An enhanced methodology for predicting protein-protein interactions between human and hepatitis C virus via ensemble learning algorithms.

Hepatitis C virus (HCV) is responsible for a variety of human life-threatening diseases, which include liver cirrhosis, chronic hepatitis, fibrosis and hepatocellular carcinoma (HCC) . Computational study of protein-protein interactions between human and HCV could boost the findings of antiviral drugs in HCV therapy and might optimize the treatment procedures for HCV infections. In this analysis, we constructed a prediction model for protein-protein interactions between HCV and human by incorporating the features generated by pseudo amino acid compositions, which were then carried out at two levels: categories and features. In brief, extra-tree was initially used for feature selection while SVM was then used to build the classification model. After that, the most suitable models for each category and each feature were selected by comparing with the three ensemble learning algorithms, that is, Random Forest, Adaboost, and Xgboost. According to our results, profile-based features were more suitable for building predictive models among the four categories. AUC value of the model constructed by Xgboost algorithm on independent data set could reach 92.66%. Moreover, Distance-based Residue, Physicochemical Distance Transformation and Profile-based Physicochemical Distance Transformation performed much better among the 17 features. AUC value of the Adaboost classifier constructed by Profile-based Physicochemical Distance Transformation on the independent dataset achieved 93.74%. Taken together, we proposed a better model with improved prediction capacity for protein-protein interactions between human and HCV in this study, which could provide practical reference for further experimental investigation into HCV-related diseases in future.Communicated by Ramaswamy H. Sarma.

Estradiol attenuates chronic restraint stress-induced dendrite and dendritic spine loss and cofilin1 activation in ovariectomized mice.

Ovarian hormone deprivation is associated with mood disorders, such as depression, and estradiol therapy is significantly more effective than placebos in treating major depression associated with menopause onset. However, the effect of estradiol on neuronal plasticity and its mechanisms remain to be further elucidated. In this study, behavioral assessments were used to examine the antidepressant effect of estradiol in ovariectomized (OVX) B6.Cg-TgN (Thy-YFP-H)-2Jrs transgenic mice on chronic restraint stress (CRS)-induced dendrite and dendritic spine loss; Yellow fluorescent protein (YFP) is characteristically expressed in excitatory neurons in transgenic mice, and its three-dimensional images were used to evaluate the effect of estradiol on the density of different types of dendritic spines. Quantification and distribution of cofilin1 and p-cofilin1 were determined by qPCR, Western blots, and immunohistochemistry, respectively. The results revealed that treatment with estradiol or clomipramine significantly improved depression-like behaviors. Estradiol treatment also significantly upregulated the dendritic density in all areas examined and increased the density of filopodia-type, thin-type and mushroom-type spines in the hippocampal CA1 and elevated the thin-type and mushroom-type spine density in the PFC. Consistent with these changes, estradiol treatment significantly increased the density of p-cofilin1 immunopositive dendritic spines. Thus, these data reveal a possible estradiol antidepressant mechanism, in that estradiol promoted the phosphorylation of cofilin1 and reduced the loss of dendrites and dendritic spines, which of these dendritic spines include not only immature spines such as filopodia-type, but also mature spines such as mushroom-type, and attenuated the depression-like behavior.

RhoA/Rock2/Limk1/cofilin1 pathway is involved in attenuation of neuronal dendritic spine loss by paeonol in the frontal cortex of D-galactose and aluminum­induced Alzheimer's disease­like rat model.

