at 200 nmol·L-1. CONCLUSIONPlicamycin inhibits rat CGN apoptosis induced by low potassium.

【Objective】To study the effect of the specific p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580 on apoptosis of cerebellar granule neurons induced by low potassium.【Methods】Apoptosis was induced by switching the cultured cerebellar granule neurons from a culture medium containing K+ 25 mmol*L-1 to a medium containing K+ 5 mmol*L-1 (cLK).Fragmentation of DNA was analyzed using agarose gel eletrophoresis.SAPK/JNK activity was measured by SAPK/JNK assay kit.【Results】Low potassium resulted in apoptosis as characterized by morphological and biochemical features,but the specific p38 MAPK inhibitor SB203580 improved the survival of cerebellar granule neurons cultured in cLK medium by blocking apoptosis in a concentration-dependent manner.The expression and phosphorylation of c-Jun increased and the activity of c-Jun N-terminal protein kinase (JNK) elevated when cerebellar granule neurons were cultured in cLK medium.But when the cerebellar granule neurons cultured in cLK medium were exposed to 25 μmol*L-1 SB203580,the expression and phosphorylation of c-Jun and the activity of JNK were both decreased evidently.【Conclusions】These results indicate that SB203580 inhibits the activation of JNK and phosphorylation of c-Jun,and therefore protects granule neurons from apoptosis induced by low potassium.

[Objective] To purify and characterize a novel factor X activator,Fve-1 from Daboia russelli siamensis (Myanmar) venom.[ Methods]F V e-1 was purified by ion-exchange chromatography and gel filtration.The hemostatic activity of F V e-1 was determined based on chromogenic substrates.The fibrinogen-clotting activity of F V e-1 was also determined.Thermal stability, pH stability,enzyme activity,and inhibition of F V e- 1 were determined by its remaining procoagulant activity.N-treminal sequence was determined by the method of automated Edman degradation.[ Results ]F V e-1 was achieved by chromatography with a molecular weight of 13,808 and an isoelectric point of 4.6. The hemostatic activity of 0.5 mg Fve-1 was equal to that of 1.5625 u thrombin or that of 54.93 ng RVV X. F V e-1 primarily activated F X, but did not affect on prothrombin and fibrinogen. The suitable pH and temperature range of F V e-1 was 6.5-7.5 and 25-60 ℃,respectively.The activity of F V e-1 was enhanced by Ca2+ and inhibited by EDTA and DTT.The N-terminal sequence of F V e-1 was NH2-N-L-Y-Q-F-G-E-M-I-N.[Conclusion] F V e-1 is a factor X-activating enzyme,which could activate FX to FX a,but have minimal effect on prothrombin and fibrinogen.

AIM: To confirm the differential expression genes in the rat ischemic brain. METHODS: The middle cerebral artery occlusion ischemic model was set up in rats. Fluorescence differential display reverse transcriptase-polymerase chain reaction (FDD RT-PCR) and reverse Northern blotting were used to fast confirm the differential expression genes. RESULTS: Nine differential expression sequence tags, including 6 known sequences and 3 unknown sequences, were confirmed. Among the known sequences, mus musculus ab1-interactor1,homo sapiens CGI-99 protein, tissue inhibitor of metalloproteinase 3 and homosapiens nuclear receptor co-repressor were up-regulated while homo-sapiens nuclear matrix protein p84 and coatomer protein complex, subunit gamma 2 were down-regulated. CONCLUSIONS: ① Combination of fluorescence differential display reverse transcriptase-polymerase chain reaction (FDD RT-PCR) and reverse Northern blotting is a method to fast-confirm the differential expression genes; ② There are differential expression genes in ischemic brain regions compared to non-ischemic parts. [

AIM To construct a non-normalized cDNA library from Agkistrodon acutus venom gland as an imtial step to develop new and more effective venom by genetic engineering technique for screening and expressing target genes. METHODS The total RNA was extracted from fresh venom gland using Trizol. mRNA was reversely transcripted to cDNA using superscriptⅡ reverse transcriptase. Second-strand synthesis was performed using DNA polymeraseⅠ. After adding EcoRⅠ adaptor, phosphorylating the end and digesting with XhoⅠ, the cDNA was collected in five fractions (<0.25 kb, 0.25-0.5 kb, 0.5-1 kb, 1-2 kb and >2 kb) using the QIAquick Gel Extraction kit and ligated to pBluescriptⅡ vectors. The five libraries obtained were plated by infecting E.coli DH10B, constructing a cDNA library of Agkistrodon acutus venom gland. Sequencing clones at random, 8696 high quality 5′ end expressed sequenced tags (ESTs) were obtained and analyzed. The initial sequences were assembled into 2855 clusters. Among which, one of the clusters (Agkihagin) consisting of 74 ESTs was identified as a novel metalloprtoteinase based on RT-PCR and sequence analysis. RESULTSThe titers of library were 2.048×106. The novel metalloproteinase belonged to PⅢ type metalloproteinase. Its open reading frame was composed of 1827 nucleotides and coded a pre-zymogen of 608 amino acid with zinc-binding domain for metalloproteinase and Asp-Glu-Cys-Asp(DECD) domain for disintegrin. CONCLUSION The capacity of cDNA library of venom gland is above the general level of cDNA library. It would be a helpful platform to construct a catalog for transcripts in the venom gland of the Agkistrodon acutus. The sequence analysis indicates that the deduced amino acid sequence of the identified gene for metalloproteinase share the highest 87% identity with the metalloproteinase genes of other snakes in the GenBank. It lays a good foundation for the study of structure-function relationships of snake venom metalloproteinases.

AIM: To observe the expression of neuronal Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) involved in neuronal apoptosis evoked by low K + and to investigate the relationship between ARNT2 and neuronal apoptosis. METHODS: After neuron apoptosis model was established, the changes of mRNA and protein of ARNT2 during apoptosis were investigated by RT-PCR and Western blotting, respectively. Immunofluorescence was analyzed by confocal microscopy to probe the subcellular localization of ARNT2. RESULTS: Induced by low K +, the expression of ARNT2 mRNA was up-regulated obviously at the point of 30 min, and peaks at the point of 1 h. This up-regulated expression lasted for 12 h, and the variation of ARNT2 protein was similar to that of mRNA. The results of immunofluorescence analyzed by confocal microscopy showed that the localization of ARNT2 protein was in the nucleus. CONCLUSION: ARNT2 locate in nuclei of normal cerebellar granule neurons of rat. During the process of apoptosis evoked by low K +, both mRNA and protein of ARNT2 are overexpressed.