The alpha2-adrenergic receptor agonist clonidine protects against cerebral ischemia/reperfusion induced neuronal apoptosis in rats.

Apoptosis is the crucial pathological mechanism following cerebral ischemic injury. Our previous studies demonstrated that clonidine, one agonist of alpha2-adrenergic receptor (α2-AR), could attenuate cerebral ischemic injury in a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). However, it's unclear whether clonidine exerts neuroprotective effects by regulating neuronal apoptosis. In this study, we elucidated whether clonidine can exert anti-apoptotic effects in cerebral ischemic injury, and further explored the possible mechanisms. Neurological deficit score was measured to evaluate the neurological function. TTC staining was used for the measurement of brain infarct size. Hematoxylin-Eosin (HE) staining was applied to examine the cell morphology. TUNEL and DAPI fluorescent staining methods were used to analyze the cell apoptosis in brain tissue. Fluorescence quantitative real-time PCR was performed to assess the gene expression of Caspase-3 and P53. Western blotting assay was applied to detect the protein expression of Caspase-3 and P53. The results showed that clonidine improved neurological function, reduced brain infarct size, alleviated neuronal damage, and reduced the ratio of cell apoptosis in the brain with MCAO/R injury. moreover, clonidine down-regulated the gene and protein expression of Caspase-3 and P53 which were over-expressed after MCAO/R injury. Whereas, yohimbine (one selective α2-AR antagonist) mitigated the anti-apoptosis effects of clonidine, accompanied by reversed gene and protein expression changes. The results indicated that clonidine attenuated cerebral MCAO/R injury via suppressing neuronal apoptosis, which may be mediated, at least in part, by activating α2-AR.

Inhibition of hyperpolarization-activated cyclic nucleotide-gated cation channel attenuates cerebral ischemia reperfusion-induced impairment of learning and memory by regulating apoptotic pathway.

Stroke is the second leading cause of death globally. Cognitive dysfunction is a common complication of stroke, which seriously affects the patient's quality of life. Previous studies have shown that the expression of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel is closely related to ischemia-reperfusion (IR) injury and subsequent cognitive impairment. We also found that ZD7288, a specific inhibitor of the HCN channel, attenuated IR injury during short-term reperfusion. Since apoptosis can induce cell necrosis and aggravate cognitive impairment after IR, the purpose of this study is to define whether ZD7288 could improve cognitive impairment after prolonged cerebral reperfusion in rats by regulating apoptotic pathways. Our data indicated that ZD7288 can ameliorate spatial cognitive behavior and synaptic plasticity, protect the morphology of hippocampal neurons, and alleviate hippocampal apoptotic cells in IR rats. This effect may be related to down-regulating the expressions of pro-apoptotic proteins such as AIF, p53, Bax, and Caspase-3, and increasing the ratio of Bcl-2/Bax. Taken together, it suggested that inhibition of the HCN channel improves cognitive impairment after IR correlated with its regulation of apoptotic pathways.

Increased intracellular Cl- concentration mediates neutrophil extracellular traps formation in atherosclerotic cardiovascular diseases.

