Enantioselective synthesis of 3a-azido-pyrroloindolines by copper-catalyzed asymmetric dearomative azidation of tryptamines.

Copper-catalyzed asymmetric dearomative azidation of tryptamines using azidobenziodoxolone as an azidating reagent was developed, which affords a variety of 3a-azido-pyrroloindolines in good to high enantioselectivities under mild reaction conditions. The azides could be readily transformed into the corresponding 3a-amino-pyrroloindolines via reduction and 1,2,3-triazole derivatives via a click reaction.

Silver-Promoted Radical Ring-Opening/Pyridylation of Cyclobutanols with N-Methoxypyridinium Salts.

The silver-promoted reaction of tertiary cyclobutanols with N-methoxypyridinium salts enables the efficient synthesis of a range of C2-substituted pyridines. The overall process likely occurs by ring-opening (via ß-scission) of the cyclobutoxy radical to generate the corresponding γ-keto alkyl radical that itself adds to the pyridinium salt. A wide range of tertiary cyclobutanols and N-methoxypyridinium salts are compatible with the reaction conditions.

Rhodium-Catalyzed Successive C-H Bond Functionalizations To Synthesize Complex Indenols Bearing a Benzofuran Unit.

An efficient rhodium-catalyzed redox-neutral annulations of N-phenoxyacetamides and ynones via successive double C-H bond activations has been developed. A series of novel and complex indenols bearing a benzofuran unit were generated with moderate to excellent regioselecetivities under mild conditions. Adding N-ethylcyclohexanamine (CyNHEt) could restrict the formation of the mono C-H bond activation byproduct, which is not the intermediate of the reaction demonstrated via the mechanistic investigations.

Enantioselective Copper-Catalyzed Electrophilic Dearomative Azidation of ß-Naphthols.

The first example of copper-catalyzed enantioselective dearomative azidation of ß-naphthols using a readily available N3-transfer reagent is reported. A series of 2-hydroxy-1-naphthamides bearing a complex N-substituent were converted to the corresponding products in high yields with up to 96% ee, and chiral 1-azido-2-hydroxy-1-naphthoates were obtained with up to 90% ee under mild reaction conditions. The azides could be further transformed into the corresponding 1,2,3-triazoles smoothly via "click" reaction.

New ecological dam for sediment and overlying water pollution treatment based on microbial fuel cell principle.

In this study, a new ecological dam based on the microbial fuel cell principle (MFCED) was designed to remove pollutants from river sediments and water bodies. Sediment organics were better removed in the MFCED mode in comparison with the other two modes (ecological dam with open circuit (OCED) and ecological dam filled with gravel in cathode chamber (GMFCED)). The difference of nitrogen source in water had little effect on the removal of chemical oxygen demand (COD) (70-80%), while nitrate was more readily removed in the MFCED. The voltage curve and power curve were measured to understand the bioelectricity generation of MFCED. During the stable operation phase of MFCED, the voltage was stabilized between 0.09-0.15 V. The results of high-throughput sequencing indicated that the anode and cathode diversities of MFCED were more than the other systems, and the species diversity of the anode was more than that of the cathode in the MFCED. Graphical abstract.

Nucleophilic Substitution of gem-Difluoroalkenes with TMSNu Promoted by Catalytic Amounts of Cs2CO3.

The efficient and practical nucleophilic cyanation and trifluoromethylation with appropriate trimethylsilyl nucleophiles were developed. Catalytic amounts of cheap and nontoxic Cs2CO3 were used to maintain a sufficiently high concentration of nucleophilic anion (CN- or CF3-) which could begin the catalytic cycle. The present methodologies provide diverse functionalized monofluoroalkenes bearing a cyano and trifluoromethyl group with excellent to moderate stereoselectivities.

Ischemic preconditioning protects the brain against injury via inhibiting CaMKII-nNOS signaling pathway.

