Oligomeric Aß25-35 induces the tyrosine phosphorylation of PSD-95 by SrcPTKs in rat hippocampal CA1 subfield.

PURPOSE: Although amyloid-ß (Aß) is one of the neuropathological hallmarks of Alzheimer's Disease (AD), the mechanisms of Aß neurotoxicity remain to be clarified. This study was aimed to evaluate the effect of Aß on postsynaptic density-95 (PSD-95) tyrosine phosphorylation. Elucidating the regulatory mechanisms underlying it may be a promising therapy in AD. METHODS: Aß25-35 oligomers (20 µg/rat) were administered intracerebroventricularly in adult male Sprague-Dawley rats. PSD-95 tyrosine phosphorylation was assessed using immunoprecipitation followed by immunoblot analysis. Immunoblot was applied for measuring the protein levels of PSD-95 and ß-actin. RESULTS: Following 3, 7, 14, 21 days after oligomeric Aß25-35 treatment, the tyrosine phosphorylation of PSD-95 increased significantly, and peaked at 3 days after oligomeric Aß25-35 treatment in hippocampal CA1 subfield. Src family protein tyrosine kinases (SrcPTKs) specific inhibitor PP2 attenuated the tyrosine phosphorylation of PSD-95 induced by Aß25-35. Amantadine [N-methyl-D-aspartate (NMDA) receptor noncompetitive antagonist], NVP-AAM077 (GluN2A-containing NMDA receptor selective inhibitor) and Ro25-6981 (GluN2B-containing NMDA receptor selective inhibitor) also suppressed the Aß25-35-induced PSD-95 tyrosine phosphorylation. CONCLUSION: These results suggest that Aß oligomers induce the tyrosine phosphorylation of PSD-95 by SrcPTKs, which is mediated by the activation of GluN2A- and GluN2B-containing NMDA receptors.

Microalgal photoautotrophic growth induces pH decrease in the aquatic environment by acidic metabolites secretion.

BACKGROUND: Microalgae can absorb CO2 during photosynthesis, which causes the aquatic environmental pH to rise. However, the pH is reduced when microalga Euglena gracilis (EG) is cultivated under photoautotrophic conditions. The mechanism behind this unique phenomenon is not yet elucidated. RESULTS: The present study evaluated the growth of EG, compared to Chlorella vulgaris (CV), as the control group; analyzed the dissolved organic matter (DOM) in the aquatic environment; finally revealed the mechanism of the decrease in the aquatic environmental pH via comparative metabolomics analysis. Although the CV cell density was 28.3-fold that of EG, the secreted-DOM content from EG cell was 49.8-fold that of CV (p-value < 0.001). The main component of EG's DOM was rich in humic acids, which contained more DOM composed of chemical bonds such as N-H, O-H, C-H, C=O, C-O-C, and C-OH than that of CV. Essentially, the 24 candidate biomarkers metabolites secreted by EG into the aquatic environment were acidic substances, mainly lipids and lipid-like molecules, organoheterocyclic compounds, organic acids, and derivatives. Moreover, six potential critical secreted-metabolic pathways were identified. CONCLUSIONS: This study demonstrated that EG secreted acidic metabolites, resulting in decreased aquatic environmental pH. This study provides novel insights into a new understanding of the ecological niche of EG and the rule of pH change in the microalgae aquatic environment.

Euendolithic Cyanobacteria and Proteobacteria Together Contribute to Trigger Bioerosion in Aquatic Environments.

