AIM To purify cardiotoxin from Naja atra venom and investigate the relationship between cardiotoxicity of cardiotoxin and coronary artery spasm induced by cardiotoxin. METHODS Cardio toxin 13 (CTX 13) was fractionated and purified by chromatography and gel filtration from Chinese cobra (Naja atra) venom. The cardiotoxicity were observed in rat in situ, its isolated heart preparation and papillary muscle preparations. RESULTS Ion exchange chromatography of lyophilized cobra venom on SP Sephadex C 50 yielded 15 fractions, of thses fractions, cardiotoxic activities were found in fraction 11, 12, 13, and 14. Gel filtration and Ion chromatography of fraction 13 on Sephadex G 50 and SP Sephadex C 25 were performed consecutively and CTX 13 was obtained. It was homogeneous on polyacrylamide gel electrophoresis with MW= 7 769 ku, and 60 amino acid residues. The iv LD 50 in mice was 0 756 mg?kg -1 . CTX 13 increased the coronary resistance and reduced the contractility of rat Langendorff heart preparations. Systolic standstill finally occurred. When the heart preparations were pretreated with nitrendipine, an calcium channel blocker, the resistance seldom increased. The contractility slightly decreased at the beginning and then significantly increased. The tonus of contraction did not occurred. CTX 13 induced dose dependent contraction of pig coronary artery ring segments. Nitrendipine inhibited the action of CTX 13 on the coronary ring segments. However, nitrendipine had no effects on the action of CTX 13 in the rat papillary muscle preparations. The MLD of CTX 13 by venoclysis was changed from (444 7?28 5) ?g?kg -1 to (541 1?23 2) ?g?kg -1 in anaesthetized rats while the rats were pretreated with nitrendipine. CONCLUTION The coronary artery spasm may be one of the causes of death due to CTX 13.

AIM This study is to observe the effects of acutobin on the activity of tissue type plasminogen activitor(t PA) and tissue plasminogen activitor inhibitor(PAI) in the cultured human umbilical vein endothelial cells, aiming at disclosing some of the mechanisms of thrombolysis of acutobin. METHODS Endothelial cells were isolated from fresh human umbilical cords by trypsin digestion of the interior surface of the umbilical vein. Cultured cells were examined by light, phase contrast and electron microscopy. The factorⅧ related antigen and CD34 of the cells were detected by AEC and DAB staining. Chromogenic assay was used to identify the activity of t PA and PAI in the medium of culture cells. Fibrin degradation products(FDPs) were measured using ELISA kit. RESULTS The cultured human umbilical endothelial cells were shown as monolayers of closely opposed, polygonal cobblestone shape by light and phase contrast microscopy. By transmission electron microscopy, cultured endothelial cells contained Weibel Palade body and showed tight junction with each other. The cells contained abundant quantities of CD34 and factorⅧ related antigen. The intercellular space among individual cell enlarged and lost polygonal cobblestone shape in the present of acutobin. Activity of t PA increased, the activity of PAI did not change significantly and FDPs increased significantly in the culture medium. CONCLUSIONS The study demonstrates the culture cells was endothelial cells according to morphologic and immunohistologic criteria. Acutobin increases the fibrinolytic activity of cultured endothelial cells and may exhibit antithrombotic effect in vivo.

Aim To investigate the effect of the specif-ic inhibitor BIX-01294 of G9a on the proliferation, ap-optosis,DNA methylation and histone modulation of a-cute leukemia cell line, Molt-4. Methods Cells were cultured with different concentrations of BIX-01294 . Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of Caspase-3, Bcl-2, Bax, P15, acetylated H3, H3 K9 me1 , H3 K9 me2 , H3 K9 me3 and H3 K27 me1 , H3K27me2 methylation were detected by Western blot. Results BIX-01294 downregulated bcl-2 , and upreg-ulated Bax, Caspase-3 and induced apoptosis in (5. 54 ± 1. 35)%, (10. 24 ± 2. 26)%, (32. 28 ± 3. 26)%, (47. 52 ± 4. 37 )% after 24 hours exposure to BIX-01294 in 0 , 1 , 2 , 4 μmol · L-1 . The difference be-tween them was statistically significant ( P <0. 05 ) . BIX-01294 inhibited DMNT1 and promoted P15 , which resulted in cell proliferation inhibition. Further studies showed that BIX-01294 decreased H3 K9 me1 , H3 K9 me2 , and H3 K27 me1 , H3 K27 me2 , and didn′t change protein expression of acetylated H3 and H3 K9 me3. Conclusion BIX-01294 inhibits G9a, resulting in downregulation of methylation of H3 K9 me1 , H3 K9 me2 , H3 K27 me1 and H3 K27 me2 and DMNT1 and P15 denovo. It inhibits cell proliferation and in-duces cell apoptosis.