Although studies have shown that cerebral ischemic preconditioning (IPC) can ameliorate ischemia/reperfusion (I/R) induced brain damage, but its precise mechanisms remain unknown. Therefore, the aim of this study was to investigate the neuroprotective mechanisms of IPC against ischemic brain damage induced by cerebral I/R and to explore whether the Calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway contributed to the protection provided by IPC. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with 3 min of IPC alone or KN62 (selective antagonist of CaMKII) treatment before IPC, after reperfusion for 3 days, 6 min ischemia was induced. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting was performed to measure the phosphorylation of CaMKII, nNOS, c-Jun and the expression of FasL. Immunoprecipitation was used to examine the binding between PSD95 and nNOS. The results showed that IPC could significantly protect neurons against cerebral I/R injury, furthermore, the combination of PSD95 and nNOS was increased, coinstantaneously the phosphorylation of CaMKII and nNOS (ser847) were up-regulated, however the activation of c-Jun and FasL were reduced. Conversely, KN62 treatment before IPC reversed all these effects of IPC. Taken together, the results suggest that IPC could diminish ischemic brain injury through CaMKII-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway.

ATPA induced GluR5-containing kainite receptor S-nitrosylation via activation of GluR5-Gq-PLC-IP(3)R pathway and signalling module GluR5·PSD-95·nNOS.

GluR5-containing kainite receptor (GluR5-KAR) plays an important role in the pathophysiology of nervous system diseases, while S-nitrosylation exerts a variety of effects on biological systems. However, the mechanism of GluR5-KAR S-nitrosylation is still unclear up to now. Here our researches found that GluR5-KAR selective agonist ATPA stimulation activated the nonclassical GluR5-KAR-Gq-PLC-IP(3)R pathway and the signalling module GluR5·PSD-95·nNOS (the former is more important), led to Ca(2+) release from intracellular calcium stores endoplasmic reticulum (ER) to cytoplasm and extracellular calcium indrawal, respectively, which further resulted in nNOS activation and GluR5-KAR S-nitrosylation, and then inhibited GluR5-mediated whole-cell current attenuation and induced apoptosis in primary cultured hippocampal neurons. Clarification of the primary mechanisms of GluR5-KAR S-nitrosylation induced by ATPA and identification of critical cysteine for GluR5-2a S-nitrosylation (Cys231 and Cys804) open up a brand-new field for revealing downstream signalling pathway of GluR5-KAR and its molecular characteristics, exploring the pathogenesis of neurological diseases and searching for promising therapies.

Cerebral ischemia-reperfusion induces GAPDH S-nitrosylation and nuclear translocation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, plays an important role in glycolysis. It was reported that GAPDH undergoes S-nitrosylation, which facilitated its binding to Siah1 and resulted in nuclear translocation and cell apoptosis. The results of this study show that GAPDH S-nitrosylation, Siah1 binding, translocation to nucleus, and concomitant neuron death occur during the early stages of reperfusion in the rat four-vessel occlusion ischemic model. N-Methyl-D-aspartate receptor antagonist MK801, neuronal nitric oxide synthase inhibitor 7-nitroindazole, or monoamine oxidase-B inhibitor (R)-(-)-deprenyl hydrochloride could inhibit GAPDH S-nitrosylation and translocation and exert neuroprotective effects.

Neuroprotection of co-activation of GABA receptors by preventing caspase-3 denitrosylation in KA-induced seizures.

Previous studies have demonstrated that kainic acid (KA)-induced seizures can cause the enhancement of excitation and lead to neuronal death in rat hippocampus. Co-activation of the inhibitory GABA receptors can attenuate the excitatory JNK3 apoptotic signaling pathway via inhibiting the increased assembly of the GluR6-PSD-95-MLK3 signaling module induced by KA in epileptic rat hippocampal CA1 and CA3 regions. Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. We designed experiments to elucidate the underlying molecular mechanisms of procaspase-3 activation and neuroprotection of co-activation of GABA receptors against neuronal death induced by KA. In this study, we show that co-activation of GABA receptors can attenuate the Fas/FasL apoptotic signaling pathway and inhibit the increased of thioredoxin reductase activity induced by KA, subsequently inhibit the activation of procaspase-3 by diminishing the denitrosylation of its active-site thiol and decreasing the cleavage of the caspase-3 zymogen to its active subunits. These results indicate that co-activation of GABA receptors results in neuroprotection by preventing caspase-3 denitrosylation in KA-induced seizure of rats.