Shellfish, mussels, snails, and other aquatic animals, which assimilate limestone (calcium carbonate, CaCO3) to build shells and skeletons, are effective carbon sinks that help mitigate the greenhouse effect. However, bioerosion, the dissolution of calcium carbonate and the release of carbon dioxide, hinders carbon sequestration process. The bioerosion of aquatic environments remains to be elucidated. In this study, the bioerosion of Bellamya spp. shells from the aquatic environment was taken as the research object. In situ microbial community structure analysis of the bioerosion shell from different geographical locations, laboratory-level infected culture, and validated experiments were conducted by coupling traditional observation and 16S rRNA sequencing analysis method. Results showed that bioeroders can implant into the CaCO3 layer of the snail shell, resulting in the formation of many small holes in the shell, which reduced the shell's density and made the shell fragile. Results also showed that bioeroders were distributed in two major phyla, namely, Cyanobacteria and Proteobacteria. Cluster analysis showed that Cyanobacteria sp. and two unidentified genera (Burkholderiaceae and Raistonia) were the key bioeroders. Moreover, results suggested that the interaction of Cyanobacteria and other bacteria promoted the biological function of "shell bioerosion." This study identified the causes of "shell bioerosion" in aquatic environments and provided some theoretical basis for preventing and controlling it in the aquatic industry. Results also provided new insights of cyanobacterial bioerosion of shells and microalgae carbon sequestration.

MiR-140-5p exerts a protective function in pregnancy-induced hypertension via mediating TGF-ß/Smad signaling pathway.

Animal experiments showed that PIH rats had increased mean arterial pressure (MAP), systolic blood pressure (SBP), and diastolic blood pressure (DBP), but decreased litter size, number of viable fetuses, fetal weight, and placental weight. The higher Flt-1 and lower VEGF was observed in PIH rats with elevated TNF-α and IL-6 levels and decreased IL-10 levels. Treatment with agomiR-140-5p improved regarding the above indicators. Cell experiments demonstrated that miR-140-5p mimic increased cell invasion and migration abilities and decreased the activity of TGF-ß/Smad pathway, while TGFBR1 can reverse the role of miR-140-5p mimic in trophoblasts.

Resveratrol ameliorates toxic effects of cadmium on placental development in mouse placenta and human trophoblast cells.

BACKGROUND: Cadmium (Cd) is a common heavy metal pollutant. Prenatal exposure to Cd results in adverse effects on fetal development. Placental apoptosis, inflammation, and epigenetic disruption have been implicated in Cd-induced placental toxicity. Resveratrol (Res) is a naturally occurring polyphenol with anti-apoptotic, anti-inflammatory, and epigenetic regulatory activities. In present study, the effects of Res on placental toxicity induced by Cd were evaluated. METHODS: Pregnant CD-1 mice were fed with base diet containing 0.2% Res started on gestational day 0 (GD0), and intraperitoneally injected with 4.5 mg/kg CdCl2 or saline once on GD9. JEG-3 cells were treated with 20 µM Res for 24 hr in the absence or presence of 20 µM CdCl2 for the second 12 hr. The fetal outcomes, the apoptosis in placenta and JEG-3 cells, the expression of inflammatory cytokines and chemokines including tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-2 (MIP-2) and chemokine (C-X-C motif) ligand 1 (KC), and expression of endoplasmic reticulum (ER) stress markers were evaluated. The expression and activities of DNA methyltransferase (DNMT), and the activation of Akt signaling pathway were detected. RESULTS: Cd exposure resulted in decreased fetal weight and crown-rump length while Res ameliorated these outcomes. Res suppressed Cd-induced apoptosis in placenta and JEG-3 cells, and decreased Cd-induced expression of TNF-α, IFN-γ, MCP-1, MIP-2, and KC in placenta. Cd greatly increased ER stress in placenta in mice, which was partially ameliorated by Res treatment. Res decreased Cd-induced upregulation of DNMT activity and suppressed Cd-induced expression of DNMT3B. Res restored estradiol secretion, enhances activity and protein levels of SIRT1 and inhibited Cd-induced activation of Akt signaling pathway. CONCLUSION: Res ameliorated Cd-induced placental toxicity and regulated DNMT3 expression and PI3K/Akt pathway activation.

The predictive value of microRNA in early hypertensive disorder complicating pregnancy (HDCP).