Alzheimer's disease (AD) has become the most prevalent neurodegenerative disorder. Given the pathogenesis of AD is unclear, there is currently no drug approved to halt or delay the progression of AD. Therefore, it is pressing to explore new targets and drugs for AD. In China, polyphenolic Chinese herbal medicine has been used for thousands of years in clinical application, and no toxic effects have been reported. In the present study, using D­galactose and aluminum­induced rat model, the effects of paeonol on AD were validated via the Morris water maze test, open field test, and elevated plus maze test. Neuronal morphology in frontal cortex was assessed using ImageJ's Sholl plugin and RESCONSTRUCT software. RhoA/Rock2/Limk1/cofilin1 signaling pathway­related molecules were determined by Western blotting. Cofilin1 and p­cofilin1 were analyzed by immunofluorescence. Results showed that pre­treatment with paeonol attenuated D­galactose and aluminum­induced behavioral dysfunction and AD­like pathological alterations in the frontal cortex. Accompanied by these changes were the alterations in the dendrite and dendritic spine densities, especially the mushroom­type and filopodia­type spines in the apical dendrites, as well as actin filaments. In addition, the activity and intracellular distribution of cofilin1 and the molecules RhoA/Rock2/Limk1 that regulate the signaling pathway for cofilin1 phosphorylation have also changed. Our data suggests that paeonol may be through reducing Aß levels to alleviate the loss of fibrillar actin and dendrites and dendritic spines via the Rho/Rock2/Limk1/cofilin1 signaling pathway in the frontal cortex, and ultimately improving AD­like behavior.

Endothelial Cdk5 deficit leads to the development of spontaneous epilepsy through CXCL1/CXCR2-mediated reactive astrogliosis.

Blood-brain barrier (BBB) dysfunction has been suggested to play an important role in epilepsy. However, the mechanism mediating the transition from cerebrovascular damage to epilepsy remains unknown. Here, we report that endothelial cyclin-dependent kinase 5 (CDK5) is a central regulator of neuronal excitability. Endothelial-specific Cdk5 knockout led to spontaneous seizures in mice. Knockout mice showed increased endothelial chemokine (C-X-C motif) ligand 1 (Cxcl1) expression, decreased astrocytic glutamate reuptake through the glutamate transporter 1 (GLT1), and increased glutamate synaptic function. Ceftriaxone restored astrocytic GLT1 function and inhibited seizures in endothelial Cdk5-deficient mice, and these effects were also reversed after silencing Cxcl1 in endothelial cells and its receptor chemokine (C-X-C motif) receptor 2 (Cxcr2) in astrocytes, respectively, in the CA1 by AAV transfection. These results reveal a previously unknown link between cerebrovascular factors and epileptogenesis and provide a rationale for targeting endothelial signaling as a potential treatment for epilepsy.

Novel antidepressant effects of Paeonol alleviate neuronal injury with concomitant alterations in BDNF, Rac1 and RhoA levels in chronic unpredictable mild stress rats.

RATIONALE: Increasing evidence has suggested that major depressive disorder (MDD) is highly associated with brain-derived neurotrophic factor (BDNF) levels, dendrites atrophy, and loss of dendritic spines, especially in emotion-associated brain regions including the hippocampus. Paeonol is a kind of polyphenols natural product with a variety of therapeutic effects. Recent studies have reported its antidepressant effects. However, it is unclear what signaling pathways contribute to improve MDD. OBJECTIVE: The present study investigated the effect of Paeonol on hippocampal neuronal morphology and its possible signaling pathways in chronic unpredictable mild stress (CUMS) rat model. METHODS: Using CUMS rat model, the antidepressant-like effect of Paeonol was validated via depression-related behavioral tests. Neuronal morphology in hippocampal CA1 and DG was assessed using ImageJ's Sholl plugin and RESCONSTRUCT software. BDNF signaling pathway-related molecules was determined by Western blotting. RESULTS: Paeonol attenuated CUMS-induced depression-like behaviors, which were accompanied by hippocampal neuronal morphological alterations. After Paeonol treatment for 4 weeks, the dendritic length and complexity and the density of dendritic spines markedly increased in the hippocampal CA1 and the dentate gyrus (DG). However, CUMS or Paeonol treatment does not selectively affect dendritic spine types. Simultaneously, administration of Paeonol deterred CUMS-induced cofilin1 activation that is essential for remolding of dendritic spines. The induction of CUMS downregulated BDNF levels and upregulated Rac1/RhoA levels; however, the tendency of these was inhibited by treatment with Paeonol. CONCLUSION: Our data suggest that BDNF-Rac1/RhoA pathway may be involved in attenuation of CUMS-induced behavioral and neuronal damage by Paeonol that may represent a novel therapeutic agent for depression.