Neutrophil extracellular traps (NETs) play crucial roles in atherosclerotic cardiovascular diseases such as acute coronary syndrome (ACS). Our preliminary study shows that oxidized low-density lipoprotein (oxLDL)-induced NET formation is accompanied by an elevated intracellular Cl- concentration ([Cl-]i) and reduced cystic fibrosis transmembrane conductance regulator (CFTR) expression in freshly isolated human blood neutrophils. Herein we investigated whether and how [Cl-]i regulated NET formation in vitro and in vivo. We showed that neutrophil [Cl-]i and NET levels were increased in global CFTR null (Cftr-/-) mice in the resting state, which was mimicked by intravenous injection of the CFTR inhibitor, CFTRinh-172, in wild-type mice. OxLDL-induced NET formation was aggravated by defective CFTR function. Clamping [Cl-]i at high levels directly triggered NET formation. Furthermore, we demonstrated that increased [Cl-]i by CFTRinh-172 or CFTR knockout increased the phosphorylation of serum- and glucocorticoid-inducible protein kinase 1 (SGK1) and generation of intracellular reactive oxygen species in neutrophils, and promoted oxLDL-induced NET formation and pro-inflammatory cytokine production. Consistently, peripheral blood samples obtained from atherosclerotic ApoE-/- mice or stable angina (SA) and ST-elevation ACS (STE-ACS) patients exhibited increased neutrophil [Cl-]i and SGK1 activity, decreased CFTR expression, and elevated NET levels. VX-661, a CFTR corrector, reduced the NET formation in the peripheral blood sample obtained from oxLDL-injected mice, ApoE-/- atherosclerotic mice or patients with STE-ACS by lowering neutrophil [Cl-]i. These results demonstrate that elevated neutrophil [Cl-]i during the development of atherosclerosis and ACS contributes to increased NET formation via Cl--sensitive SGK1 signaling, suggesting that defective CFTR function might be a novel therapeutic target for atherosclerotic cardiovascular diseases.

Platelet CFTR inhibition enhances arterial thrombosis via increasing intracellular Cl- concentration and activation of SGK1 signaling pathway.

Platelet hyperactivity is essential for thrombus formation in coronary artery diseases (CAD). Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients with cystic fibrosis elevates intracellular Cl- levels ([Cl-]i) and enhanced platelet hyperactivity. In this study, we explored whether alteration of [Cl-]i has a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl-]i was increased in platelets from CAD patients. In a FeCl3-induced mouse mesenteric arteriole thrombosis model, platelet-specific Cftr-knockout and/or pre-administration of ion channel inhibitor CFTRinh-172 increased platelet [Cl-]i, which accelerated thrombus formation, enhanced platelet aggregation and ATP release, and increased P2Y12 and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl-]i, thereby decreasing thrombosis formation. Our results showed that clamping [Cl-]i at high levels or Cftr deficiency-induced [Cl-]i increasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y12 and PAR4 expression via NF-κB signaling. Constitutively active mutant S422D SGK1 markedly increased P2Y12 and PAR4 expression. The specific SGK1 inhibitor GSK-650394 decreased platelet aggregation in wildtype and platelet-specific Cftr knockout mice, and platelet SGK1 phosphorylation was observed in line with increased [Cl-]i and decreased CFTR expression in CAD patients. Co-transfection of S422D SGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl-]i is a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl-]i-sensitive SGK1 signaling pathway. Therefore, [Cl-]i in platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.

Clonidine ameliorates cerebral ischemia-reperfusion injury by up-regulating the GluN3 subunits of NMDA receptor.

This study aimed to investigate the protective effects of the alpha-2 adrenergic receptor (α2-AR) agonist, clonidine, on the cerebral ischemia-reperfusion (I/R) injury and elaborate the underlying mechanisms. Cerebral I/R model was established by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 4 h in adult male SD rats. Saline, clonidine and yohimbine (an α2-AR antagonist) were intraperitoneally administered each day for one week before surgery. Neurological deficit was evaluated just before decapitation. TTC staining was applied for correlation of cerebral infarction volume. HE staining was performed to observe the neuron morphology. Immunohistochemical staining was performed to detect the localization and expression of GluN3 proteins. Western blot analysis also was used to detect the expression levels of GluN3 proteins. Our data showed that clonidine ameliorated neurological deficit and reduced the cerebral infarction volume of the rats with cerebral I/R. It is worth noting that treatment with clonidine up-regulated the protein expression of GluN3 in the rats with the cerebral I/R, especially in the cell membrane. Moreover, clonidine also up-regulated the transposition from cytoplasm to cell membrane of GluN3 after cerebral I/R. In addition, yohimbine abolished the neuroprotective effects of clonidine. The results indicated that clonidine played a protective role in cerebral I/R injury through regulation of the protein expression of GluN3 subunits of N-methyl-D-aspartate (NMDA) receptor.