OBJECTIVE: To examine the predictive value of microRNA (miRNA) in hypertensive disorder complicating pregnancy (HDCP). METHODS: 102 pregnant women with HDCP admitted to our hospital from March 2017 to June 2019 were recruited as the study cohort and randomly divided into an HDCP group, a mild preeclampsia group, and a severe preeclampsia group, with 34 patients in each group. In addition, 34 healthy pregnant women who underwent pregnancy tests in our hospital were recruited as the normal group. The relative expressions of plasma miR-19a, miR-126, and miRNA-210 in were measured. A Pearson correlation analysis was used to analyze the correlations between the miR-19a, miR-181b, and miRNA-210 expressions and the severity of HDCP. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic efficacy of the miR-19a, miR-126, and miRNA-210 expressions. RESULTS: The miR-19a and miRNA-210 expressions were higher in the HDCP group, the mild preeclampsia group, and the severe preeclampsia group than they were in the normal group, and the miR-126 expression was lower (all P<0.05). The miR-19a, miR-126, and miRNA-210 expressions were different among the four groups (P<0.05). The miR-19a and miRNA-210 expression levels in the severe preeclampsia group were higher than they were in the HDCP group, and the miR-126 expression was lower (P<0.05). A Pearson correlation analysis showed the miR-19a and miR-210 levels in the HDCP patients were positively correlated with the severity of the disease (P<0.05), and the miR-126 level is negatively correlated with disease severity (P<0.05). Our ROC curve analysis demonstrated that the miR-19a, miR-126, and miR-210 levels have a predictive value for HDCP. The areas under the curve were 0.800, 0.633, and 0.723, the sensitivities were 81.2%, 71.4%, and 80.2%, and the specificities were 73.5%, 67.5%, 81.5%. Additionally, the area under the curve of the combination of the three was 0.896, and the sensitivity and specificity were 90.5% and 93.9% respectively. CONCLUSION: miR-19a, miR-126, and miR-210 are strongly connected to the severity of HDCP and can be used as a sensitive indicator to predict HDCP patients clinically.

Water reuse and growth inhibition mechanisms for cultivation of microalga Euglena gracilis.

BACKGROUND: Microalgae can contribute to more than 40% of global primary biomass production and are suitable candidates for various biotechnology applications such as food, feed products, drugs, fuels, and wastewater treatment. However, the primary limitation for large-scale algae production is the fact that algae requires large amounts of fresh water for cultivation. To address this issue, scientists around the world are working on ways to reuse the water to grow microalgae so that it can be grown in successive cycles without the need for fresh water. RESULTS: In this study, we present the results when we cultivate microalgae with cultivation water that is purified and reused. Specifically, we purify the cultivation water using an ultrafiltration membrane (UFM) treatment and investigate how this treatment affects: the biomass and biochemical components of the microalgae; characteristics of microalgae growth inhibitors; the mechanism whereby potential growth inhibitors are secreted (followed using metabolomics analysis); the effect of activated carbon (AC) treatment and advanced oxidation processes (AOPs) on the removal of growth inhibitors of Euglena gracilis. Firstly, the results show that E. gracilis can be only cultivated through two growth cycles with water that has been filtered and reused, and the growth of E. gracilis is significantly inhibited when the water is used a third time. Secondly, as the number of reused water cycles increases, the Cl- concentration gradually increases in the cultivation water. When the Cl- concentration accumulates to a level of fivefold higher than that of the control, growth of E. gracilis is inhibited as the osmolality tolerance range is exceeded. Interestingly, the osmolality of the reused water can be reduced by replacing NH4Cl with urea as the source of nitrogen in the cultivation water. Thirdly, E. gracilis secretes humic acid (HA)-which is produced by the metabolic pathways for valine, leucine, and isoleucine biosynthesis and by linoleic acid metabolism-into the cultivation water. Because HA contains large fluorescent functional groups, specifically extended π(pi)-systems containing C=C and C=O groups and aromatic rings, we were able to observe a positive correlation between HA concentration and the rate of inhibition of E. gracilis growth using fluorescence spectroscopy. Moreover, photosynthetic efficiency is adversely interfered by HA, thereby reductions in the synthetic efficiency of paramylon and lipid in E. gracilis. In this way, we are able to confirm that HA is the main growth inhibitor of E. gracilis. Finally, we verify that all the HA is removed or converted into nutrients efficiently by AC or UV/H2O2/O3 treatments, respectively. As a result of these treatments, growth of E. gracilis is restored (AC treatment) and the amount of biomass is promoted (UV/H2O2/O3 treatment). CONCLUSIONS: These studies have important practical and theoretical significance for the cyclic cultivation of E. gracilis and for saving water resources. Our work may also provide a useful reference for other microalgae cultivation.

