16 research outputs found

    Copper-Catalyzed Oxidative Cyclization of Maleimides with Amines and Alkyne Esters: Direct Access to Fully Substituted Dihydropyrroles and Pyrrole Derivatives

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    An efficient and practical Cu­(I)-catalyzed oxidative cyclization cascade reaction of diverse amines, alkyne esters and maleimides has been developed. The reactions can afford 4,6-dioxopyrrolo­[3,4-<i>b</i>]­pyrrole-2,3-dicarboxylates and related derivatives with satisfactory yields by altering the reaction conditions slightly. The substrate scope highlights the flexibility of the catalyst, and a reaction mechanism is also proposed

    Phosphoproteome-based kinase activity profiling reveals the critical role of MAP2K2 and PLK1 in neuronal autophagy

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    <p>Recent studies have demonstrated that dysregulation of macroautophagy/autophagy may play a central role in the pathogenesis of neurodegenerative disorders, and the induction of autophagy protects against the toxic insults of aggregate-prone proteins by enhancing their clearance. Thus, autophagy has become a promising therapeutic target against neurodegenerative diseases. In this study, quantitative phosphoproteomic profiling together with a computational analysis was performed to delineate the phosphorylation signaling networks regulated by 2 natural neuroprotective autophagy enhancers, corynoxine (Cory) and corynoxine B (Cory B). To identify key regulators, namely, protein kinases, we developed a novel network-based algorithm of <i>in silico</i> Kinome Activity Profiling (iKAP) to computationally infer potentially important protein kinases from phosphorylation networks. Using this algorithm, we observed that Cory or Cory B potentially regulated several kinases. We predicted and validated that Cory, but not Cory B, downregulated a well-documented autophagy kinase, RPS6KB1/p70S6K (ribosomal protein S6 kinase, polypeptide 1). We also discovered 2 kinases, MAP2K2/MEK2 (mitogen-activated protein kinase kinase 2) and PLK1 (polo-like kinase 1), to be potentially upregulated by Cory, whereas the siRNA-mediated knockdown of <i>Map2k2</i> and <i>Plk1</i> significantly inhibited Cory-induced autophagy. Furthermore, Cory promoted the clearance of Alzheimer disease-associated APP (amyloid β [A4] precursor protein) and Parkinson disease-associated SNCA/α-synuclein (synuclein, α) by enhancing autophagy, and these effects were dramatically diminished by the inhibition of the kinase activities of MAP2K2 and PLK1. As a whole, our study not only developed a powerful method for the identification of important regulators from the phosphoproteomic data but also identified the important role of MAP2K2 and PLK1 in neuronal autophagy.</p

    HMGB1 is involved in autophagy inhibition caused by SNCA/α-synuclein overexpression

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    <p>SNCA/α-synuclein and its rare mutations are considered as the culprit proteins in Parkinson disease (PD). Wild-type (WT) SNCA has been shown to impair macroautophagy in mammalian cells and in transgenic mice. In this study, we monitored the dynamic changes in autophagy process and confirmed that overexpression of both WT and SNCA<sup>A53T</sup> inhibits autophagy in PC12 cells in a time-dependent manner. Furthermore, we showed that SNCA binds to both cytosolic and nuclear high mobility group box 1 (HMGB1), impairs the cytosolic translocation of HMGB1, blocks HMGB1<b>-</b>BECN1 binding, and strengthens BECN1<b>-</b>BCL2 binding. Deregulation of these molecular events by SNCA overexpression leads to autophagy inhibition. Overexpression of BECN1 restores autophagy and promotes the clearance of SNCA. siRNA knockdown of <i>Hmgb1</i> inhibits basal autophagy and abolishes the inhibitory effect of SNCA on autophagy while overexpression of HMGB1 restores autophagy. Corynoxine B, a natural autophagy inducer, restores the deficient cytosolic translocation of HMGB1 and autophagy in cells overexpressing SNCA, which may be attributed to its ability to block SNCA<b>-</b>HMGB1 interaction. Based on these findings, we propose that SNCA<b>-</b>induced impairment of autophagy occurs, in part, through HMGB1, which may provide a potential therapeutic target for PD.</p

    Effects of Huanglian-Jie-Du-Tang and Its Modified Formula on the Modulation of Amyloid-β Precursor Protein Processing in Alzheimer's Disease Models

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    <div><p>Huanglian-Jie-Du-Tang (HLJDT) is a famous traditional Chinese herbal formula that has been widely used clinically to treat cerebral ischemia. Recently, we found that berberine, a major alkaloid compound in HLJDT, reduced amyloid-β (Aβ) accumulation in an Alzheimer’s disease (AD) mouse model. In this study, we compared the effects of HLJDT, four single component herbs of HLJDT (Rhizoma coptidis (RC), Radix scutellariae (RS), Cortex phellodendri (CP) and Fructus gardenia (FG)) and the modified formula of HLJDT (HLJDT-M, which is free of RS) on the regulatory processing of amyloid-β precursor protein (APP) in an <i>in vitro</i> model of AD. Here we show that treatment with HLJDT-M and its components RC, CP, and the main compound berberine on N2a mouse neuroblastoma cells stably expressing human APP with the Swedish mutation (N2a-SwedAPP) significantly decreased the levels of full-length APP, phosphorylated APP at threonine 668, C-terminal fragments of APP, soluble APP (sAPP)-α and sAPPβ-Swedish and reduced the generation of Aβ peptide in the cell lysates of N2a-SwedAPP. HLJDT-M showed more significant APP- and Aβ- reducing effects than berberine, RC or CP treatment alone. In contrast, HLJDT, its component RS and the main active compound of RS, baicalein, strongly increased the levels of all the metabolic products of APP in the cell lysates. The extract from FG, however, did not influence APP modulation. Interestingly, regular treatment of TgCRND8 APP transgenic mice with baicalein exacerbated the amyloid plaque burden, APP metabolism and Aβ production. Taken together, these data provide convincing evidence that HLJDT and baicalein treatment can increase the amyloidogenic metabolism of APP which is at least partly responsible for the baicalein-mediated Aβ plaque increase in the brains of TgCRND8 mice. On the other hand, HLJDT-M significantly decreased all the APP metabolic products including Aβ. Further study of HLJDT-M for therapeutic use in treating AD is warranted.</p></div

