Author Correction: Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys.

Following the publication of our paper (Zhang et al., 2020), it has come to our attention that we erroneously listed two funding sources unrelated to this study in the "ACKNOWLEDGEMENTS" section. Hereby, we wish to update the "ACKNOWLEDGEMENTS" section as a correction.

Suppression of CCDC6 sensitizes tumor to oncolytic virus M1.

Oncolytic virus is an effective therapeutic strategy for cancer treatment, which exploits natural or manipulated viruses to selectively target and kill cancer cells. However, the innate antiviral system of cancer cells may resistant to the treatment of oncolytic virus. M1 virus is a newly identified oncolytic virus belonging to alphavirus species, but the molecular mechanisms underlying its anticancer activity are largely unknown. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. RNA seq analysis was used to analyze the gene alternation after M1 virus infection. Small interfering RNAs transfection for gene knockdown was used for gene functional tests. Caspase-3/7 activity was detected by Caspase-Glo Assay Systems. A mice model of orthotopic bladder tumor was established to determine the oncolytic effectiveness of the M1 virus. The expression of cleaved-Caspase 3 as well as Ki-67 in tumor cells were detected by immunohistochemical analysis. To further define the molecular factors involved in M1 virus-mediated biological function, we knocked down genes related to alphavirus' activity and found that CCDC6 plays an important role in the oncolytic activity of M1 virus. Moreover, knocked down of CCDC6 augments the reproduction of M1 virus and resulted in endoplasmic reticulum (ER) stress-induced cell apoptosis in vitro as well as in vivo orthotopic bladder cancer model. Our research provides a rational new target for developing new compounds to promote the efficacy of oncolytic virus therapy.

Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys.

Hypobaric hypoxia (HH) exposure can cause serious brain injury as well as life-threatening cerebral edema in severe cases. Previous studies on the mechanisms of HH-induced brain injury have been conducted primarily using non-primate animal models that are genetically distant to humans, thus hindering the development of disease treatment. Here, we report that cynomolgus monkeys ( Macacafascicularis) exposed to acute HH developed human-like HH syndrome involving severe brain injury and abnormal behavior. Transcriptome profiling of white blood cells and brain tissue from monkeys exposed to increasing altitude revealed the central role of the HIF-1 and other novel signaling pathways, such as the vitamin D receptor (VDR) signaling pathway, in co-regulating HH-induced inflammation processes. We also observed profound transcriptomic alterations in brains after exposure to acute HH, including the activation of angiogenesis and impairment of aerobic respiration and protein folding processes, which likely underlie the pathological effects of HH-induced brain injury. Administration of progesterone (PROG) and steroid neuroprotectant 5α-androst-3ß,5,6ß-triol (TRIOL) significantly attenuated brain injuries and rescued the transcriptomic changes induced by acute HH. Functional investigation of the affected genes suggested that these two neuroprotectants protect the brain by targeting different pathways, with PROG enhancing erythropoiesis and TRIOL suppressing glutamate-induced excitotoxicity. Thus, this study advances our understanding of the pathology induced by acute HH and provides potential compounds for the development of neuroprotectant drugs for therapeutic treatment.

A Novel Synthetic Steroid of 2ß,3α,5α-Trihydroxy-androst-6-one Alleviates the Loss of Rat Retinal Ganglion Cells Caused by Acute Intraocular Hypertension via Inhibiting the Inflammatory Activation of Microglia.

Neuroinflammation has been well recognized as a key pathological event in acute glaucoma. The medical therapy of acute glaucoma mainly focuses on lowering intraocular pressure (IOP), while there are still scarce anti-inflammatory agents in the clinical treatment of acute glaucoma. Here we reported that ß,3α,5α-trihydroxy-androst-6-one (sterone), a novel synthetic polyhydric steroid, blocked neuroinflammation mediated by microglia/macrophages and alleviated the loss of retinal ganglion cells (RGCs) caused by acute intraocular hypertension (AIH). The results showed that sterone significantly inhibited the morphological changes, the up-regulation of inflammatory biomarker ionized calcium-binding adapter molecule 1 (Iba-1), and the mRNA increase of proinflammatory tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) in BV2 microglia and RAW264.7 macrophages. Moreover, immunofluorescence and western blotting analysis revealed that sterone markedly abrogated the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB) p65 subunit. Furthermore, sterone significantly suppressed the inflammatory microglial activation and RGCs' reduction caused by retinal ischemia/reperfusion (I/R) injury in a rat AIH model. These results suggest sterone may be a potential candidate in the treatment of acute glaucoma caused by microglial activation-mediated neuroinflammatory injury.

Association of Low Zinc Finger Antiviral Protein Expression with Progression and Poor Survival of Patients with Hepatocellular Carcinoma.

