[Show abstract][Hide abstract] ABSTRACT: Hyperacute rejection (HAR) is a main barrier in xenotransplantation, which is mediated by the combination of natural antibody to the xenograft and complement activation. Current therapies have focus on the inhibition of complement by development of complement inhibitor and transgenic animal organ. Here, we investigated the effects of rF II, a recombinant fibrinogenase from Agkistrodon acutus venom, on complement and HAR. The degradation effect of rF II on complement was tested by SDS-PAGE, CH50 examination, ELISA Kit and cofocal immunofluorescence microscopy in vitro and in vivo. An ex-vivo rat-to-human perfusion model and a vivo guinea-pig-to-rat heat HAR model were used to determine the protection of rF II against HAR. Our investigation indicated that rF II could significantly degrade human C5, C6, and C9, decrease the activity of complement, and inhibit the MAC deposition on HUVECs membrane in vitro. In addition, serum levels of C1q, C3 and C4 in rat were gradually reduced after infusion of rF II. Importantly, in an ex vivo rat-to-human perfusion model, the survival of rat hearts perfused with human serum treated with rF II (83.36±16.63min) were significantly longer than that of hearts perfused with fresh human serum(15.94±4.75min). At the time of 15minutes after perfusion, functions of hearts added with 50ug/ml rF II sustained well with heart rates at 283±65.32 beats/minute and LVDP at 13.70±5.45Kpa, while that of hearts perfused with fresh human serum were severely damaged by HAR with heart rates at 107.77±40.31 beats/minute and LVDP at 1.01±0.83 Kpa. We also found that rF II significantly decreased the levels of C1q, C3 and C4 in human fresh serum perfusate. In a vivo guinea-pig-to-rat heat HAR model, the survival of rat hearts treated with rF II were significantly longer than that of hearts perfused with normal saline; and relieved heart damage by complete activation. Our finding demonstrates the anti-complement property of rF II and its protection against HAR, indicating that rF II might be as a potential therapeutic agent for xenotransplantation.
[Show abstract][Hide abstract] ABSTRACT: The incidence of disseminated intravascular coagulation (DIC), which leads to multiple organ dysfunction and high mortality, has remained constant in recent years. At present, treatments of DIC have focused on preventing cytokine induction, inhibiting coagulation processes and promoting fibrinolysis. Recent clinical trials have supported the use of antithrombin and activated protein C supplementation in DIC. To better understand the mechanism of treatment on DIC, we here report a novel fibrinogenase from Agkistrodon acutus (FIIa) that effectively protected against LPS-induced DIC in a rabbit model, and detected the tissue factors expression in HUVE cells after using FIIa. In vivo, administration of FIIa reduced hepatic and renal damage, increased the concentration of fibrinogen, the activities of protein C, the platelet count, APTT, PT, FDP, the level of AT-III and t-PA, decreased the level of PAI-1, and increased survival rate in LPS-induced DIC rabbits. In vitro experiments, we further confirmed that FIIa up-regulated the expression of t-PA and u-PA, down-regulated the expression of PAI-1, and directly activated protein C. Our findings suggest that FIIa could effectively protect against DIC via direct degradation of microthrombi and activation of protein C as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.
[Show abstract][Hide abstract] ABSTRACT: Glioma is the most common and fatal primary brain tumor. Thus far, therapeutic strategies to efficiently and specifically antagonize glioma are limited and poorly developed. Here we report that glia-enriched miR-135a, a microRNA that is dramatically downregulated in malignant glioma and correlated with the pathological grading, is capable of inducing mitochondria-dependent apoptosis of malignant glioma by regulating various genes including STAT6, SMAD5 and BMPR2, as well as affecting the signaling pathway downstream. Moreover, this lethal effect is selectively towards malignant glioma cells, but not neurons and glial cells, through a novel mechanism. Our findings suggest an important role of miR-135a in glioma etiology and provide a potential candidate for malignant glioma therapy.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the effects of the fibrinolytic enzyme FII(a) from Agkistrodon acutus venom on acute pulmonary thromboembolism (APT) in animal models.
