Expression of endonuclease G in the brain tissue of rats with chronic fluorosis / 中华地方病学杂志

Objective To investigate the possible mechanism of endonuclease G (Endo G)-mediated non-Caspase-dependent apoptotic pathway in brain neuronal apoptosis in chronic fluorosis rats.Methods Sixty Sprague Dawley (SD) rats (half male and half female) were randomly divided into two groups:control group fed with tap water with fluoride content ＜ 0.5 mg/L and fluorine group in which sodium fluoride was added into drinking water with fluoride content of 50.0 mg/L.Both groups were fed with standard food with fluorine content ＜ 0.5 mg/kg.The experiment period was 10 months.At the end of the experiment,all the animals were sacrificed,and brain tissue was taken.Flow cytometry was used to examine apoptosis rate,immune-histochemistry was employed to detect the distribution of Endo G in brain tissue;Western blotting was used to test the protein expression of Endo G.Results Compared to the apoptosis rate of control group [(1.3 ± 0.6)％,(1.9 ± 0.3)％],the apoptosis rate in hippocampus and cortex of rats with chronic fluorosis [(2.6 ± 0.6)％,(3.1-± 0.7)％] was significantly increased (t =3.1,3.4,all P ＜ 0.05).The Endo G positive neurons and their degree of staining in CA1,CA2,CA3 and CA4 of hippocampus,frontal cortex as well as the upper layer of parietal cortex [(11.1 ± 2.2),(10.2 ± 1.9),(9.8 ± 3.1),(9.9 ± 1.6),(10.6 ± 2.9),(8.2 ± 2.4),(11.1 ± 2.8) scores] in rats with chronic fluorosis were significantly higher than those in the control group [(5.8 ± 1.8),(6.7 ± 2.6),(5.2 ± 2.4),(7.2 ± 2.1),(7.7 ± 2.6),(6.1 ± 1.9),(8.1 ± 2.6) scores,t =2.9,2.5,2.4,2.3,2.2,2.5,2.3,P ＜ 0.01 or ＜ 0.05].The protein level of Endo G in the mitochondria of rat brains with chronic fluorosis [(86.4 ± 7.2)％,(83.9 ± 6.8)％] was significantly lower than that of control group [(100.0 ± 6.1)％,(100.0 ± 5.5)％,t =2.6,2.3,all P ＜ 0.05].Meanwhile,the protein level of Endo G in the nucleus of neurons from chronic fluorosis rats [(117.5 ± 6.4)％,(115.2 ± 6.2)％] was significantly higher than that of the control [(100.0 ± 5.2)％,(100.0 ± 5.5)％,t =2.5,2.2,all P ＜ 0.05].Conclusion The high expression of Endo G and nuclear transfer are related to the neuron apoptosis in chronic fluorosis rat,which may be one of the mechanisms of brain injury of the disease.

Mechanism of apoptosis of bladder cancer cells BIU87 induced by emodin through Caspase-independent pathway / 中华泌尿外科杂志