Evaluation of Endotracheal Intubation with a Flexible Fiberoptic Bronchoscope in Lateral Patient Positioning: A Prospective Randomized Controlled Trial.

BACKGROUND: There is an unmet need for a reliable method of airway management for patients in the lateral position. This prospective randomized controlled two-center study was designed to evaluate the feasibility of intubation using a flexible fiberoptic bronchoscope in the lateral position during surgery. METHODS: Seventy-two patients scheduled for elective nonobstetric surgery in the lateral decubitus position requiring tracheal intubation under general anesthesia at Lishui Central Hospital of Zhejiang Province and Jiaxing First Hospital of Zhejiang Province from April 1, 2015, to September 30, 2015, were enrolled in this study. Patients were randomly assigned to the supine position group (Group S, n = 38) and the lateral position group (Group L, n = 34). Experienced anesthetists performed tracheal intubation with a fiberoptic bronchoscope after general anesthesia. The time required for intubation, intubation success rates, and hemodynamic changes was recorded. Between-group differences were assessed using the Student's t-test, Mann-Whitney U-test, or Chi-square test. RESULTS: The median total time to tracheal intubation was significantly longer in Group S (140.0 [135.8, 150.0] s) compared to Group L (33.0 [24.0, 38.8] s) (P < 0.01). The first-attempt intubation success rate was significantly higher in Group L (97%) compared to Group S (16%). Hemodynamic changes immediately after intubation were more exaggerated in Group S compared to Group L (P = 0.02). CONCLUSION: Endotracheal intubation with a flexible fiberoptic bronchoscope may be an effective and timesaving technique for patients in the lateral position. TRIAL REGISTRATION: Chinese Clinical Trial Register, ChiCTR-IIR-16007814; http://www.chictr.org.cn/showproj.aspx?proj=13183.

[Macrocalin A induces apoptosis of multiple myeloma U266 cells through inhibiting the proteasome].

This study was purposed to investigate the inhibitory effect of macrocalin A (MA) on proteasome of multiple myeloma U266 cells in vitro and molecular mechanism of MA-inducing apoptosis. U266 cells in vitro were incubated with different concentrations (2, 4, 8 µg/mL) of MA, the Hochest staining and Annexin-V/PI double staining were used to detect the apoptosis of U266 cells. The expressions of protein ß1, ß1i, ß2, ß2i, ß5, ß5i, ubiquitous, 19S subunit S6', and BAD,BCL-2, FAS, FAS-L,MAPK, PARP, Pro-caspase 3, cleaved-caspase 3 were detected by Western blot technique. The results showed that along with time prolonging and dose increasing of MA, the small and compact fluorescent particles were observed in cytoplasm and nucleus of U266 cells stained with Hoechst 33258, the Annexin V(+)/PI(-) cells and the total apoptosis cells (Annexin V(+)/PI(-) and Annexin V(+)/PI(+)) increased. MA could elevate the ubiquitylation level in U266 cells, suppress the expression of ß1i,ß2, ß5i and 19S subunit S6', meanwhile the expression of BCJ-2, MAPK, PARP and pro-caspase 3 were down-regulated along with increasing of drug concentrations, but the expressions of BAD, FAS, FAS-L cleaved-caspase 3 were enhanced. It is concluded that MA can inhibit the effect of proteasome, and the mitochondrial pathway and death receptor pathway may play important roles in apoptosis of U266 cells induced by MA.

[Apoptosis of human lung carcinoma cell line EBC-1 induced by N, N'-di-(m-methylphenyl)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide and its molecular mechanism].