Neuroprotection of GluK1 kainate receptor agonist ATPA against ischemic neuronal injury through inhibiting GluK2 kainate receptor-JNK3 pathway via GABA(A) receptors.

It is well known that GluK2-containing kainate receptors play essential roles in seizure and cerebral ischemia-induced neuronal death, while GluK1-containing kainate receptors could increase tonic inhibition of post-synaptic pyramidal neurons. This research investigated whether GluK1 could inhibit activation of c-Jun N-terminal kinase 3 (JNK3) signaling pathway mediated by the GluK2 in cerebral ischemia-reperfusion. The results show that GluK1 activation by (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) at 1nmol per rat could inhibit the assembly of GluK2·Postsynaptic density 95·mixed lineage kinase 3 signaling module, activation of JNK3 and its downstream signal molecules. However, the inhibition of ATPA could be prevented by GluK1 antagonist NS3763, GluK1 antisense, and GABA(A) receptor antagonist bicuculline. In addition, ATPA played a neuroprotective role against cerebral ischemia. In sum, the findings indicate that activation of GluK1 by ATPA at specific dosages may promote GABA release, which then suppresses post-synaptic GluK2-JNK3 signaling-mediated cerebral ischemic injury via GABA(A)R.

nNOS downregulation attenuates neuronal apoptosis by inhibiting nNOS-GluR6 interaction and GluR6 nitrosylation in cerebral ischemic reperfusion.

Glutamate receptor 6 (GluR6) is well documented to play a pivotal role in ischemic brain injury, which is mediated by the GluR6·PSD95·MLK3 signaling module and subsequent c-Jun N-terminal kinase (JNK) activation. Our recent studies show that GluR6 is S-nitrosylated in the early stages of ischemia-reperfusion. NO (Nitric Oxide) is mainly generated from neuronal nitric oxide synthase (nNOS) in cerebral neurons during the early stages of reperfusion. Here, the effect of nNOS downregulation on GluR6 S-nitrosylation and GluR6-mediated signaling was investigated in cerebral ischemia and reperfusion. Administration of nNOS oligonucleotides confirmed that GluR6 nitrosylation is induced by nNOS-derived endogenous NO and further activates the GluR6·PSD95·MLK3 signaling module and JNK signaling pathway. Moreover, this study revealed for the first time that nNOS can bind with GluR6 during ischemic reperfusion, and PSD95 is involved in this interaction. In summary, our results suggest that nNOS binds with GluR6 via PSD95 and then produces endogenous NO to S-nitrosylate GluR6 in cerebral ischemia-reperfusion, which provides a new approach for stroke therapy.

S-nitrosylation of c-Src via NMDAR-nNOS module promotes c-Src activation and NR2A phosphorylation in cerebral ischemia/reperfusion.

Previous studies suggested that activated c-Src promote the tyrosine phosphorylation of NMDA receptor subunit NR2A, and thus aggravate the injury induced by transient cerebral ischemia/reperfusion (I/R) in rat hippocampus CA1 region. In this study, we examined the effect of nitric oxide (NO) on the activation of c-Src and the tyrosine phosphorylation of NMDA receptor NR2A subunit. The results show that S-nitrosylation and the phosphorylation of c-Src were induced after cerebral I/R in rats, and administration of nNOS inhibitor 7-NI, nNOS antisense oligonucleotides and exogenous NO donor sodium nitroprusside diminished the increased S-nitrosylation and phosphorylation of c-Src during cerebral I/R. The cysteine residues of c-Src modified by S-nitrosylation are Cys489, Cys498, and Cys500. On the other hand, NMDAR antagonist MK-801 could attenuate the S-nitrosylation and activation of c-Src. Taken together, the S-nitrosylation of c-Src is provoked by NO derived from endogenous nNOS, which is activated by Ca(2+) influx from NMDA receptors, and promotes the auto-phosphorylation at tyrosines and further phosphorylates NR2A. The molecular mechanism we outlined here is a novel postsynaptic NMDAR-nNOS/c-Src-mediated signaling amplification, the 'NMDAR-nNOS â†’ NO â†’ SNO-c-Src â†’ p-c-Src â†’ NMDAR-nNOS' cycle, which presents the possibility as a potential therapeutic target for stroke treatment.