Objective To study the effects of emodin on apoptosis and mRNA and protein of apoptosis inducing factor (AIF) and Endonuclease G (Endo G) in human bladder cancer cells BIU87,and to investigate the anticancer mechanism of emodin. Methods The BIU87 cells were divided into 4 groups,control group,Z-VAD-FMK group,emodin group,emodin combined with Z-VAD-FMK group.The effects of different concentrations of emodin at different action time on cells proliferation of BIU87 in vitro culture were measured by methylthiazole (MTT) chromatometry,the cells apoptosis were detected by flow cytometry,and expression of AIF and Endo G were examined by reverse transcription PCR (RT-PCR) and Western blot.Results MTT assay demonstrated that the higher concentration of emodin and the longer action time,the more significant inhibition of tumor cell growth.Based on the IC50 value,80 μmol/L and 72 h of emodin intervention were selected as an intervention condition.The apoptosis rate in emodin group (44.57 ± 1.52 ) ％was significantly higher than that in emodin combined with Z-VAD-FMK group (35.58 ± 1.61 ) ％ ( P ＜0.01).RT-PCR and Western blot showed that the mRNA and protein of AIF in emodin combined with Z-VAD-FMK group,emodin group,control group,Z-VAD-FMK group were ( 1.74 ± 0.11 ) and (2.59 ±0.13),(1.36±0.08) and (1.89±0.14),(0.37 ±0.02) and (0.53±0.11),(0.42 ±0.06) and (0.44 ± 0.07),respectively.There were significant differences between emodin group and the other groups (P ＜0.01 ).The mRNA and protein of Endo G in emodin combined with Z-VAD-FMK group,emodin group,control group,Z-VAD-FMK group were (2.28±0.15) and (3.31 ±0.36),(1.85 ±0.13) and (2.15 ±0.27),(0.53 ±0.07) and (0.71 ±0.16),(0.61 ±0.04) and (0.67 ±0.22),respectively.The differences were significant between emodin group and the other groups ( P ＜ 0.01 ). Coneltusion Emodin can upgrade the expression of AIF and Endo G in bladder cancer cells BIU87,which can induce apoptosis through Caspase-independent pathway.

Leishmania (Viannia) panamensis expresses a nuclease with molecular and biochemical features similar to the Endonuclease G of higher eukaryotes / Leishmania (Viannia) panamensis expresa una nucleasa molecular y bioquímicamente similar a la Endonucleasa G de eucariotas superiores

Objective: To characterize the molecular and biochemical features of the Endonuclease G of Leishmania (Viannia) panamensis.Methods: The gene of the putative L. (V.) panamensis Endonuclease G was amplified, cloned, and sequenced. The recombinant protein was produced in a heterologous expression system and biochemical assays were run to determine its ion, temperature, and pH preferences.Results: The L. (V.) panamensis rENDOG has biochemical features similar to those found in other trypanosomatids and higher eukaryotes. In addition, phylogenetic analysis revealed a possible evolutionary relationship with metazoan ENDOG.Conclusions: L. (V.) panamensis has a gene that codifies an ENDOG homologous to those of higher organisms. This enzyme can be produced in Escherichia coli and is able to degrade covalently closed circular double-stranded DNA. It has a magnesium preference, can be inhibited by potassium, and is able to function within a wide temperature and pH range.

Objetivo: Caracterizar molecular y bioquímicamente la Endonucleasa G (EndoG) de Leishmania (Viannia) panamensis.Métodos: El gen de la putativa Endonucleasa G de L. (V.) panamensis fue amplificado, clonado y secuenciado. La proteína recombinante se produjo en un sistema de expresión heterólogo y la proteína activa se sometió a pruebas bioquímicas para determinar la preferencia de iones, temperatura y pH.Resultados: La rEndoG de L. (V.) panamensis muestra características bioquímicas similares a aquellas descritas en otros trypanosomatidos y en eucariotas superiores. Además, los análisis filogenéticos muestran una posible relación evolutiva con la Endonucleasa G de metazoos.Conclusiones: Leishmania (V.) panamensis posee un gen que codifica para una endonucleasa homóloga a la EndoG de otros organismos superiores, que se puede producir de forma recombinante en Escherichia coli y que es capaz de degradar ADN circular cerrado de doble cadena. Tiene una preferencia por los iones magnesio y manganeso para usarlos como cofactor y es inhibida por el potasio. Además, funciona en un amplio rango de pH y temperatura.

Effect of Ginsenoside Rd Pretreatment on the Expressions of NR2B Receptor and Endonuclease G After Focal Cerebral Ischemia-Reperfusion in Rats / 国际脑血管病杂志