Both rabbit and dog APT models were used. For the rabbit APT model, the thrombi weight before and after administration was measured. Central venous pressure (CVP) and mean arterial pressure (MAP) were measured before and 15, 30, 60, and 120 min after the injection of the blood clot. Partial thromboplastin time (APTT), prothrombin time (PT), platelet count, and fibrinogen concentration were measured using auto analyzers. Plasminogen activity was measured based on chromogenic substrates. In the dog APT model, pulmonary blood flow was recorded using pulmonary angiography.
Intravenous administration of FIIa (0.1-5.0 mg/kg) improved the APT-induced hemodynamic derangements and reduced thrombi weight. The angiography evidence also showed that the pulmonary emboli had almost disappeared after FII(a) infusion. FII(a) (0.1, 0.5, or 1.0 mg/kg) did not impair the coagulation pathways, although very high doses of FII(a) (5.0 mg/kg) could stimulate the production of plasminogen and result in impairment of the pathways.
FII(a) could effectively protect against APT via degradation of thrombi with less activation of plasminogen, and may provide a novel fibrinolytic enzyme for targeting the main pathological processes of the disease.
[Show abstract][Hide abstract] ABSTRACT: To investigate the mechanism of bleomycin (BLM)-induced pulmonary fibrosis.
Cultured human fetal lung fibroblast (HLF) cells were exposed to bleomycin (BLM) at 0-30 microg/mL for 24 h. Western blot analysis was used to detect lysyl oxidase (LO) protein expression. Real-time RT-PCR was used to detect LO mRNA level. LO catalytic activity was measured using diaminopentane as a substrate and Amplex red as a hydrogen peroxide probe. Copper (Cu) concentration was detected by flame atomic absorption spectrophotometry.
Exposure of HLF cells to BLM at 10 microg/mL and 30 microg/mL increased LO catalytic activity to 130% and 158% of the control in the conditioned media. The expression of LO mRNA was increased to 5.5-fold of the control in HLF cells exposure to BLM at 3 microg/mL. BLM at 3 microg/mL also increased the expression of 46 kDa preproLO, 50 kDa proLO and 32 kDa mature LO to 219%, 130%, and 135% of the control, respectively. The Cu concentrations in conditioned media of cultured HLF cells exposed to BLM (10 and 30 microg/mL) were increased significantly to 1.48 and 2.46-fold of the control, respectively.
Bleomycin induces upregulation of LO in cultured human fetal lung fibroblasts, which may be the mechanism of bleomycin-induced pulmonary fibrosis.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the role of glycogen synthase kinase-3beta (GSK-3beta) in the induced differentiation of human glioblastoma cells.
Cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation assay. The protein level of p-GSK-3beta, GSK-3beta, glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) were determined using Western blots. The overexpression of mutant GSK-3beta was analyzed by immunocytochemistry.
The biotoxin cholera toxin is capable of inducing differentiation of U87-MG human glioblastoma cells, which is characterized by morphological changes to astrocytic phenotype, increase in differentiation marker protein GFAP and decrease in proliferation. GSK-3beta activation is induced during this differentiation. Small interfering RNA against GSK-3beta suppresses the induced-differentiation in U87-MG cells. Conversely, overexpression of a constitutively active form of human GSK-3beta (pcDNA3-GSK-3beta-S9A) mutant leads to differentiation of U87-MG cells.
Our findings suggest that GSK-3beta plays an important role in astrocytic differentiation of human glioblastoma cells and may be a novel therapeutic target in the malignant tumor.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether aspirin is able to augment gemcitabine-induced cytotoxicity in human pancreatic cancer cells.