Rapid Screening and Identification of Antitumor Ingredients from the Mangrove Endophytic Fungus Using an Enzyme-Immobilized Magnetic Nanoparticulate System.

As a consequence of recent progression in biomedicine and nanotechnology, nanoparticle-based systems have evolved as a new method with extensive applications in responsive therapy, multimodal imaging, drug delivery and natural product separation. Meanwhile, the magnetic nanoparticulate system has aroused great interest for separation and purification because of its excellent magnetic properties. Phospholipase A2 (PLA2) is a highly expressed regulator to promote the growth of various cancers and is an ideal target to treat cancers. In this study, a novel strategy based on ligand-receptor interactions to discover novel PLA2 inhibitors was established, in which PLA2-functionalized Fe3O4@PLGA-PEG-NH2 magnetic nanoparticles were used as a supporting material combined with high-performance liquid chromatography-mass spectrometry, aiming to accelerate the discovery of novel PLA2 inhibitors from natural sources such as mangrove endophytic fungi. Under the optimized ligand fishing conditions, six target compounds were ultimately fished and identified to be cyclic peptides (1-3) and sterols (4-6), which compounds 1, 2 and 4-6 have well-documented cytotoxicities. Compound 3 exerted better inhibitory effect on A549 cells by experiment. In conclusion, PLA2-functionalized Fe3O4@PLGA-PEG-NH2 magnetic nanoparticles-based ligand fishing provided a feasible, selective and effective platform for the efficient screening and identification of antitumor components from natural products.

The diagnostic value and regulatory mechanism of miR-200a targeting ZEB1 in pregnancy-induced hypertension.

OBJECTIVE: To investigate the diagnostic value and regulatory mechanism of miR-200a targeting ZEB1 in pregnancy-induced hypertension (PIH). METHODS: The expression of miR-200a and ZEB1 was detected in the placenta of PIH patients, and then the human trophoblastic cell line JEG-3 was transfected and divided into different groups: control group, NC group, ZEB1 siRNA group, miR-200a inhibitor group and miR-200a inhibitor group + ZEB1 siRNA group. After transfection, cell proliferation and migration/invasion were evaluated byMTT and Transwell assays, respectively, whereas apoptosis was assessed byflow cytometry. MiR-200a was measured by qRT-PCR, while ZEB1 was detectedby Western blotting. RESULTS: The expression of miR-200a was gradually increased in the placenta of patients with hypertension and mild or severe preeclampsia, while the mRNA and protein levels of ZEB1 were downregulated. A dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-200a andZEB1.Compared to the control, the miR-200a inhibitor caused a strongdecrease in miR-200a andan upregulation of ZEB1, with a significant enhancement ofcell proliferation, migration and invasion and a decrease in apoptosis. However, no significant alteration was observedin the miR-200a level after administration of ZEB1 siRNA, while ZEB1 was downregulated, with significant suppression of growth. CONCLUSION: MiR-200a was upregulated in PIH patients, andinhibition of miR-200a may improve disease progression, as it could facilitatetrophoblastproliferation, migration and invasionandinhibitapoptosisby targeting ZEB1.

Hyperbaric oxygen on rehabilitation of brain tumors after surgery and effects on TNF-α and IL-6 levels.