Aim To purify L-amino acid oxidase(LAAO) from the venom of Naja atra and study its effect on endothelial cells.Methods The NAV-LAAO was purified by ion-exchange chromatography and affinity chromatography.The MTT assay and Western blot were used to detect the viability and apoptosis of HUVEC.The tubule-forming was used to study the angiogenesis of cells.Results The NAV-LAAO was purified successfully from the venom of Naja atra.The molecular weight of NAV-LAAO was determined to be 58 ku by SDS-PAGE.NAV-LAAO effectively inhibited the growth and tubule-forming of HUVEC,and the 50% inhibitory concentration(IC50) was 21.42 mg?L-1.Compared with control,the levels of caspase-3 and caspase-8 increased in HUVEC treated with NAV-LAAO.Conclusions The NAV-LAAO is purified successfully from Naja atra venom by ion-exchange chromatography and affinity chromatography.The NAV-LAAO inhibits the growth and tubule-forming capacity of HUVEC in a dose-dependent manner.

Objective To investigate the effect of phenylhexyle isothiocyanate (PHI) on demethylation and activation of transcription gene p15 in acute leukemia cell line Molt-4. Methods DNA sequencing and modified methylation specific PCR (MSP) were used to screen p15-M and p15-U mRNA after Moh-4 cells were treated with PHI. P15 mRNA was measured by RT-PCR. Pl5 protein was detected by Western blotting. Results Hypermethylation of gene pl5 was apparently attenuated and activation of transcription p15 gene was de novo after 5 days exposure to PHI. PHI enhanced both the expression of p15 mRNA and p15 protein in a concentration-dependent manner. The ratio of the gray scale of p15 mRNA strap was 0.17±0.12 in control, 0.29±0.14 in PHI 10 μmol/L, 0.55±0.07 in PHI 20 μmol/L, 0.93±0.13 in PHI 40 μmol/L. Conclusion PHI could active demethylation and transcription of gene p15.

Objective To evaluate the role of phosphatase and tensin homolog deleted on ehromo-some ten (PTEN) protein in sevoflurane postconditioning-induced mitigation of focal cerebral ischemia-reperfusion (I/R) injury via activating PI3K/Akt pathway in rats.Methods One hundred and eight Sprague-Dawley rats,aged 2-3 months,weighing 250-280 g,were randomly assigned into 6 groups (n =18 each) using a random number table:sham operation group (group S),group I/R,I/R + sevoflurane postconditioning group (group I/R + S),I/R + normal saline group (group I/R + NS),I/R + selective PTEN inhibitor pic group (group I/R + P),and I/R + pic + sevofluraue postconditioning group (group I/R + P+ S).Focal cerebral I/R was induced by right middle cerebral artery occlusion (MCAO).The animals were anesthetized with intraperitoneal chloral hydrate.In I/R + P and I/R + P + S group,pic 20 μg/100 g (0.4 ml/100 g) was injected intraperitoneally every 3 h for 4 times before MCAO,and the equal volume of normal saline was given instead of pic in I/R + NS group.The rats in all sevoflurane postconditioning groups inhaled 2.5 ％ sevoflurane for 30 min starting from the onset of reperfusion,and the rats in the other groups inhaled oxygen for 30 min instead.At 24 h of reperfusion,neurological deficit scores (NDSs) were measured and the rats were then sacrificed.Their brains were removed for determination of infarct size (by TTC),cell apoptosis (by TUNEL),and expression of phosphorylated PTEN (p-PTEN) protein and phosphorylated Akt (p-Akt) (by Western blot).Apoptosis index was calculated.Results Compared with S group,the NDSs,percentage of cerebral infarct size and apoptosis index were significantly increased,and the expression of p-Akt and p-PTEN protein was up-regulated in the other 5 groups.Compared with I/R and I/R + NS groups,the NDSs,percentage of cerebral infarct size and apoptosis index were significantly decreased,and the expression of p-Akt and p-PTEN protein was up-regulated in I/R + S,I/R + P and I/R + P + S groups.There were no significant changes in the parameters mentioned above between I/R + S,I/R + P and I/R + P + S groups,and between I/R and I/R + NS groups.Conclusion Sevoflurane postconditioning can activate PI3K/Akt pathway via inhibiting PTEN protein,thus mitigating focal cerebral I/R injury in rats.