Knock-down of postsynaptic density protein 95 expression by antisense oligonucleotides protects against apoptosis-like cell death induced by oxygen-glucose deprivation in vitro.

OBJECTIVE: Postsynaptic density protein 95 (PSD-95) plays important roles in the regulation of glutamate signaling, such as that of N-methyl-D-aspartate receptors (NMDARs). In this study, the functional roles of PSD-95 in tyrosine phosphorylation of NMDAR subunit 2A (NR2A) and in apoptosis-like cell death induced by oxygen-glucose deprivation (OGD) in cultured rat cortical neurons were investigated. METHODS: We used immunoprecipitation and immunoblotting to detect PSD-95 protein level, tyrosine phosphorylation level of NR2A, and the interaction between PSD-95 and NR2A or Src. Apoptosis-like cells were observed by 4,6-diamidino-2-phenylindole staining. RESULTS: Tyrosine phosphorylation of NR2A and apoptosis-like cell death were increased after recovery following 60-min OGD. The increases were attenuated by pretreatment with antisense oligonucleotides against PSD-95 before OGD, but not by missense oligonucleotides or vehicle. PSD-95 antisense oligonucleotides also inhibited the increased interaction between PSD-95 and NR2A or Src, while NR2A expression did not change under this condition. CONCLUSION: PSD-95 may be involved in regulating NR2A tyrosine phosphorylation by Src kinase. Inhibition of PSD-95 expression can be neuroprotective against apoptosis-like cell death after recovery from OGD.

S-nitrosylation of mixed lineage kinase 3 contributes to its activation after cerebral ischemia.

Previous studies in our laboratory have shown that mixed lineage kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-D-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein kinase kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.

N-methyl-D-aspartate receptor-dependent denitrosylation of neuronal nitric oxide synthase increase the enzyme activity.

Our laboratory once reported that neuronal nitric oxide synthase (nNOS) S-nitrosylation was decreased in rat hippocampus during cerebral ischemia-reperfusion, but the underlying mechanism was unclear. In this study, we show that nNOS activity is dynamically regulated by S-nitrosylation. We found that overexpressed nNOS in HEK293 (human embryonic kidney) cells could be S-nitrosylated by exogenous NO donor GSNO and which is associated with the enzyme activity decrease. Cys(331), one of the zinc-tetrathiolate cysteines, was identified as the key site of nNOS S-nitrosylation. In addition, we also found that nNOS is highly S-nitrosylated in resting rat hippocampal neurons and the enzyme undergos denitrosylation during the process of rat brain ischemia/reperfusion. Intrestingly, the process of nNOS denitrosylation is coupling with the decrease of nNOS phosphorylation at Ser(847), a site associated with nNOS activation. Further more, we document that nNOS denitrosylation could be suppressed by pretreatment of neurons with MK801, an antagonist of NMDAR, GSNO, EGTA, BAPTA, W-7, an inhibitor of calmodulin as well as TrxR1 antisense oligonucleotide (AS-ODN) respectively. Taken together, our data demonstrate that the denitrosylation of nNOS induced by calcium ion influx is a NMDAR-dependent process during the early stage of ischemia/reperfusion, which is majorly mediated by thioredoxin-1 (Trx1) system. nNOS dephosphorylation may be induced by the enzyme denitrosylation, which suggest that S-nitrosylation/denitrosylation of nNOS may be an important mechanism in regulating the enzyme activity.