Hyperbaric oxygenation (HBO) on postoperative rehabilitation of brain tumors and effects on tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were explored. A retrospective analysis of 132 patients with brain tumors treated in the People's Hospital of Rizhao from October 2014 to October 2017 was performed. There were 62 patients in the observation group and 70 patients in the control group. Patients in the control group were treated with conventional drugs, and patients in the observation group were treated with HBO on the basis of conventional drug therapy. Levels of serum TNF-α and IL-6 were measured by ELISA before and after treatment. Cerebral arterial flow velocity and spasticity were measured by cranial color Doppler ultrasonography. Neurological function deficit (NFD) and activities of daily living (ADL) were used to evaluate the clinical recovery of the patients. Clinical efficacy was compared and analyzed. There were no significant differences between the two groups before treatment (P>0.05). After treatment, serum TNF-α and IL-6 levels were significantly lower than pretreatment levels (P<0.05), and serum TNF-α and IL-6 levels in the observation group were lower than those in the control group (P<0.05). Cerebral arterial flow velocity in observation group after treatment was significantly lower than that in the control group. The number of patients with cerebral arterial spasm after treatment in the observation group was significantly smaller than that in the control group. NFD scores in the observation group were lower than those in the control group after treatment. After treatment, ADL scores in the observation group were significantly higher than those in the control group (P<0.05). The comprehensive treatment effect of HBO is significant. It can inhibit the expression of inflammatory factors in serum and reduce cerebral arterial flow velocity and effectively reduce the number of patients with cerebral arterial spasm. It can reduce NFD and improve the quality of life of patients. Therefore, it is worthy of clinical popularization.

Structure Characterization and Otoprotective Effects of a New Endophytic Exopolysaccharide from Saffron.

Saffron, a kind of rare medicinal herb with antioxidant, antitumor, and anti-inflammatory activities, is the dry stigma of Crocus sativus L. A new water-soluble endophytic exopolysaccharide (EPS-2) was isolated from saffron by anion exchange chromatography and gel filtration. The chemical structure was characterized by FT-IR, GC-MS, and 1D and 2D-NMR spectra, indicating that EPS-2 has a main backbone of (1→2)-linked α-d-Manp, (1→2, 4)-linked α-d-Manp, (1→4)-linked α-d-Xylp, (1→2, 3, 5)-linked ß-d-Araf, (1→6)- linked α-d-Glcp with α-d-Glcp-(1→ and α-d-Galp-(1→ as sidegroups. Furthermore, EPS-2 significantly attenuated gentamicin-induced cell damage in cultured HEI-OC1 cells and increased cell survival in zebrafish model. The results suggested that EPS-2 could protect cochlear hair cells from ototoxicity exposure. This study could provide new insights for studies on the pharmacological mechanisms of endophytic exopolysaccharides from saffron as otoprotective agents.

Roles of miR-210 in the pathogenesis of pre-eclampsia.

INTRODUCTION: This study aimed to explore the bio-function of miR-210 in the pathogenesis of pre-eclampsia and provide new insights into the diagnosis and treatment of pre-eclampsia. MATERIAL AND METHODS: A JAR cell line cultured in standard or hypoxic conditions was used in this study. Expression levels of miR-210 and PTPN2 were determined using real-time polymerase chain reaction (RT-PCR). Protein and phosphorylation levels were assessed using western blotting. Proliferation of JAR cells was evaluated using MTT assay. Migration and invasion were measured using transwell assay. RESULTS: Expression of miR-210 increased significantly in a time-dependent manner after hypoxia treatment within 36 h (p < 0.05). miR-210 inhibitor significantly decreased the cell proliferation, migration, and invasion (p < 0.05), while miR-210 mimic reversed these findings (p < 0.05). Hypoxia significantly suppressed the expression of PTPN2; however, this elevation was abolished by miR-210 inhibitor (p < 0.05). Inhibition of PTPN2 or hypoxia significantly increased the proliferation, migration, and invasion of JAR cells, while miR-210 inhibitor significantly reversed these changes (p < 0.05). The phosphorylation levels of PDGFR, Akt, and Erk were markedly upregulated by hypoxia or si-PTPN2, but this effect was abolished by miR-210 inhibitor (p < 0.05). CONCLUSIONS: miR-210 can promote proliferation, migration, and invasion via downregulating PTPN2 in the PDGFR-Akt pathway.

Current Trends for ST-segment Elevation Myocardial Infarction during the Past 5 Years in Rural Areas of China's Liaoning Province: A Multicenter Study.