BACKGROUND/AIMS: Zinc finger antiviral protein (ZAP) has been reported to be expressed in hepatocellular carcinoma (HCC), and ZAP expression is associated with apoptotic signaling in cancer cells. This study aimed at investigating the expression of ZAP in HCC cells and its significance in clinical pathology. METHODS: Real-time quantitative PCR and western blot assays were employed to detect ZAP RNA and protein expression in normal human hepatocytes, HCC cells, and five primary HCC cell lines. Immunohistochemistry was performed to detect ZAP expression in 147 paraffin-embedded HCC tissues and adjacent normal tissues. The clinical significance of ZAP expression was analyzed in tissue samples from patients with or without infection by hepatitis B virus (HBV). RESULTS: ZAP expression in HCC cells and human primary HCC cell lines was significantly lower than that of normal human hepatocytes. Among 147 HCC samples, ZAP expression was lower in HCC tissues than in adjacent normal tissues for 107 (77.0%) samples. In patients with HCC and HBV infection, ZAP expression was related to pathological grade (P < 0.05); in HBV-negative patients with HCC, ZAP expression was associated with tumor size (P < 0.05) and clinical stage (P < 0.05). The overall survival time in patients with low ZAP expression was significantly shorter than survival times of those with high ZAP expression (P < 0.05), especially for patients with moderately to well-differentiated HCC (Grade 1-2) and HCC at stage T1 and T2 (P < 0.05). Cox multivariate analysis showed that ZAP expression was an independent predictor of survival of patients with HCC (P < 0.01). CONCLUSION: Low ZAP expression is closely associated with disease progression and poor prognosis for patients with HCC.

Intravenous injections of the oncolytic virus M1 as a novel therapy for muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is associated with low survival and high recurrence rates even in cases in which patients receive systemic treatments, such as surgery and chemotherapy. Here, we found that a naturally existing alphavirus, namely, M1, selectively kills bladder cancer cells but not normal cells, findings supported by our observations of changes in viral replication and MIBC and patient-derived MIBC cell apoptosis. Transcriptome analysis revealed that interferon-stimulated genes (ISGs) are expressed at low levels in sensitive bladder cancer cells and high levels in resistant cells. Knocking down ZC3HAV1 (ZAP), an antiviral factor in ISGs, restores M1 virus reactivity in resistant cells, and overexpressing ZAP partially reverses M1 virus-induced decreases in cell viability in sensitive cells. In orthotopic MIBC mice, tail vein injections of M1 significant inhibit tumor growth and prolong survival period, antitumor effects of M1 are stronger than those of the first-line chemotherapy agent cisplatin (CDDP). Treated tumors display enhanced cleaved-caspase-3 signals, which are representative of cell apoptosis, and decreased Ki-67 signals, which are representative of cell proliferation. Moreover, tissue microarray (TMA) analyses of clinical tumor specimens revealed that up to 45.6% of cases of MIBC presented with low ZAP expression, a finding that is prevalent in advanced MIBC. Our results indicate that the oncolytic virus M1 is a novel agent capable of functioning as a precise and effective therapy for MIBC.

Evolutionary trajectories of snake genes and genomes revealed by comparative analyses of five-pacer viper.

Snakes have numerous features distinctive from other tetrapods and a rich history of genome evolution that is still obscure. Here, we report the high-quality genome of the five-pacer viper, Deinagkistrodon acutus, and comparative analyses with other representative snake and lizard genomes. We map the evolutionary trajectories of transposable elements (TEs), developmental genes and sex chromosomes onto the snake phylogeny. TEs exhibit dynamic lineage-specific expansion, and many viper TEs show brain-specific gene expression along with their nearby genes. We detect signatures of adaptive evolution in olfactory, venom and thermal-sensing genes and also functional degeneration of genes associated with vision and hearing. Lineage-specific relaxation of functional constraints on respective Hox and Tbx limb-patterning genes supports fossil evidence for a successive loss of forelimbs then hindlimbs during snake evolution. Finally, we infer that the ZW sex chromosome pair had undergone at least three recombination suppression events in the ancestor of advanced snakes. These results altogether forge a framework for our deep understanding into snakes' history of molecular evolution.

Characterization of a Synthetic Steroid 24-keto-cholest-5-en-3ß, 19-diol as a Neuroprotectant.