Depolarization induces NR2A tyrosine phosphorylation and neuronal apoptosis.

BACKGROUND: Cytosol Ca2+ overload plays a vital role in ischemic neuronal damage, which is largely contributed by the Ca2+ influx through L-type voltage-gated calcium channels (L-VGCCs) and N-methyl-D-aspartate (NMDA) type glutamate receptors. In this article, L-VGCCs were activated by depolarization to investigate the cross-talk between NMDA receptors and L-VGCCs. METHODS: Depolarization was induced by 20 minutes incubation of 75 mM KCl in cultured rat cortical neuron. Apoptosis-like neuronal death was detected by DAPI staining. Tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A), interactions of Src and NR2A were detected by immunoblot and immunoprecipitation. RESULTS: Depolarization induced cortical neuron apoptosis-like cell death after 24 hours of restoration. The apoptosis was partially inhibited by 5 mM EGTA, 100 µM Cd2+, 10 µM nimodipine, 100 µM genistein, 20 µM MK-801, 2 µM PP2 and combined treatment of nimodipine and MK-801. NR2A tyrosine phosphorylation increased after depolarization, and the increase was inhibited by the drugs listed above. Moreover, non-receptor tyrosine kinase Src bound with NR2A after depolarization and restoration. The binding was also inhibited by the drugs listed above. CONCLUSIONS: The results indicated that depolarization-induced neuronal death might be due to extracellular Ca2+ influx through L-VGCCs and subsequently Src activationmediated NR2A tyrosine phosphorylation.

Blocking Daxx trafficking attenuates neuronal cell death following ischemia/reperfusion in rat hippocampus CA1 region.

Previous studies have shown that the death-associated protein (Daxx) shuttles between nucleus and cytoplasm under ischemic stress, and the subcellular localization of Daxx plays an important role in ischemic neuron death. In this study, by blocking the Daxx trafficking, the rat hippocampus CA1 neurons were protected against cerebral ischemia/reperfusion, and the molecular mechanism underlying this neuroprotection was studied. We found that pretreatment of SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), or an anti-oxidant, N-acetylcysteine (NAC), could not only prevent Daxx from trafficking but also increase the number of the surviving CA1 pyramidal cells of hippocampus at 5days of reperfusion. Furthermore, knock-down of endogenous Daxx exerted similar neuroprotective effect during ischemia/reperfusion. We found the treatment of SP600125 or NAC could decrease the activation of Ask1 during ischemia/reperfusion and suppress the assembly of the Fas·Daxx·Ask1 signaling module, and in succession inhibit JNK activation and c-Jun phosphorylation. This study provides the Daxx trafficking as a new potential therapeutic target for ischemic brain injury.

K252a suppresses neuronal cells apoptosis through inhibiting the translocation of Bax to mitochondria induced by the MLK3/JNK signaling after transient global brain ischemia in rat hippocampal CA1 subregion.

It is demonstrated that the c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Our previous studies have suggested that K252a can obviously inhibit JNK activation induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. Here, we further discussed the potential mechanism of ischemic brain injury induced by the activation of JNK after 15?min of transient global cerebral ischemia. As a result, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and 14-3-3 protein (a cytoplasmic anchor of Bax) induced by the activation of JNK, K252a decreased the release of Bax from Bcl-2/Bax and 14-3-3/Bax dimers, further attenuating the translocation of Bax from cytosol to mitochondria and the release of cytochrome c induced by ischemia/reperfusion, which related to mitochondria-mediated apoptosis. Importantly, pre-infusion of K2525a 20?min before ischemia showed neuroprotective effect against neuronal cells apoptosis. These findings imply that K252a induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 subregion via inhibiting the mitochondrial apoptosis pathway induced by JNK activation.

Activation of GluR6-containing kainate receptors induces ubiquitin-dependent Bcl-2 degradation via denitrosylation in the rat hippocampus after kainate treatment.

We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.