Two gemcitabine-insensitive human pancreatic cancer cell lines, PANC-1 and Capan-1, were used. Cells were treated with either aspirin or gemcitabine alone or both of them. Cell growth and apoptosis were determined by MTT assay, Annexin V or Hoechest 33258 staining. Cell cycle distribution was examined by flow cytometry. Western blot with specific phosphorylated protein antibodies was used to detect the activation of protein kinase. RT-PCR and Western blot were applied to assess the transcription and protein level for cyclin D1 and Bcl-2.
Aspirin alone significantly inhibits the proliferation of PANC-1 cells by causing cell cycle arrest at G(1) phase. Aspirin potentiates the anti-survival effect of gemcitabine as well as its pro-apoptotic effect in PANC-1 cells, although aspirin per se does not trigger apoptosis. Aspirin inhibits GSK-3beta activation and suppresses the expression of its downstream gene products (cyclin D1 and Bcl-2), which are implicated in proliferation, survival and chemoresistance of pancreatic cancer. The effects of aspirin on Capan-1, were similar to that on PANC-1.
Our results suggest that aspirin inhibits the proliferation of gemcitabine-resistant pancreatic cancer cells and augments the antisurvival effect of gemcitabine, probably by suppressing the activity of GSK-3beta and its downstream gene products.
[Show abstract][Hide abstract] ABSTRACT: To investigate a possible regulator gene involved in the cholera toxin-induced differentiation of rat C6 glioma cells.
The global changes in the mRNA expression pattern induced by cholera toxin were analyzed using gene chip microarray. The selected gene was then silenced by RNA interference or overexpressed with an ORF plasmid to determine its necessity in this process.
Nur77, a member of the orphan nuclear receptor family (NR4A), was markedly up-regulated during the process of differentiation. Furthermore, RNAi of nur77 attenuated the induction effect of cholera toxin on C6 cells, whereas overexpression of nur77 led to similarly differentiated behavior, including morphologic and biomarker changes, as well as cell cycle arrest.
Nur77 participated actively and essentially as an important regulator in the cholera toxin-induced differentiation of C6 cells.
[Show abstract][Hide abstract] ABSTRACT: Despite the use of a series of preventive measures, a high incidence of severe acute respiratory syndrome (SARS) was observed among health care workers (HCWs) during the SARS epidemic. This study aimed to determine which preventive measures may have been effective in protecting HCWs from infection, and which were not effective.
A retrospective study was performed among 758 'frontline' health care workers who cared for SARS patients at the Second Affiliated Hospital and the Third Affiliated Hospital of Sun Yat-sen University. The HCWs with IgG against SARS and those without IgG against SARS were respectively defined as the "case group" and the "control group", and logistic regression was conducted to explore the risk factors for SARS infection in HCWs.
After adjusting for age, gender, marital status, educational level, professional title, and the department in which an individual worked, the results of a multivariate logistic regression analysis indicated that incidence of SARS among HCWs was significantly and positively associated with: performing tracheal intubations for SARS patients, methods used for air ventilation in wards, avoiding face-to-face interaction with SARS patients, the number of pairs of gloves worn by HCWs, and caring for serious SARS cases.
Some measures, particularly good air ventilation in SARS wards, may be effective in minimizing or preventing SARS transmission among HCWs in hospitals.
BMC Public Health 02/2009; 9:81. · 2.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As a model of the reperfusion injury found in stroke, we treated cerebellar granule neurons (CGNs) with hypoxia followed by reoxygenation. Hypoxia for 3h followed by 24h reoxygenation (H/R) induced a typical apoptosis of CGNs. CGNs exposed to H/R responded by activating JNK, increasing the expression of p38 and ultimately caused CGNs dying. Furthermore, apoptosis of CGNs induced by H/R was inhibited by pre-treatment with SB203580 or SP600125, and the inhibitory effect of SB203580 was greater than that of SP600125. Additionally, we also found that H/R temporally activated Akt and inactivated glycogen synthesis kinase-3beta (GSK-3beta), two proteins the functions of which were important in cell survival and energy metabolism. These findings demonstrated that H/R-induced apoptosis in CGNs by enhancing JNK and p38 activity, which contributed at least in part to H/R-induced apoptosis of CGNs.