OBJECTIVE: To study whether N, N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) inhibits proliferation and induces apoptosis in human lung carcinoma cell line EBC-1 cells and its molecular mechanism. METHODS: Different concentrations of ZGDHu-1 and different times of culture were used to treat EBC-1 cells in vitro. The inhibition of proliferation was measured by BrdU-ELISA. Cell apoptosis was detected by Annexin V/PI staining and cellular DNA fragmentation ELISA. Phosphorylated p38MAPK and STAT3 were examined by flow cytometry. The protein expressions of bcl-2, bax, p53, Fas, and caspase-3 were detected by Western blot analysis. RESULTS: ZGDHu-1 inhibited EBC-1 cell proliferation within a certain range of treating times and does, with a 24 h IC(50) of (295 ± 25) ng/ml, 48 h of (112 ± 8) ng/ml and 72 h of (23 ± 2) ng/ml. The EBC-1 cell apoptosis was confirmed by Annexin V/PI labeling and cellular DNA fragmentation ELISA in a dose-related manner. When EBC-1 cells were treated with 50, 200, and 500 ng/ml ZGDHu-1 for 48 h, the expression rates of phosphor-p38MAPK protein were 67.4%, 88.2%, 91.1%, respectively, and that of the control was 10.6%. That of STAT3 protein were 56.5%, 43.6% and 34.6%, respectively, and that of the control was 89.1%. The expression of bax, p53 and Fas protein was significantly increased, that of bcl-2 was not changed, and that of caspase-3 was significantly decreased by the ZGDHu-1 treatment. CONCLUSION: ZGDHu-1 can inhibit proliferation and induce apoptosis in EBC-1 cells. The mitochondrial pathway mediated by Fas may be one of its mechanisms. The apoptosis of EBC-1 cells may associate with up-regulation of phosphor-p38MAPK and down-regulation of phosphor-STAT3 in the cells.

[Immunohistochemical studies of estrogen receptor in the heart of Gekko swinhonis].

AIM: To observed the expression of estrogen receptor (ER alpha and ER beta) in the heart of Gekko swinhonis. METHODS: The immunohistochemical technique for the estrogen receptor was used. RESULTS: The positive ER alpha and beta cells existed in cardiac myocytes and fibroblasts of the atria and the ventricles of Gekko swinhonis and had no sexual difference. The difference of ER alpha between the atria (11.56 +/- 1.67) and ventricles (6.68 +/- 1.88) was observed in both sexes (P < 0.01). CONCLUSION: The atria are probably the main target tissue of estrogen through ER alpha pathway while some functions of whole heart will be regulated by estrogen through ER beta pathway. The sexual differences aren' t related to the content of ER. It may be involved in the state of activity and function of ER under the physiological conditions.

[ZGDHu-1-inducing apoptosis of SHI-1 leukemia cells and its molecular mechanism].

The aim of study was to investigate the mechanism of N, N'-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) inducing apoptosis in SHI-1 human leukemia cell line. Different concentrations of ZGDHu-1 and different times of culture were used to treat SHI-1 cells; the apoptosis of SHI-1 cells was analyzed by morphology, DNA agarose gel electrophoresis, DNA content detection, Annexin-V/PI and Hoechst33258 labeling method, the mitochondrial transmembrane potential (Delta Psi m) were measured by dihydrorhodamin 123, and expressions of bcl-2, bax, Fas, p53 and mitochondrial membrane protein were analyzed by flow cytometry, while the bcl-2, bax and p53 gene were analyzed by RT-PCR. The transcriptional level of hTERT-mRNA was measured by real-time fluorescence quantitative RT-PCR. The results showed that after exposure to ZGDHu-1, SHI-1 cells were induced to apoptosis in a time-and does-dependent manner. SHI-1 cell apoptosis was confirmed by typical cell morphology, DNA fragmentation, sub-G(1) phase, Hoechst33258 and Annexin-V/PI labeling etc. The expression of bax, bax/bcl-2, p53 and Fas gene significantly increased and bcl-2 slightly decreased. ZGDHu-1 could increased the expression of mitochondrial membrane protein in a dose-dependent manner while Delta Psi m reduced. The expression of hTERT-mRNA significantly decreased. It is concluded that ZGDHu-1 can up-regulate the expression of p53, bax and bax/bcl-2. The mitochondrial pathway mediated by descent of mitochondrial transmembrane potential may be one of the mechanisms inducing apoptosis by ZGDHu-1, in which Fas gene also participates. Telomerase may be an effective gene target for anti-tumour effect of ZGDHu-1.