BACKGROUND: Since 2010, two versions of National Guidelines aimed at promoting the management of ST-segment elevation myocardial infarction (STEMI) have been formulated by the Chinese Society of Cardiology. However, little is known about the changes in clinical characteristics, management, and in-hospital outcomes in rural areas. METHODS: In the present multicenter, cross-sectional study, participants were enrolled from rural hospitals located in Liaoning province in Northeast China, during two different periods (from June 2009 to June 2010 and from January 2015 to December 2015). Data collection was conducted using a standardized questionnaire. In total, 607 and 637 STEMI patients were recruited in the 2010 and 2015 cohorts, respectively. RESULTS: STEMI patients in rural hospitals were older in the second group (63 years vs. 65 years, P = 0.039). We found increases in the prevalence of hypertension, prior percutaneous coronary intervention (PCI), and prior stroke. Over the past 5 years, the cost during hospitalization almost doubled. The proportion of STEMI patients who underwent emergency reperfusion had significantly increased from 42.34% to 54.47% (P < 0.0001). Concurrently, the proportion of primary PCI increased from 3.62% to 10.52% (P < 0.0001). The past 5 years have also seen marked increases in the use of guideline-recommended drugs and clinical examinations. However, in-hospital mortality and major adverse cardiac events did not significantly change over time (13.01% vs. 10.20%, P = 0.121; 13.34% vs. 13.66%, P = 0.872). CONCLUSIONS: Despite the great progress that has been made in guideline-recommended therapies, in-hospital outcomes among rural STEMI patients have not significantly improved. Therefore, there is still substantial room for improvement in the quality of care.

Involvement of P38MAPK activation by NMDA receptors and non-NMDA receptors in amyloid-ß peptide-induced neuronal loss in rat hippocampal CA1 and CA3 subfields.

Oligomeric amyloid-ß peptide (Aß) has been found to be associated with the pathogenesis of Alzheimer's disease (AD). Numerous studies have reported Aß neurotoxicity, but the underlying molecular mechanisms remain to be fully illuminated. In the present study, we investigated the Aß-induced activation and regulation of P38MAPKs in rat hippocampus in vivo. The results showed that intracerebroventricular injection of oligomeric Aß25-35 increased the activation (phosphorylation) of P38MAPKs, and the level of cleaved caspase-3, but decreased the number of neurons in rat hippocampal CA1 and CA3 subfields. Downregulation of P38MAPK activity by SB239063 protected against the Aß neurotoxicity. Pretreatment with NMDA and non-NMDA receptor antagonists respectively suppressed P38MAPK activation induced by Aß25-35 oligomers and presented neuroprotective effect. Taken together, these data suggest that P38MAPK activation via NMDA and non-NMDA receptors is a key signal cascade in Aß-induced neuronal death. Inhibition of P38MAPK cascades may be a promising treatment in AD.

Application of accelerated solvent extraction coupled with high-performance counter-current chromatography to extraction and online isolation of chemical constituents from Hypericum perforatum L.

Accelerated solvent extraction (ASE) coupled with high-performance counter-current chromatography (HPCCC) was successfully used for the extraction and online isolation of five chemical constituents from the plant Hypericum perforatum L. The upper phase of the solvent system of ethyl acetate-methanol-water (5:2:5, v:v:v) was used as both the ASE solvent and the HPCCC stationary phase. Two hydrophobic compounds including 28.4 mg of hyperforin with a HPLC purity of 97.28% and 32.7 mg of adhyperforin with a HPLC purity of 97.81% were isolated. The lower phase of ethyl acetate-methanol-n-butanol-water (5:2:2.5:12, v:v:v:v) was used as both the ASE solvent and CCC stationary phase. Three hydrophilic compounds of 12.7 mg of 3,4,5-O-tricaffeoylquinic acid with a HPLC purity of 98.82%, 15.2 mg of 1,3,5-O-tricaffeoylquinic acid with a HPLC purity of 99.46% and 42.5mg of 3-O-caffeoylquinic acid with a HPLC purity of 96.90%, were obtained in a one-step extraction-separation process with less than 3h from 10.02 g of raw material of H. perforatum. The targeted compounds isolated, collected and purified by HPCCC were analyzed by high performance liquid chromatography (HPLC), the chemical structures of all five compounds above mentioned were identified by UV, MS and NMR.

Comprehensive separation and identification of chemical constituents from Apocynum venetum leaves by high-performance counter-current chromatography and high performance liquid chromatography coupled with mass spectrometry.