Antiplatelet and Antithrombotic Effects of Isaridin E Isolated from the Marine-Derived Fungus via Downregulating the PI3K/Akt Signaling Pathway.

Isaridin E, a cyclodepsipeptide isolated from the marine-derived fungus Amphichorda felina (syn. Beauveria felina) SYSU-MS7908, has been demonstrated to possess anti-inflammatory and insecticidal activities. Here, we first found that isaridin E concentration-dependently inhibited ADP-induced platelet aggregation, activation, and secretion in vitro, but did not affect collagen- or thrombin-induced platelet aggregation. Furthermore, isaridin E dose-dependently reduced thrombosis formation in an FeCl3-induced mouse carotid model without increasing the bleeding time. Mechanistically, isaridin E significantly decreased the ADP-mediated phosphorylation of PI3K and Akt. In conclusion, these results suggest that isaridin E exerts potent antithrombotic effects in vivo without increasing the risk of bleeding, which may be due to its important role in inhibiting ADP-induced platelet activation, secretion and aggregation via the PI3K/Akt pathways.

Piracetam ameliorated oxygen and glucose deprivation-induced injury in rat cortical neurons via inhibition of oxidative stress, excitatory amino acids release and P53/Bax.

Our previous work has demonstrated that piracetam inhibited the decrease in amino acid content induced by chronic hypoperfusion, ameliorated the dysfunction of learning and memory in a hypoperfusion rat model, down-regulated P53, and BAX protein, facilitated the synaptic plasticity, and may be helpful in the treatment of vascular dementia. To explore the precise mechanism, the present study further evaluated effects of piracetam on Oxygen and glucose deprivation (OGD)-induced neuronal damage in rat primary cortical cells. The addition of piracetam to the cultured cells 12 h before OGD for 4 h significantly reduced neuronal damage as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release experiments. Piracetam also lowered the levels of malondialdehyde, nitrogen monoxidum, and xanthine oxidase which was increased in the OGD cells, and enhanced the activities of superoxide dismutase and glutathione peroxidase, which were decreased in the OGD cells. We also demonstrated that piracetam could decrease glutamate and aspartate release when cortical cells were subjected to OGD. Furthermore, Western blot study demonstrated that piracetam attenuated the increased expression of P53 and BAX protein in OGD cells. These observations demonstrated that piracetam reduced OGD-induced neuronal damage by inhibiting the oxidative stress and decreasing excitatory amino acids release and lowering P53/Bax protein expression in OGD cells.

Imposex effects on the veined rapa whelk (Rapana venosa) in Bohai Bay, China.

Environmentally relevant concentrations of organotin compounds (OTs) may trigger sex changes in marine invertebrates and pose a threat to the marine ecosystem. In this study, we investigated organotin levels and the biological responses of wild veined rapa whelk (Rapana venosa) from Lüjuhe district (LJH), Dashentang district (DST), and Nanpaihe district (NPH) in Bohai Bay, China. We found that 11.11 and 22.95 % of the veined rapa whelks from DST and NPH exhibited imposex characteristics with a relative penis size index (RPSI) of 12.50 and 12.31, respectively. The RNA/DNA ratio was significantly lower in females from DST than those from LJH (p < 0.05), and a slight increase in DNA damage was observed in females and imposex individuals compared to males. Moreover, less genetic distance occurred between LJH and NPH (0.016) than between LJH and DST (0.028), although they belonged to the same regional population. OTs analysis showed that triphenyltin chloride concentrations (41.45 ng/g dried weight) were significantly higher than tributyltin concentrations (9.51 ng/g dried weight) in tissues (p < 0.05), but no significant differences were observed in sediments (p > 0.05). In conclusion, the occurrence of imposex individuals and biological responses of the wild veined rapa whelk from Bohai Bay suggest that the marine ecosystem might be at risk.

Construction and characterization of a normalized cDNA library from the river snail Bellamya aeruginosa after exposure to copper.