[Effect of ZGDHu-1 on proliferation and apoptosis of A549 cells in vitro and antitumor activity in vivo].

This study is to explore the mechanism and effect of N, N'-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation and apoptosis of A549 cells in vitro and on A549 xenograft tumor in nude mice. With different concentrations of ZGDHu-1 at different times were used to treat A549 cells in vitro. The proliferation was determined by living cell count, SRB assay and Brdu-ELISA. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin V/PI and Hoechst 33258 labeling method. The nude mice model of A549 xenograft tumor was established by subcutaneous inoculation. The suppression activity of ZGDHu-1 by intraperitoneal injection on xenograft mice model was detected. The expressions of bcl-2, bax and p53 gene and protein were analyzed by RT-PCR and flow cytometry. ZGDHu-1 can inhibit A549 cell proliferation viability within a certain range of treating time and does, and a majority of A549 cells were arrested in G2-M phase. The A549 cells apoptosis was confirmed by typical cell morphology, DNA fragment, Sub G1 phase, Hoechst 33258 and Annexin V/PI labeling method with a time and dose related manner. When the xenograft tumor mice model were treated with 10, 20 and 40 mg x kg(-1) ZGDHu-1 for 14 days, the tumor growth inhibition rate were 43.7%, 56.9% and 60.0%, respectively. The expression of bax, bax/bcl-2 and p53 gene and protein increased significantly and bcl-2 decreased slightly by the treatment of ZGDHu-1. ZGDHu-1 can significantly suppress the growth of A549 xenograft tumor in vivo and inhibited proliferation by inducing tumor cell apoptosis in vitro. The mechanism may associate with its up-regulation of bax and p53 during the apoptosis process.

[Effects of N, N-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1,2,4, 5-tetrazine-1,4-dicarboxamide (ZGDhu-1) on SHI-1 leukemia cells in vitro].

OBJECTIVE: To study the effect of ZGDHu-1 on proliferation, differentiation and apoptosis in SHI-1 human leukemia cell line and explore its possible mechanism. Methods SHI-1 cells were cultured with different concentration of ZGDHu-1 and for different time. The cell proliferation was analysed by cell counting, alive cell count, MTT assay and Brdu-ELISA. Cell apoptosis was analysed by morphology, DNA content, Annexin-V/PI and Hoechst 33258 labeling method. Cell differentiation were assayed by morphology,expression of CD11b,CD14 and CD64 and NBT reduction. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry. RESULTS: ZGDHu-1 inhibited SHI-1 cell proliferation in a time and dose dependent manner, the IC50- 48 h and IC50- 72 h were 250 ng/ml and 85 ng/ml, respectively. The majority of SHI-1 cells were arrested in G2/M phase. 48h after treated with 200 ng/ml ZGDHu-1, and those in G2/M phase accounted for (48.4 +/- 2.1)%. The SHI-1 cells apoptosis was increased with a time- and does-dependent manner. The morphology of SHI-1 cells cultured with 2-50 ng/ml ZGDHu-1 for three days become more mature with higher NBT positivity and up-regulated expressions of CD11b,CD14 and CD64. The expression of phosphor-p38MAPK was increased and phosphor-STAT3 down-regulated by the treatment of ZGDHu-1. CONCLUSION: ZGDHu-1 can inhibit SHI-1 cell proliferation and induce the cell differentiation and apoptosis. The mechanism may associate with its up-regulation of phosphor-p38MAPK and down-regulation phosphor-STAT3.

[Effects of N, N'-Di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide on proliferation, apoptosis and differentiation of NB4 leukemia cells in vitro].