High-performance counter-current chromatography (HPCCC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) was efficiently utilized for the separation and identification of the chemical components with a wide range of polarity from the mixed extract of Chinese medicinal herb Apocynum venetum. For HPCCC separation, four sets of solvent systems, n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:4.5, v:v:v:v), ethyl acetate-methanol-water (5:2:5, v:v:v) and n-butanol-methanol-water (5:1:5, v:v:v) were used for the one-step separation by four stages. The HPCCC separation was initiated by filling the column with the lower phase of n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:5, v:v:v:v) as a stationary phase followed by elution with the upper phase of n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:5, v:v:v:v) to separate the hydrophobic compounds (tail to head). Then the mobile phase was switched to the upper phase of ethyl acetate-acetonitrile-water (5:3:7, v:v:v) to eluted the moderate hydrophobic compounds, then the mobile phase was switched to the upper phase of ethyl acetate-methanol-water (5:2:5, v:v:v) to eluted the moderate hydrophilic compounds, and finally the hydrophilic compounds still retained in the column was eluted by the upper phase of n-butanol-methanol-water (5:1:5, v:v:v). A total of 16 named compounds including adhyperforin, hyperforin, amentoflavone, biapigenin, quercetin, avicularin, acetylated isoquercetin, acetylated hyperoside, astragalin, trifolin, isoquercetin, hyperoside, querciturone, rutin, chlorogenic acid and quercetin-3-O-ß-D-glucosyl-ß-D-glucopyranoside were successfully separated via the four sets of solvent systems in one step operation for 130 min. The compounds separated by HPCCC were identified by comparing with mixed standards data of HPLC-MS as well as NMR data.

Solvent gradient elution for comprehensive separation of constituents with wide range of polarity in Apocynum venetum leaves by high-speed counter-current chromatography.

A novel gradient elution was efficiently utilized for the separation of the chemical components with a wide range of polarity from the mixed extract of the Chinese medicinal herb Apocynum venetum or mixed standards by high-speed counter-current chromatography. Three sets of solvent systems, n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v), ethyl acetate-methanol-water (5:2:5 v/v/v) and n-butanol-methanol-water (5:1:5 v/v/v) were used for the one-step elution. The separation was initiated by filling the column with the lower phase of n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v) as a stationary phase followed by elution with the upper phase of n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v) to separate the hydrophobic compounds (tail to head). Then the mobile phase was switched to the upper phase of ethyl acetate-methanol-water (5:2:5 v/v/v) to elute the moderate hydrophobic compounds, and finally the hydrophilic compounds still retained in the column were fractionated by eluting the column with the upper phase of n-butanol-methanol-water (5:1:5 v/v/v). A total of 13 compounds including adhyperforin, hyperforin, amentoflavone, biapigenin, quercetin, astragalin, trifolin, isoquercetin, hyperside, acetyled hyperside, rutin, chlorogenic acid and quercetin-3-O-ß-D-glucosyl-ß-D-glucopyranoside were successfully separated via the three sets of solvent systems in one-step operation for 90 min.

Oligomerized Abeta25-35 induces increased tyrosine phosphorylation of NMDA receptor subunit 2A in rat hippocampal CA1 subfield.

Amyloid-beta peptide (Abeta) plays a causal role in the pathogenesis of Alzheimer's disease (AD). To elucidate the mechanisms underlying the over-activation of NMDA receptors in AD, we investigated the alteration of NR2A tyrosine phosphorylation after intracerebroventricular infusion of Abeta25-35 oligomers. Abeta25-35 treatment resulted in the elevated tyrosine phosphorylation of NR2A in rat hippocampal CA1 subfield and facilitated the interactions of NR2A or PSD-95 with Src kinases. PP2, a specific inhibitor of Src family protein tyrosine kinases (SrcPTKs), not only attenuated the Abeta25-35-induced increases in the tyrosine phosphorylation of NR2A and in the associations among Src, NR2A, and PSD-95, but also protected against neuronal loss in the CA1 region. Preapplication of a noncompetitive NMDA receptor antagonist amantadine, an NR2A-selective NMDA receptor antagonist NVP-AAM077, or an NR2B-selective NMDA receptor antagonist Ro25-6981 inhibited the increased tyrosine phosphorylation of NR2A and prevented the associations among Src, NR2A, and PSD-95, but Ro25-6981 had less contribution. These results suggest that the activation of NMDA receptors after Abeta treatment promotes the formation of NR2A-PSD-95-Src complex and thus increases the tyrosine phosphorylation of NR2A by Src kinases, which up-regulates the function of NMDA receptors. Such positive feedback mediates the Abeta-induced over-activation of NMDA receptors and is involved in neuronal impairment.