    LC–MS-Based Urinary Metabolite Signatures in Idiopathic Parkinson’s Disease

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    Increasing evidence has shown that abnormal metabolic phenotypes in body fluids reflect the pathogenesis and pathophysiology of Parkinson’s disease (PD). These body fluids include urine; however, the relationship between, specifically, urinary metabolic phenotypes and PD is not fully understood. In this study, urinary metabolites from a total of 401 clinical urine samples collected from 106 idiopathic PD patients and 104 normal control subjects were profiled by using high-performance liquid chromatography coupled to high-resolution mass spectrometry. Our study revealed significant correlation between clinical phenotype and urinary metabolite profile. Metabolic profiles of idiopathic PD patients differed significantly and consistently from normal controls, with related metabolic pathway variations observed in steroidogenesis, fatty acid beta-oxidation, histidine metabolism, phenylalanine metabolism, tryptophan metabolism, nucleotide metabolism, and tyrosine metabolism. In the fruit fly <i>Drosophila melanogaster</i>, the alteration of the kynurenine pathway in tryptophan metabolism corresponded with pathogenic changes in the alpha-synuclein overexpressed <i>Drosophila</i> model of PD. The results suggest that LC–MS-based urinary metabolomic profiling can reveal the metabolite signatures and related variations in metabolic pathways that characterize PD. Consistent PD-related changes across species may provide the basis for understanding metabolic regulation of PD at the molecular level

    Extracts of HLJDT constituent herbs alter the processing of APP in N2a-SwedAPP cells.

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    <p>Conditioned medium and cell lysates were prepared from N2a-SwedAPP cells that were treated with (A) RC, (B) CP, (C) FG or (D) RS at various doses as indicated for 48 h. Western blotting was used to detect sAPPα and sAPPβ-sw in conditioned medium, and to detect Fl-APP, pAPPThr668 and β-actin in cell lysates. Bars represent mean±S.E.M. for three experiments. One-way ANOVA revealed significant differences due to treatment at various doses of components of HLJDT: *p<0.05; **p<0.01; ***p<0.001.</p

    Modulation of APP processing by HLJDT and HLJDT-M in N2a-SwedAPPcells.

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    <p>Conditioned medium and cell lysates were prepared from N2a-SwedAPP cells that were treated with (A) HLJDT or (B) HLJDT-M at various doses as indicated for 48 h. Western blotting was used to detect sAPPα and sAPPβ-sw in conditioned medium, and to detect Fl-APP, pAPPThr668, CTFs and β-actin in cell lysates. Bars represent mean±S.E.M. for three experiments. One-way ANOVA revealed significant differences due to treatment at various doses of HLJDT or HLJDT-M: *p<0.05; **p<0.01; ***p<0.001.</p

    <i>RBMS3</i> arrests cell cycle at the G1/S checkpoint.

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    <p>(<b>A</b>) Representative and summary of DNA content detected by flow cytometry showed that the percentage of cells in the S phase was lower while the percentage of cells in the G1 phase was higher in SUNE1-RBMS3 cells than that in SUNE1-V1 cells. (* p<0.05, Student’s <i>t</i>-test). Values were expressed as mean ± SD of three independent experiments. (<b>B</b>) Protein expressions of cyclin D1, cyclin E, CDK2, p53, p21, Rb and Rb (Ser780) were compared between <i>RBMS3</i>- and empty vector-transfected NPC cells. β-actin was used as a loading control.</p

    Downregulation of RBMS3 in nasopharyngeal carcinoma (NPC).

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    <p>(<b>A</b>) Expression of <i>RBMS3</i> in 15 primary NPC cases was compared using qPCR between tumor tissues (T) and their paired normal tissue (N). GAPDH was set as an internal control. (<b>B</b>) <i>RBMS3</i> expression was normalized by internal control <i>GAPDH</i>. Statistical analysis confirmed the qPCR results (p<0.001). (<b>C</b>) qPCR analysis of <i>RBMS3</i> expression in three NPC cell lines (C666, CNE2 and SUNE1). The fold changes of <i>RBMS3</i> expression were compared with the immortalized NP cell line, NP460. (<b>D</b>) Immunohistochemical detection of RBMS3 protein in NPC tissue samples and non-tumor nasopharyngeal tissues. Normal: strong positive staining for RBMS3 protein in normal nasopharyngeal epitheliums (arrowhead). NPC1: weak positive staining for RBMS3 protein in NPC tissues (arrowhead). NPC2: negative staining for RBMS3 protein in NPC tissues (arrowhead).</p
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