The purpose of this study was to explore the effect of N, N'-di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation, differentiation and apoptosis in NB4 human leukemia cell line and its possible mechanism. Different concentrations of ZGDHu-1 and the different time of cultivation were used to treat NB4 cells. The proliferation inhibition of NB4 cells was analysed by cell counting, alive cell count, MTT assay. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin-V/PI and Hoechst 33258 labeling method. The analysis of cell morphological change, expression of CD11b, CD13 and NBT reduction were performed to evaluate the differentiation of NB4 cells. The expressions of bcl-2, bax and phosphorylated p38MAPK or STAT3 were detected by flow cytometry. While the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that ZGDHu-1 could inhibit NB4 cell proliferation viability within a certain range of treating time and does, IC(50) values at 48 and 72 hours were 450 ng/ml and 200 ng/ml respectively. A majority of NB4 cells were arrested in G(2/M) phase and a progressive decline of cells was seen in G(0/1). The NB4 cells apoptosis was confirmed by cell typical cell morphology, DNA fragments and sub-G(1) phase peak as well as Hoechst33258 and Annexin-V/PI labeling method with a time-dose-related manner. The morphology of NB4 cells cultured in the presence of 2 - 100 ng/ml ZGDHu-1 for three days was more mature with higher NBT positivity and expressions of CD11b and CD13 than those in control. The expression of phosphor-p38MAPK and bax was increased while phosphor-STAT3 and bcl-2 were unchanged by the treatment of ZGDHu-1. ZGDHu-1 could decrease the expression of hTERT-mRNA in a dose-dependent manner. It is concluded that ZGDHu-1 can inhibit proliferation, induce differentiation and apoptosis of NB4 cells. The mechanism may be associated with up-regulation of bax expression, enhancement of phosphor-p38MAPK activation and inhibition of hTERT-mRNA.

[Effects of sodium nitroprusside on P38MAPK/STAT3 activation and telomerase reverse transcriptase mRNA expression in inducing apoptosis of K562 cell line].

This study was aimed to investigate the activation of P38MAPK/STAT3 and expression of telomerase reverse transcriptase during sodium nitroprusside (SNP) inducing apoptosis of human leukemia cell line K562 and to explore the molecular mechanisms of SNP-inducing apoptosis in K562 cells. The K562 cell were treated with different concentrations of SNP and were cultured for different time. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and Annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantitate the in situ cell apoptosis. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry, while the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that SNP inhibited K562 cell growth. The K562 cell apoptosis was confirmed by typical cell morphology and DNA fragment, peak of sub-G1 phase, TUNEL and Annexin-V/PI labeling. A majority of K562 cells were arrested in G0/G1 phase. After treatment with SNP at 0.5-3.0 mmol/L, the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 increased first and decreased afterwards. Incubation of K562 cell with SNP (2 mmol/L) could increase the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 at 12 hours and 24 hours respectively, and down-regulated at 72 hours and 48 hours. SNP could decrease the expression of hTERT-mRNA in time-and dose-dependent manner. It is concluded that SNP can significantly induce K562 cells apoptosis, its mechanism may be related to the activation of P38MAPK and suppression of phosphorylated-STAT3 and hTRET-mRNA.

[Mechanism of sodium nitroprusside-induced apoptosis in K562 cell line].

To study the molecular mechanisms of nitric oxide donor sodium nitroprusside (SNP) -induced apoptosis in K562 human leukemia cell line, the different concentrations of SNP and different time of culture were used to treat K562 cell. At the same time, potassium ferricyamide (PFC) was used as control, blank was designed in experiment. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantify in situ cell apoptosis. Reactive oxygen species (ROS) in cells and mitochondrial transmembrane potential (DeltaPsim) were labeled by dihydrorhodamin 123, 2', 7'-dichlorodihydrofluorescein diacetate and rhodamin 123/PI. bcl-2, bax, bad, p53 gene proteins and mitochondrial membrane protein were analysed by flow cytometry. The results showed that the K562 cell apoptosis was confirmed by typical cell morphology, DNA fragment, sub-G(1) phase, TUNEL and annexin-V/PI labeling. A majority of K562 cells were arrested in G(0)/G(1) phase. During the process of SNP-induced apoptosis in K562 cell, the mean fluorescence intensity of ROS in cells was significantly higher than those in blank and PFC control, while the DeltaPsim reduced. The expression of p53, bax, bad, Fas protein and mitochondrial membrane protein increased and bcl-2 protein decreased after SNP treatment. It is concluded that SNP induces K562 cell apoptosis through increasing ROS in cells, expressing the p53, bax, bad, Fas protein and mitochondrial membrane protein and decreasing bcl-2 protein, opening the mitochondrial permeability transition pore and reducing DeltaPsim. Furthermore, the Fas was activated during the apoptosis process.