Phosphorylation and hypoxia-induced heme oxygenase-1 gene expression in cardiomyocytes.

BACKGROUND: Heme oxygenase-1 (HO-1) is a stress protein and the rate-limiting enzyme in heme degradation. We sought to examine the notion that protein kinases and phosphatases through phosphorylation and dephosphorylation modulate the HO-1 expression in cardiomyocytes under hypoxic conditions. METHODS AND RESULTS: Exposure of neonatal rat cardiomyocytes to hypoxia markedly induced the HO-1 expression, as assessed by Northern blot, Western blot, and transfection assay. The hypoxia-induced HO-1 expression was blocked by the kinase inhibitors staurosporine and SB202190 in a dose-dependent manner. Hypoxia decreased the activity of phosphatase-1 (PP-1). To examine the effect of PP-1 inhibition on HO-1 expression we used the phosphatase inhibitor okadaic acid (OA) and an antisense vector. OA treatment or overexpression of the antisense PP-1 transcript markedly induced HO-1 expression. Furthermore, transfection assay using HO-1 promoter constructs revealed the involvement of the nuclear factor kB (NF-kB) and Activator protein-1 (AP-1) in the hypoxia-induced activation of the HO-1 gene. The HO-1 promoter activity was modulated by OA under normoxic conditions or staurosporine under hypoxia. CONCLUSIONS: Our results suggest that activation of protein kinases and downregulation of PP-1 activity contribute to the hypoxia-induced HO-1 gene expression and that the proximal HO-1 promoter region containing NF-kB and AP-1 binding sites is likely to play a role in the transcriptional activation of the HO-1 gene in cardiomyocytes in response to hypoxic stress.

Mitochondrial Ca2+ flux and respiratory enzyme activity decline are early events in cardiomyocyte response to H2O2.

Oxidative stress is involved in mitochondrial apoptosis, and plays a critical role in ischemic heart disease and cardiac failure. Exposure of cardiomyocytes to H(2)O(2) leads to oxidative stress and mitochondrial dysfunction. In this study, we investigated the temporal order of mitochondrial-related events in the neonatal rat cardiomyocyte response to H(2)O(2) treatment. At times ranging from 10 to 90 min after H(2)O(2) treatment, levels were determined for respiratory complexes I, II, IV and V, and citrate synthase activities, mitochondrial Ca(2+) flux, intracellular oxidation, mitochondrial membrane potential and apoptotic progression. Complexes II and IV activity levels were significantly reduced within 20 min of H(2)O(2) exposure while complexes I and V, and citrate synthase were unaffected. Mitochondrial membrane potential declined after 20 and 60 min of H(2)O(2) exposure while intracellular oxidation, declining complex I activity and apoptotic progression were detectable only after 60 min. Measurement of mitochondrial Ca(2+) ([Ca(2+)](m)) using rhodamine 2 detected an early accumulation of [Ca(2+)](m) occurring between 5 and 10 min. Pretreatment of cardiomyocytes with either ruthenium red or cyclosporin A abrogated the H(2)O(2)-induced decline in complexes II and IV activities, indicating that [Ca(2+)](m) flux and onset of mitochondrial permeability transition pore opening likely precede the observed early enzymatic decline. Our findings suggest that [Ca(2+)](m) flux represents an early pivotal event in H(2)O(2)-induced cardiomyocyte damage, preceding and presumably leading to reduced mitochondrial respiratory activity levels followed by accumulation of intracellular oxidation, mitochondrial membrane depolarization and apoptotic progression concomitant with declining complex I activity.