The construction of a normalized cDNA library is a popular tool for identifying novel biomarkers for monitoring environmental pollution. In the present study, a normalized cDNA library was constructed from the river snail Bellamya aeruginosa after exposure to Cu(2+) by using the SMART technique. The titer of the cDNA library was 1.78 × 10(6) pfu/ml, with a recombinant efficiency of 95.8%. In addition, from 6,000 randomly selected and sequenced clones, 5,473 high-quality ESTs were identified. After processing the sequences, 3,961 unigenes representing 897 contigs and 3,064 singlets were obtained with 27.6% redundancy. Analysis of expressed sequenced tags using COG and GO annotation and KEGG pathway data showed that a large group of genes related to growth and development, signal transduction, and defense mechanisms were present in the cDNA library. Based on our findings, this normalized cDNA library will provide a valuable resource for further research on functional genes and ecotoxicology in B. aeruginosa.

Syntheses and cell-based phenotypic screen of novel 7-amino pyrido[2,3-d]pyrimidine-6-carbonitrile derivatives as potential antiproliferative agents.

A series of N-3-substituted 7-aminopyrido[2,3-d]pyrimidin-6-carbonitrile derivatives was readily synthesized and their anti-proliferative activities on five types of tumor cells were evaluated through a cell-based phenotypic screening approach. Compound 3k was found to be potent on human colon cancer SW620 cells with an IC(50) value of 12.5 mM. Structural optimization of compound 3k led to compound 4a with improved anti-proliferative potency on SW620 cells with an IC(50) value of 6.9 mM. Further cell-cycle analyses suggested that compound 4a induced apoptosis of SW620 cells in a concentration-dependent manner.

Different oxidants and PKC isozymes mediate the opposite effect of inhibition of Q(i) and Q(o) site of mitochondrial complex III on calcium currents in rat cortical neurons.

The inhibition of the complex III of the mitochondrial respiratory chain under hypoxia-ischemia has been observed. However, the downstream events of this inhibition remain to be studied. In this paper, we used the Q(i) site inhibitor antimycin A and the Q(o) site inhibitor myxothiazol to inhibit the Q(i) site and the Q(o) site of the complex III and studied the effect and mechanism of the inhibition of these sites on voltage-gated Ca(2+) currents (I(Ca)) in rat prefrontal neurons with whole cell patch-clamp method in slices. The results showed that antimycin A inhibited I(Ca), but myxothiazol increased it. Further mechanism study showed that antimycin A inhibited I(Ca) via the H(2)O(2)-hydroxyl radicals/cPKC (mainly PKCbetaI) pathway, whereas myxothiazol increased I(Ca) via the superoxide anion/nPKC (mainly the PKCdelta) pathway.

Synthesis, antiproliferative activities and in vitro biological evaluation of novel benzofuransulfonamide derivatives.

In a cell-based screen of novel antiproliferative agents, the hit compound 1a, which bears a benzofuransulfonamide scaffold, exhibited broad-spectrum antiproliferative activities against a panel of tumor cell lines. The promising in vitro antiproliferative activity and structural novelty of 1a prompted us to investigate the synthesis of five analogs of 1a and test their antiproliferative activities. The most potent analogue, 1h, exhibited enhanced antiproliferative activities compared with the parent 1a, and exhibited an IC(50) value against NCI-H460 cells of 4.13 µM compared with 4.52 µM for the positive control cisplatin. Flow cytometric analysis revealed that 1h induces significant levels of apoptosis in NCI-H460 cells in vitro at low micromolar concentrations. These results suggest that 1a and analogs based on its benzofuransulfonamide scaffold may constitute a novel class of antiproliferative agents, which deserve further study.

2,2-Dimethyl-5-{[(4-nitro-phen-yl)amino]-methyl-idene}-1,3-dioxane-4,6-dione.