Aim To isolate and purify a novel plasminogen activator(PA)from Gloydius brevicaudus venom(GBV)and study characterization and biological activities of GBV-PA.Methods Affinity chromatography in Benzamidine Sepharose 6B(AC)and Lichrospher C-18 4.6/250 reversed phase chromatography(RPC)were used for isolation and purification;SDS-PAGE was used to detect molecular weight(MW);Disc polyacrylamide gel eletrophoresis was used to measure the point of isoelectric(pI);Chromogenic substrate method was used to observe the biological activities.Results A novel GBV-PA which its purification reached the homogeneity level was isolated and purified from GBV by AC and RPC;The MW of the novel GBV-PA was 3.26?104 and the pI was 5.2;The novel GBV-PA activated human plasminogen specifically and the special activity was 2.87 t-PA IU?mg-1;Moreover,our results indicated that this novel GBV-PA was a serine proteinase which had no affinity to fibrin.Conclusion A novel GBV-PA that can be isolated and purificated from GBV by AC and RPC was proved to be a serine protease and has no affinity to fibrin.

Objective:To investigate the role of endoscopic ultrasonography (EUS) in differentiating between autoimmune pancreatitis (AIP) and pancreatic cancer (PC).Methods:Data of 133 patients with AIP and 113 patients with PC who underwent EUS because of obstructive jaundice at Peking Union Medical College Hospital from January 2013 to December 2018 were retrospectively analyzed in the study, and were randomly divided into either a derivation sample or a validation sample using 1∶1 allocation according to the random number. In the derivation sample, 10 EUS characteristics were used to construct a prediction model to distinguish between AIP and PC, in which predictors were identified by multivariate stepwise logistic regression analysis and predictive efficacy was evaluated by receiver operating characteristics (ROC) curve analysis. The predictive efficacy was assessed in the validation sample. In view of the subjectivity in the judgment of diffuse/focal hypoechogenicity, 2 prediction models were designed in order to avoid bias.Results:By multivariate stepwise logistic regression analysis, diffuse hypoechogenicity ( OR=591.0, 95% CI: 98.8->999.9, P<0.001) and vessel involvement ( OR=11.9, 95% CI: 1.4-260.2, P=0.023) were identified as statistically significant predictors for distinguishing AIP from PC. EUS characteristics excluding diffuse/focal hypoechogenicity were stepped by logistic regression, which showed that hyperechoic foci/strands ( OR=177.3, 95% CI: 18.7->999.9, P<0.001), pancreatic duct dilation ( OR=60.5, 95% CI: 6.2->999.9, P=0.004), bile duct wall thickening ( OR=35.4, 95% CI: 3.7->999.9, P=0.009), lymphadenopathy ( OR=16.8, 95% CI: 1.7-475.2, P=0.038) and vessel involvement ( OR=22.7, 95% CI: 2.0-725.7, P=0.028) were statistically significant predictors to distinguish the two diseases. Both prediction models were built in the derivation sample, with area under the ROC curve of 0.995 and 0.979 respectively. In the validation sample, sensitivity, specificity, accuracy, positive predictive value and negative predictive value of both prediction models were all >90% by using the optimal cutoff value. Even for discrimination between focal AIP and PC, sensitivity and accuracy of both models were >90%, and specificity, positive predictive value and negative predictive value were all >85%. Conclusion:The 2 prediction models have good differential predictive value, and EUS is a useful tool to differentiate between AIP and PC.

Objective:To analyze clinical characteristics of primary pancreatic lymphoma (PPL) patients.Methods:Clinical features of 22 patients diagnosed as PPL admitted to Peking Union Medical College Hospital from January 2002 to May 2023 were analyzed retrospectively.Results:The median age was 56.4±13.3 years. The median time from onset to diagnosis was 1.0 (1.0, 3.0) months. The main clinical manifestations were abdominal pain (15/22), weight loss (14/22) and jaundice (10/22). Elevated lactate dehydrogenase (LDH) was observed in 15/20 (75%) patients. Only 2 (2/9, 22.2%) patients had increased CA199 levels and 2 (2/9, 22.2%) patients had increased CEA levels. The maximum tumor diameter was 5.0 (3.8, 6.9) cm. Contrast-enhanced CT mostly showed low enhancement lesions. Major pancreatic duct dilatation were rare on CT scan (4/20). Fifteen patients were confirmed by pancreatic pathology, of which 8 were obtained by surgery, 4 were obtained by CT or ultrasound-guided percutaneous biopsy, and 3 were obtained by EUS-FNA. The main pathological type was diffuse large B-cell lymphoma (14/22). 19 patients received chemotherapy, and 6 patients died with a median follow-up of 5.0 (1.5, 35.5) months.Conclusions:PPL is rare and easy to be misdiagnosed. Elevated LDH levels, normal tumor markers, and non-dilatation of main pancreatic duct are important diagnostic clues. It is important to obtain pathology by EUS-FNA and other methods for definite diagnosis.