[Estrogen receptor alpha and colocalization of Bcl-2 in hippocampus of Alzheimer's disease].

OBJECTIVE: To investigate the possible mechanism by which estrogen regulates apoptosis through the estrogen receptor. METHODS: By means of fluorescence immunocytochemistry, the present study investigated the distribution of Bcl-2 and the colocolization of Bcl-2 and ERalpha immunoreactivity in the hippocampus of 10 Alzheimer's disease (AD) patients and 10 aged controls. RESULTS: Bcl-2 immunoreactivity was widely distributed in neurons, concentrating predominantly on the subfields CA3 and CA4 in the stratum pyramidale of hippocampus both in controls and in AD patients. Bcl-2 staining in the labeled neuron was observed mainly in the cytoplasm and neuritic processes, but a few nuclei were also positive. Bcl-2 labeling was also detected in the astrocytes mainly in AD, but sparsely in controls. Double-labeled fluorescence immunocytochemistry showed that most Bcl-2-immunolabeled neurons also exhibited positive staining for ERalpha. CONCLUSIONS: Estrogen may function as a regulator of apoptosis to modulate the expression of Bcl-2 in neurons and astrocytes in hippocampus of AD through ERalpha.

Colocalization and alteration of estrogen receptor-alpha and -beta in the hippocampus in Alzheimer's disease.

The human hippocampus is severely affected in Alzheimer's disease (AD). Because postmenopausal estrogen use may decrease the risk and delay the onset and progression of AD, possibly by a direct action on the hippocampal neurons, we used fluorescence immunocytochemistry to examine the colocalization of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) in the hippocampus of elderly human controls and AD patients. Double-labeling cells (DLCs) of ERalpha and ERbeta can be divided into 3 types: double-cytoplasm-staining cells (DCCs), double-nucleus-staining cells (DNCs), and ERalpha nucleus-staining and ERbeta cytoplasm-staining cells (NCCs). There was no difference in the percentage of DLCs in total ERalpha-positive cells or in total ERbeta-positive cells in the CA1 to CA4 subfields of the hippocampus between controls and AD patients. Interestingly, the ratio of DNCs to the total ERalpha-positive cells (2.6% +/- 0.5%) or to the total ERbeta-positive cells (1.8% +/- 0.3%) in the CA1 subfield of the AD hippocampus was significantly decreased in comparison with controls (5.0% +/- 0.7% and 3.9% +/- 0.6%, respectively; P<0.001), suggesting that changes in the compartmentalization of these receptors could play a role in the pathogenesis of AD.

Estrogen receptor alpha-immunoreactive astrocytes are increased in the hippocampus in Alzheimer's disease.

Postmenopausal estrogen use may decrease the risk, and delay the onset and progression, of Alzheimer's disease (AD). By means of fluorescence immunocytochemistry, the present study investigated the distribution of estrogen receptor alpha (ERalpha) in the human hippocampus in controls and in AD cases. ERalpha immunoreactivity was observed in neurons and glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes in the hippocampus both in controls and AD cases. The number and density of GFAP- and ERalpha-positive astrocytes was increased in AD. The number of GFAP-immunoreactive astrocytes, the number of nuclear ERalpha-staining astrocytes, and cytoplasmic ERalpha-staining astrocytes per unit area (1 mm(2)) significantly increased (P < 0.001, P < 0.05, P < 0.05, respectively) in CA1 in AD patients, while the percentage of ERalpha-immunoreactive astrocytes of the two groups did not differ (P > 0.05). These data suggest an important role for ERalpha-mediated effects of estrogens on neurons and astrocytes in the hippocampus of human and AD patients.