In the title compound, C(13)H(12)N(2)O(6), the dihedral angle between the benzene ring and the amino-methyl-ene unit is 5.42 (16)°, while the angle between the amino-methyl-ene unit and the dioxane ring is 3.06 (43)°. The dioxane ring shows a half-boat conformation, in which the C atom between the dioxane ring O atoms is 0.464 (10) Šout of the plane. An intra-molecular N-H⋯O hydrogen bond stabilizes the mol-ecular conformation. In the crystal, a three-dimensional framework is built up via inter-molecular N-H⋯O hydrogen bonds.

Expression and activation of the reprogramming transcription factors.

Recently, a series of exciting reports have revealed that terminally differentiated somatic cells can be reprogrammed to generate induced pluripotent stem (iPS) cells via overexpression of a cocktail of transcription factors such as Oct3, Sox2, Klf4, and c-Myc or Oct3, Sox2, Nanog, and Lin28. Most recently, these iPS cells has been used to generate viable, live-born progeny by tetraploid complementation. Reprogramming of iPS cells inaugurates a new era of biology and medicine, it inevitably brings new challenges, e.g., how these factors induce reprogramming and how their expression is regulated. To facilitate iPS cell research, this review focuses on how expression and activation of these transcription factors are regulated.

N-Ethyl-N-phenyl-N'-tosyl-formamidine.

The title compound, C(16)H(18)N(2)O(2)S, was obtained as an unexpected product while attempting to form carbon-nitro-gen bonds by catalytic amidation. The mol-ecule displays an E conformation about the C=N double bond. The planes of the two aromatic rings in the mol-ecule form a dihedral angle of 47.06 (9)°.

(E)-Methyl 2,6-dichloro-N-cyano-benzimidate.

The mol-ecule of the title compound, C(9)H(6)Cl(2)N(2)O, displays an E conformation about the C=N double bond. The N-cyano-imidate fragment is substanti-ally planar [maximum deviation 0.010 (4) Å] and perpendicular to the benzene ring [dihedral angle = 88.50 (14)°]. In the crystal packing, inter-molecular Cl⋯Cl inter-actions [3.490 (2) Å] are observed.

Fibroblast Growth Factor 21 Exerts its Anti-inflammatory Effects on Multiple Cell Types of Adipose Tissue in Obesity.

OBJECTIVE: Obesity-related, chronic, low-grade inflammation has been identified as a key factor in the development of many metabolic diseases, such as type 2 diabetes and cardiovascular diseases. Adipocytes, preadipocytes, and macrophages have been implicated in initiating inflammation in adipose tissue. This study aims to investigate the effects of fibroblast growth factor-21 (FGF-21) on obesity-related inflammation and its mechanisms in vivo and in vitro. METHODS: Monosodium glutamate (MSG) was used to induce obesity in mice and subsequently treated the mice with or without FGF-21. Primary adipocytes and stromal vascular fraction cells were isolated from MSG-obesity mice for additional experiments. RESULTS: Results obtained by ELISA and real-time polymerase chain reaction showed that FGF-21 efficiently ameliorated obesity-related inflammation in MSG-obesity mice. This study demonstrated that preadipocytes and adipocytes responded to anti-inflammatory effects of FGF-21. In vitro, 3 T3-L1 preadipocytes lacking ß-klotho did not respond to FGF-21 under glucose uptake. Interestingly, the treatment of 3 T3-L1 preadipocytes with FGF-21 significantly attenuated lipopolysaccharide-induced inflammatory response. CONCLUSIONS: Our study showed that FGF-21-induced glucose uptake and FGF-21-related anti-inflammatory effects are mediated by different signaling pathways. Moreover, FGF-21 showed anti-inflammatory effects on preadipocytes; these effects are mediated by the fibroblast growth factor receptor substrate 2/ERK1/2 signaling pathway.

Randomized comparison of early inflammatory response after sirolimus-eluting stent vs bare metal stent implantation in native coronary lesions.