Experimental and toxicologic pathology: official journal of the Gesellschaft fur Toxikologische Pathologie 09/2008; 61(2):137-43. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel fibrinolytic enzyme, FII(a), was isolated from Agkistrodon acutus venom, which can degrade fibrin/fibrinogen and dissolve thrombus without activating plasminogen or influencing the activities of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1). In this study, we evaluated the effect of FII(a) on lipopolysaccharide (LPS)-induced experimental disseminated intravascular coagulation (DIC) in rabbits, through the continuous infusion of 100-microg/kg/h LPS for a period of 6 h. Seven groups were established: LPS control, FII(a) (0.1, 0.3, and 0.6 mg/kg/h, respectively), heparin control (100 IU/kg/h), heparin + FII(a) (heparin 100 IU/kg/h associated with FII(a) 0.3 mg/kg/h), and a saline control group. A continuous injection of LPS induced a gradual impairment in hemostatic parameters, kidney fibrin deposition, and a high mortality rate. The intravenous administration of FII(a) improved the concentration of fibrinogen, the activities of protein C, plasminogen, t-PA, antithrombin III (ATIII), and PAI-1. Kidney fibrin deposition and the mortality also decreased. In the in vitro experiments, FII(a) can degrade fibrin/fibrinogen and high-dose FII(a) enhanced the activity of protein C. These findings suggest that the effects of FII(a) on LPS-induced DIC were from fibrinogen degradation and enhanced protein C activity. The simultaneous administration of FII(a) and heparin further improved all the hemostatic parameters, including decreased kidney fibrin deposition, and none of the rabbits died within 24 h, which indicates that the effects were mediated by degradation of fibrin/fibrinogen together with thrombin inhibition. We conclude that FII(a) may be useful in the treatment of DIC.
Translational Research 12/2007; 150(5):295-302. · 3.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To purify and characterize the coagulant protein FIa from Daboia russelli siamensis (Myanmar) venom.
FIa was purified from Daboia russelli siamensis (Myanmar) venom by ion-exchange chromatography on CM-Sephadex C-50, and gel filtration on Sephadex G-75 and a Superdex 75 column. The hemostatic activity of FIa was determined by the method of Williams and Esnouf. The specific chromogenic substrates were used respectively to determine the activation of factor X and prothrombin. The fibrinogen-clotting activity of FIa was determined by the method of Gao et al. Normal saline was used as a negative control while factor Xa and thrombin were used as positive controls, respectively.
FIa, a coagulant protein, was achieved by ion-exchange chromatography and gel filtration with a molecular weight of 34,479 and an isoelectric point of 7.2. FIa was shown to have strong hemostatic activity. The hemostatic activity of 0.5 mg FIa was equal to that of 1.5625 u thrombin. FIa primarily activated factor X, however, had no influence on prothrombin, nor did it cleave or clot fibrinogen.
FIa is a factor X-activating enzyme, which could activate factor X to factor Xa, but has no effect on prothrombin and fibrinogen.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to investigate whether the activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is required for thermal preconditioning to protect rat cerebellar granule neurons (CGN) against apoptosis induced by low potassium, and to explore the possibility of a link between the upregulated heat shock protein (HSP)70 expression and Akt activation in the acquisition of neuroprotection induced by thermal preconditioning.