Objective:To observe the effect of ginsenoside Rd pretreatment on the expressions of N-methyl-D-aspartate(NMDA)receptor subunit NR2 B protein and endonuclease G(EndoG)in basal ganglia region after cerebral focal ischemia-reperfusion in rats and to investigate possible mechanism of ginsenoside Rd in the treatment of ischemic stroke.Methods:A rat model of middle cerebral artery occlusion(MCAO)was induced by intraluminal filament method.The expressions of NR2B and EndoG in basal ganglia region for focal cerebral iSChemia 1 hour,and 1,6,24 and 72 hours reperfusion were detected by immunohistochemical staining and image analysis method.The effects of ginsenoside Rd on the expressions of FaxioG and NR2B and the volume of cerebral infarction were evaluated.Results:The positive expression of NR2B in basal ganglia region on the ischemic side in ischemia-reperfusion group was increased significantly.The expression of EndoG in the nucleus was notable;the positive expressions of NR2B and EndoG at different reperfusion time points in ginsenoside Rd pretreatment group were decreased significantly(P<0.05 or P<0.01),and the volume of cerebral infarction was reduced significantly(P<0.01).Conclusions:The expressions of NMDA receptor subunit NR2B protein and apoptosis-inducing factor EndoG were increased significantly after cerebral focal ischemia reperfusion;ginsenoside Rd pretreatment may significantly reduce the expressions of NR2B and EndoG.It reduces the volume of cerebral infarction by inhibiting excitatory neurotoxicity and blocking neuronal apoptosis,and thus plays a role in neuroprotection.

Treatment with MnTBAP Protects Against Early Nuclear Translocation of Endonuclease G and Reduces Cerebral Infarction after Focal Cerebral Ischemia/Reperfusion in Mice

BACKGROUND: Reactive Oxygen Species (ROS) have been implicated in the pathophysiology of brain injury after ischemia/reperfusion. Recently, it has been reported that endonuclease G (EndoG), a mitochondrial protein, is activated by neuronal excitotoxicity and translocated into nucleus inducing apoptosis. However, it is not elucidated whether ROS are involved in the nuclear translocation of EndoG in focal cerebral ischemia/reperfusion in mice. We investigated whether treatment of manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP) protects against early nuclear translocation of EndoG and reduces cerebral infarction after ischemia/reperfusion in mice METHODS: Adult male mice were subjected to middle cerebral artery occlusion (MCAO) for 60 min, followed by reperfusion. Immunohistochemistry and Western blot analysis for EndoG were performed at various time points after ischemia/reperfusion. Double staining with EndoG and Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) was also performed. MnTBAP was used to determine whether the production of ROS could inhibit translocation of EndoG into the nucleus. RESULTS: Western blot analysis and Immunohistochemistry of EndoG showed that nuclear EndoG was detected as early as 4 hrs after reperfusion, and mitochondrial EndoG was significantly reduced at the same time. Double staining with EndoG and TUNEL showed a spatial relationship between EndoG expression and DNA fragmentation. MnTBAP-treated mice showed that the translocation of EndoG was attenuated in comparison with the vehicle- treated mice and decreased infarction volume after ischemia/reperfusion. CONCLUSIONS: MnTBAP reduced the generation of ROS, and inhibited the early translocation of EndoG, which was followed by the reduction of infarction volume in the ischemic brain after ischemia/reperfusion.

Up-regulation of Bax and endonuclease G, and down-modulation of Bcl-X(L) involved in cardiotoxin III-induced apoptosis in K562 cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced K562 cell apoptosis was confirmed by DNA fragmentation (DNA ladder, sub-G1 formation) and phosphatidylserine (PS) externalization with an IC50 value of 1.7 mug/ml at 48 h. A mechanistic analysis demonstrated that CTX III-induced apoptotic cell death was accompanied by up-regulation of both Bax and endonuclease G (Endo G), and downregulation of Bcl-X(L). CTX III had no effect on the levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. CTX III treatment caused loss of the mitochondrial membrane potential (delta psi m), release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and -3. CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. However, CTX III did not generate reactive oxygen species (ROS) and antioxidants, including N-acetylcysteine and catalase, did not block CTX III-induced apoptosis in K562 cells. Modulation of Bax, Bcl-X(L), and the Endo G proteins, release of mitochondrial cytochome c, and activation of caspase-3 and -9 all are involved in the CTX III-triggered apoptotic process in human leukemia K562 cells.