BACKGROUND: The clinical significance of early inflammatory response after coronary stent implantation has been controversial. Sirolimus-eluting stent (SES) has been shown to be better outcomes compared with bare metal stent (BMS). We prospectively investigated the early inflammatory response after SES or BMS implantation in patients with single-vessel lesion, and evaluated the relationship between inflammation and late clinical outcomes in a randomized design. METHODS: Forty-eight patients with single-vessel disease were randomized into SES or BMS implantation group (n=24 respectively). Blood samples were taken before stenting, 1 h, 24 h and 8 months afterward. The plasma concentrations of C-reactive protein (CRP) and interleukin-6 (IL-6) were determined by ELISA. The clinical and angiographic follow-up were performed at 8 months after stenting. RESULTS: There was no difference in baseline characteristics, plasma CRP and IL-6 concentrations between the 2 groups. However, plasma IL-6 concentrations at 1 h after stenting were higher in both groups than in baseline (p<0.01). In addition, the plasma CRP and IL-6 concentrations at 24 h after stenting were significantly higher in both groups compared with baseline (p<0.01 respectively). Likewise, plasma CRP and IL-6 concentrations were significantly higher in BMS group compared with SES group at 24 h after stenting (p<0.05 respectively). At the follow-up (mean 8 months after stenting), the rate of in-stent restenosis (ISR) and target lesion revascularization (TLR) were higher in BMS group than in SES group (p<0.05 respectively) although the plasma CRP and IL-6 concentrations are similar between the groups. CONCLUSIONS: Single coronary stenting could trigger an early inflammatory response. However, patients undergoing SES implantation has less augmentation of early inflammatory markers after stenting compared to patients treated with BMS, which was positively related the incidence of ISR and TLR at follow-up. This may reflect the potential impact of SES implantation on the early inflammatory response and late clinical outcomes.

trans-4-[(2,6-Dimethyl-phen-oxy)methyl]cyclo-hexa-necarboxylic acid.

The title compound, C(16)H(22)O(3), is a useful inter-mediate in the synthesis of poly(amido-amine) dendrimers. The cyclo-hexane ring adopts a chair conformation. In the crystal structure, mol-ecules are linked into centrosymmetric dimers by pairs of O-H⋯O hydrogen bonds.

Elevated circulating inflammatory markers in female patients with cardiac syndrome X.

BACKGROUND: The pathophysiological mechanism in cardiac syndrome X has been suggested as impairment in normal endothelial function of the coronary microvasculature, resulting in inadequate flow reserve. However, despite the extensive studies, the precise mechanisms in cardiac syndrome X remain unclear. PURPOSE: The present study was, therefore, to investigate whether inflammatory cells and markers such as C-reactive protein (CRP) and interleukin-6 (IL-6) might be involved in the pathogenesis of cardiac syndrome X. METHODS: Thirty-six female patients with cardiac syndrome X and 30 sex-matched normal controls were prospectively enrolled in this study. Blood samples were drawn for measuring white blood and monocyte cells, inflammatory markers such as CRP and IL-6, and data were compared between patients with cardiac syndrome X and normal controls. RESULTS: The data showed that increased numbers of white blood and monocyte cells were found in patients with cardiac syndrome X compared with normal controls (white blood cells: 7072+/-1146/mm(3) vs. 6138+/-1079/mm(3); monocyte cells: 612+/-186/mm(3) vs. 539+/-190/mm(3)p<0.05, respectively). Moreover, patients with cardiac syndrome X were detected to have significantly higher plasma CRP and IL-6 levels in comparison with patients with normal controls (CRP: 0.48+/-0.26 mg/L vs. 0.22+/-0.15 mg/L; IL-6: 13.4+/-1.2 pg/dl vs. 6.2+/-0.6 pg/dl, p<0.01, respectively). The multivariate analysis showed that CRP was the independent variable most strongly associated with cardiac syndrome X. CONCLUSIONS: Our data suggested that low-grade, chronic inflammation might contribute to the development of cardiac syndrome X manifested by increased plasma levels of inflammatory cells and inflammatory markers.