CGN cultured for 8 d in vitro were switched to 5K medium for 24 h after thermal preconditioning (TP; 43.5 degree for 90 min, then 37 degree for 1 h). To study the role of the PI3-K/Akt pathway, a PI3-K inhibitor, LY294002 (20 micromol/L) was added into the cultures 1 h before TP. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay and fluorescein diacetate staining were used to determine cell viability. Hoechst 33258 staining and agar gel electrophoresis were used to test the morphological and biological characters of CGN. Western blot analysis was employed to detect the levels of phospho-Akt, phospho-glycogen synthase kinase 3beta (GSK3beta) Akt, GSK3beta, and HSP70.
TP protected CGN against apoptosis induced by low potassium. LY294002 inhibited the neuroprotective effect on CGN induced by TP. TP induced a robust activation of Akt and the inactivation of GSK3beta via PI3-K. Furthermore, the activation of the PI3-K/Akt pathway by TP persisted for 24 h in the 5K cultures. LY294002 (20 micromol/L) failed to inhibit the upregulated HSP70 expression induced by TP.
The activation of the PI3-K/Akt pathway is required for TP to protect CGN against apoptosis induced by low potassium, but the neuroprotective effect by Akt activation is not mediated through the downstream induction of HSP70 expression.
[Show abstract][Hide abstract] ABSTRACT: A novel metalloproteinase, recombinant fibrinogenase IV (rFIVa), was expressed and purified from Agkistrodon acutus venom. It is a single-chain protein with an apparent molecular weight of 27 kDa. Western blot showed that it had a good immunological reaction against anti-FIVa rabbit serum. The kinetic parameters Km and Kcat of rFIVa on the substrate T6140 were 7.471 x 10(-4) mol/l and 5.103 x 10(-5) s(-1). RFIVa cleaved preferentially the alpha-chain, and the beta- and gamma-chains of fibrinogen were also cleaved when the incubation time was prolonged. The administration of rFIVa (1.8 and 5.4 mg/kg) to animals with acute blood-stasis model produced a decrease in fibrinogen to control values. To our knowledge, this is the first report of the expression, purification, and evaluation of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from A. acutus venom.
Applied Microbiology and Biotechnology 09/2006; 72(1):72-6. · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To analyze YZ-2, a novel protein associated with cerebral ischemia and prepares its polyclonal antibody.
According to the bioinformatics analysis and prediction of the possible high structure, hydrophilicity and antigenicity of YZ-2, a 22-amino acid residue partial peptide of YZ-2 was synthesized. The synthesized peptide was then used to immunize. And the properties of anti-YZ2 were analyzed by ELISA and Western blot.
The hydrophilicity and antigenicity were predicted by methods of bioinformatics. The polyclonal antibody of YZ-2 was successfully obtained and its specificity and sensitivity were conformed by ELISA and Western blot.
By the bioinformatics analysis and prediction, the hydrophilicity and antigenicity of YZ2 were analyzed. The antibody of YZ-2 was successfully obtained.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2006; 22(2):205-7.
[Show abstract][Hide abstract] ABSTRACT: A novel metalloproteinase, recombinant fibrinogenase IV (rFIV(a)), was expressed and purified from Deinakistrodon acutus venom. It was a single chain protein with an apparent molecular weight 27 kDa and an isoeletric point of pH 7.1. RFIV(a) cleaved preferentially the Aalpha-chain and also cleaved Bbeta, gamma-chains of fibrinogen when the incubation time was prolonged. The proteolytic activity was inhibited by EDTA, l-cysteine, and DTT, indicating rFIV(a) was a metalloproteinase requiring disulfide bonds for its activity. It kept above 85% of the initial activity from pH 4.5-11, showed an equal maximum activity at the temperature range from 30 to 50 degrees C, and was inactivated by Zn2+, Cu2+ and Cd2+. Homology modeling of rFIV(a) showed that two highly conserved disulfide bonds (Cys159-Cys164 and Cys117-Cys197) was maintained from its structure, and it exhibited the characteristic conserved motif H142E143XXH146XXGXXH152, whose three histidine residues were involved in binding of the catalytically essential zinc ion. This work demonstrates the expression, purification and characterization of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from D. acutus venom.