BACKGROUND: Neuroactive steroids represent promising candidates for the treatment of neurological disorders. Our previous studies identified an endogenous steroid cholestane-3ß, 5α, 6ß-triol (Triol) as a novel neuroprotectant. AIM: We aimed to identify a potent candidate for stroke treatment through a screening of Triol analogs. METHODS: Hypoxia- and glutamate-induced neuronal injury models in vitro, middle cerebral artery occlusion (MCAO)-induced cerebral ischemia model in vivo, fluorescein diacetate (FDA) for alive and propidium iodide (PI) for dead staining, LDH assay, and calcium imaging techniques were used. RESULTS: 24-keto-cholest-5-en-3ß, 19-diol (Diol) showed the most potent neuroprotective effect among the screened structurally related compounds. FDA and PI staining showed that Diol concentration dependently increased the survival rate of cerebellar granule neurons (CGNs) challenged with glutamate or hypoxia, with an effective threshold concentration of 2.5 µM. Consistently, the quantitative LDH release assay showed the same concentration-dependent protection in both models. Diol, at 10 µM, potently decreased glutamate- and hypoxia-induced LDH release from 51.6 to 18.2% and 62.1 to 21.7%, respectively, which values are close to the normal LDH release (~16-18%). Moreover, we found Diol effectively decreased MCAO-induced infarction volume in mice from ~23% to 7%, at a dose of 6 mg/kg. We further explored the underlying mechanism and found that Diol attenuated NMDA-induced intracellular calcium ([Ca(2+) ]i ) increase in cortical neurons, suggesting a negative modulatory effect on NMDA receptor. CONCLUSION: Taken together, we identified Diol as a potent neuroprotectant. It may represent a novel and promising neuroprotectant for stroke intervention.

A novel recombinant fibrinogenase of Agkistrodon acutus venom protects against hyperacute rejection via degradation of complements.

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 rFII, a recombinant fibrinogenase from Agkistrodon acutus venom, on complement and HAR. The degradation effect of rFII 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 rFII against HAR. Our investigation indicated that rFII 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 rFII. Importantly, in an ex vivo rat-to-human perfusion model, the survival of rat hearts perfused with human serum treated with rFII (83.36 ± 16.63 min) were significantly longer than that of hearts perfused with fresh human serum(15.94 ± 4.75 min). At the time of 15 minutes after perfusion, functions of hearts added with 50 ug/ml rFII sustained well with heart rates at 283 ± 65.32 beats/minute and LVDP at 13.70 ± 5.45 Kpa, 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 rFII 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 rFII 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 rFII and its protection against HAR, indicating that rFII might be as a potential therapeutic agent for xenotransplantation.

A novel fibrinogenase from Agkistrodon acutus venom protects against DIC via direct degradation of thrombosis and activation of protein C.

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.

The effect of the fibrinolytic enzyme FIIa from Agkistrodon acutus venom on acute pulmonary thromboembolism.

AIM: To evaluate the effects of the fibrinolytic enzyme FII(a) from Agkistrodon acutus venom on acute pulmonary thromboembolism (APT) in animal models. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Bleomycin induces upregulation of lysyl oxidase in cultured human fetal lung fibroblasts.

AIM: To investigate the mechanism of bleomycin (BLM)-induced pulmonary fibrosis. METHODS: 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. RESULTS: 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. CONCLUSION: Bleomycin induces upregulation of LO in cultured human fetal lung fibroblasts, which may be the mechanism of bleomycin-induced pulmonary fibrosis.

Glycogen synthase kinase-3beta regulates astrocytic differentiation of U87-MG human glioblastoma cells.

AIM: To evaluate the role of glycogen synthase kinase-3beta (GSK-3beta) in the induced differentiation of human glioblastoma cells. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Aspirin inhibits proliferation of gemcitabine-resistant human pancreatic cancer cells and augments gemcitabine-induced cytotoxicity.

AIM: To investigate whether aspirin is able to augment gemcitabine-induced cytotoxicity in human pancreatic cancer cells. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Orphan nuclear receptor Nur77 is required for the differentiation of C6 glioma cells induced by cholera toxin.

AIM: To investigate a possible regulator gene involved in the cholera toxin-induced differentiation of rat C6 glioma cells. METHODS: 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. RESULTS: 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. CONCLUSION: Nur77 participated actively and essentially as an important regulator in the cholera toxin-induced differentiation of C6 cells.

Which preventive measures might protect health care workers from SARS?

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Some measures, particularly good air ventilation in SARS wards, may be effective in minimizing or preventing SARS transmission among HCWs in hospitals.

JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons.

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.

The effect of fibrinolytic enzyme FIIa from Agkistrodon acutus venom on disseminated intravascular coagulation in rabbits.

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.

Purification and partial characterizations of coagulant protein FIa from Daboia russelli siamensis (Myanmar) venom.

AIM: To purify and characterize the coagulant protein FIa from Daboia russelli siamensis (Myanmar) venom. METHODS: 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. RESULTS: 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. CONCLUSION: FIa is a factor X-activating enzyme, which could activate factor X to factor Xa, but has no effect on prothrombin and fibrinogen.

Activation of PI3-K/Akt pathway for thermal preconditioning to protect cultured cerebellar granule neurons against low potassium-induced apoptosis.

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.