[Show abstract][Hide abstract] ABSTRACT: To study the enzymological characterization of a fibrinolytic enzyme (FII(a)) from Agkistrodon acutus venom.
The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FII(a) was determined by atomic absorption spectroscopy.
After incubation with FII(a) (0.25 g/L), Aalpha-, Bbeta- and gamma-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h , respectively. The molecular weights of major degradation products were 45,000 and 41,000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FIIa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171+/-25 mg/kg), potassium (489+/-17 mg/kg) and calcium (319+/-13 mg/kg) were found in FIIa. Zn2+, Ca2+ and Mg2+ could recover the fibrinogenolytic activity of FIIa, which was inhibited by EDTA. Only Ca2+ could recover the fibrinogenolytic activity inhibited by EGTA.
FIIa can degrade the Aalpha-, Bbeta- and gamma-chains of fibrinogen. FII(a) is a metalloproteinase, and Zn2+, Ca2+, and disulfide bonds are necessary for its fibrinogenolytic activity.
[Show abstract][Hide abstract] ABSTRACT: To investigate the fibrin(ogen)olytic character of FIIa isolated from Agkistrodon acutus venom in vitro and in vivo.
125I-labeled human plasma clot lysis was measured in vitro and rabbit carotid artery thrombosis was as an in vivo model.
In vitro, urokinase (UK) at 25, 35, 40, 45, 60 kU/L and FIIa at 0.08, 0.23, 0.4, 0.5, and 0.7 g/L resulted an equivalent clot lysis (20%, 40%, 50%, 60%, and 80%). UK at 25 to approximately 60 kU/L induced 27.3%+/-3.6%, 35.2%+/-2.3%, 39.3%+/-2.4%, 44.2%+/-4.6%, and 51.1%+/-1.2% fibrinogen degradation. But FIIa at 0.08 approximately -0.7 g/L induced 95.4%+/-0.3%, >95.6%, >95.6%, >95.6%, >95.6% fibrinogen degradation respectively. In vivo, UK 40 kU/kg and FIIa 1.0 mg/kg reduced the weight of residual thrombus to 9.0+/-2.5 mg and 7.8+/-3.5 mg compared with negative control group (30.0+/-5.4 mg). But the fibrinogen degradation rate after UK 40 kU/kg and FIIa 1.0 mg/kg treatment was 24.4%+/-6.2% and 4.1%+/-7.8%, respectively (P<0.05, n=6). The order of the lysis speed after UK 125 kU/L treatment was platelet poor plasma (PPP) clots>the whole blood clots>platelet rich plasma (PRP) clots. The sequence for FIIa 0.4 g/L was PRP >PPP >whole blood clots.
At the same percentage of clot lysis, FIIa degraded more fibrinogen than UK did in vitro but less fibrinogen than UK did in vivo. The order of the lysis speed was PPP>whole blood clots>PRP clots for UK and PRP>PPP>whole blood clots for FIIa.
[Show abstract][Hide abstract] ABSTRACT: To determine the prevalence of inapparent infection with severe acute respiratory syndrome (SARS) among healthcare workers, we performed a serosurvey to test for immunoglobulin (Ig) G antibodies to the SARS coronavirus (SARS-CoV) among 1,147 healthcare workers in 3 hospitals that admitted SARS patients in mid-May 2003. Among them were 90 healthcare workers with SARS. As a reference group, 709 healthcare workers who worked in 2 hospitals that never admitted any SARS patients were similarly tested. The seroprevalence rate was 88.9% (80/90) for healthcare workers with SARS and 1.4% (15/1,057) for healthcare workers who were apparently healthy. The seroprevalence in the reference group was 0.4% (3/709). These findings suggest that inapparent infection is uncommon. Low level of immunity among unaffected healthcare workers reinforces the need for adequate personal protection and other infection control measures in hospitals to prevent future epidemics.