Circulating microRNA-1a is a biomarker of Graves' disease patients with atrial fibrillation.

PURPOSE: It has been increasingly suggested that specific microRNAs expression profiles in the circulation and atrial tissue are associated with the susceptibility to atrial fibrillation. Nonetheless, the role of circulating microRNAs in Graves' disease patients with atrial fibrillation has not yet been well described. The objective of the study was to identify the role of circulating microRNAs as specific biomarkers for the diagnosis of Graves' disease with atrial fibrillation. METHODS: The expression profiles of eight serum microRNAs, which are found to be critical in the pathogenesis of atrial fibrillation, were determined in patients with Graves' disease with or without atrial fibrillation. MicroRNA expression analysis was performed by real-time PCR in normal control subjects (NC; n = 17), patients with Graves' disease without atrial fibrillation (GD; n = 29), patients with Graves' disease with atrial fibrillation (GD + AF; n = 14), and euthyroid patients with atrial fibrillation (AF; n = 22). RESULTS: Three of the eight serum microRNAs,i.e., miR-1a, miR-26a, and miR-133, had significantly different expression profiles among the four groups. Spearman's correlation analysis showed that the relative expression level of miR-1a was positively correlated with free triiodothyronine (FT3) and free thyroxine (FT4), and negatively related to thyroid stimulating hormone. Spearman's correlations analysis also revealed that the level of miR-1a was negatively correlated with a critical echocardiographic parameter (left atrial diameter), which was dramatically increased in GD + AF group compared to GD group. Furthermore, the receiver-operating characteristic curve analysis indicated that, among the eight microRNAs, miR-1a had the largest area under the receiver-operating characteristic curves not only for discriminating between individuals with and without Graves' disease, but also for predicting the presence of atrial fibrillation in patients with Graves' disease. CONCLUSIONS: Our findings showed that the levels of serum miR-1a were significantly decreased in GD + AF group compared with GD group, suggesting that serum miR-1a might serve as a novel biomarker for diagnosis of atrial fibrillation in patients with Graves' disease.

SIRT1 suppresses self-renewal of adult hippocampal neural stem cells.

The balance between self-renewal and differentiation of adult neural stem cells (aNSCs) is essential for the maintenance of the aNSC reservoir and the continuous supply of new neurons, but how this balance is fine-tuned in the adult brain is not fully understood. Here, we investigate the role of SIRT1, an important metabolic sensor and epigenetic repressor, in regulating adult hippocampal neurogenesis in mice. We found that there was an increase in SIRT1 expression during aNSC differentiation. In Sirt1 knockout (KO) mice, as well as in brain-specific and inducible stem cell-specific conditional KO mice, the proliferation and self-renewal rates of aNSCs in vivo were elevated. Proliferation and self-renewal rates of aNSCs and adult neural progenitor cells (aNPCs) were also elevated in neurospheres derived from Sirt1 KO mice and were suppressed by the SIRT1 agonist resveratrol in neurospheres from wild-type mice. In cultured neurospheres, 2-deoxy-D-glucose-induced metabolic stress suppressed aNSC/aNPC proliferation, and this effect was mediated in part by elevating SIRT1 activity. Microarray and biochemical analysis of neurospheres suggested an inhibitory effect of SIRT1 on Notch signaling in aNSCs/aNPCs. Inhibition of Notch signaling by a γ-secretase inhibitor also largely abolished the increased aNSC/aNPC proliferation caused by Sirt1 deletion. Together, these findings indicate that SIRT1 is an important regulator of aNSC/aNPC self-renewal and a potential mediator of the effect of metabolic changes.

Involvement of arterial baroreflex in the protective effect of dietary restriction against stroke.

Dietary restriction (DR) protects against neuronal dysfunction and degeneration, and reduces the risk of ischemic stroke. This study examined the role of silent information regulator T1 (SIRT1) and arterial baroreflex in the beneficial effects of DR against stroke, using two distinct stroke models: stroke-prone spontaneously hypertensive rats (SP-SHRs) and Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Sirt1 knockout (KO) mice were used to examine the involvement of sirt1. Sinoaortic denervation was used to inactivate arterial baroreflex. Dietary restriction was defined as 40% reduction of dietary intake. Briefly, DR prolonged the life span of SP-SHRs and reduced the infarct size induced by MCAO. Dietary restriction also improved the function arterial baroreflex, decreased the release of proinflammatory cytokines, and reduced end-organ damage. The beneficial effect of DR on stroke was markedly attenuated by blunting arterial baroreflex. Lastly, the infarct area in sirt1 KO mice was significantly larger than in the wild-type mice. However, the beneficial effect of DR against ischemic injury was still apparent in sirt1 KO mice. Accordingly, arterial baroreflex, but not sirt1, is important in the protective effect of DR against stroke.

Induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemia.

Recent reports indicate that autophagy serves as a stress response and may participate in pathophysiology of cerebral ischemia. Nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin), the rate-limiting enzyme in mammalian NAD (+) biosynthesis, protects against ischemic stroke through inhibiting neuronal apoptosis and necrosis. This study was taken to determine the involvement of autophagy in neuroprotection of Nampt in cerebral ischemia. Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation (OGD) in cultured cortical neurons were performed. Nampt was overexpressed or knocked-down using lentivirus-mediated gene transfer in vivo and in vitro. Immunochemistry (LC3-II), electron microscope and immunoblotting assays (LC3-II, beclin-1, mammalian target of rapamycin [mTOR], S6K1 and tuberous sclerosis complex-2 [TSC2]) were performed to assess autophagy. We found that overexpression of Nampt increased autophagy (LC3 puncta immunochemistry staining, LC3-II/beclin-1 expression and autophagosomes number) both in vivo and in vitro at 2 hours after MCAO. At the early stage of OGD, autophagy inducer rapamycin protected against neuronal injury induced by Nampt knockdown, whereas autophagy inhibitor 3-methyladenine abolished the neuroprotective effect of Nampt partly. Overexpression or knockdown of Nampt regulated the phosphorylation of mTOR and S6K1 signaling pathway upon OGD stress through enhancing phosphorylation of TSC2 at Ser1387 but not Thr1462 site. Furthermore, in cultured SIRT1-knockout neurons, the regulation of Nampt on autophagic proteins LC3-II and beclin-1 was abolished. Our results demonstrate that Nampt promotes neuronal survival through inducing autophagy via regulating TSC2-mTOR-S6K1 signaling pathway in a SIRT1-dependent manner during cerebral ischemia.

Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphate-activated kinase pathway.

OBJECTIVE: Stroke is a leading cause of mortality and disability. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in mammalian nicotinamide adenine dinucleotide (NAD)(+) biosynthesis and contributes to cell fate decisions. However, the role of Nampt in brain and stroke remains to be investigated. METHODS: We used lentivirus-mediated Nampt overexpression and knockdown to manipulate Nampt expression and explore the effects of Nampt in neuronal survival on ischemic stress both in vivo and in vitro. We also used adenosine monophosphate (AMP)-activated kinase-α2 (AMPKα2) and silent mating type information regulation 2 homolog 1 (SIRT1) knockout mice to investigate the underlying mechanisms of Nampt neuroprotection. RESULTS: Nampt inhibition by a highly-specific Nampt inhibitor, FK866, aggravated brain infarction in experimentally cerebral ischemia rats, whereas Nampt overexpression in local brain and Nampt enzymatic product nicotinamide mononucleotide (NMN) reduced ischemia-induced cerebral injuries. Nampt overexpression and knockdown regulated neuron survival via the AMPK pathway. Neuroprotection of Nampt was abolished in AMPKα2(-/-) neurons. In neurons, Nampt positively modulated NAD(+) levels and thereby controlled SIRT1 activity. SIRT1 coprecipitated with serine/threonine kinase 11 (LKB1), an upstream kinase of AMPK, and promoted LKB1 deacetylation in neurons. Nampt-induced LKB1 deacetylation and AMPK activation disappeared in SIRT1(-/-) neurons. In contrast, Ca(2+) /calmodulin-dependent protein kinase kinase-ß (CaMKK-ß), another upstream kinase of AMPK, was not involved in the neuroprotection of Nampt. More important, Nampt overexpression-induced neuroprotection was abolished in SIRT1(+/-) and AMPKα2(-/-) mice. INTERPRETATION: Our findings reveal that Nampt protects against ischemic stroke through rescuing neurons from death via the SIRT1-dependent AMPK pathway and indicate that Nampt is a new therapeutic target for stroke.

A pair of windmill-shaped enantiomers from Lindera aggregata with activity toward improvement of insulin sensitivity.

(+)-Linderaspirone A and (-)-linderaspirone A, a pair of natural windmill-shaped enantiomers, were isolated from the traditional Chinese medicine plant Lindera aggregate by HPLC using a chiral column, achieving over 98% ee. Their structures and absolute configurations were determined on the basis of extensive analysis of NMR spectra, crystal X-ray diffraction, and calculation of the optical rotations (OR). They have an unprecedented carbon skeleton and showed significant activity against glucosamine-induced insulin resistance.

A novel class of antagonists for the FFAs receptor GPR40.

The free fatty acid receptor, GPR40, is implicated in the pathophysiology of type 2 diabetes, and is a new potential drug target for the treatment of type 2 diabetes. Its antagonist is thought to be not only a useful chemical probe for further exploring the function of GPR40 but also a lead structure for drug development. With virtual screening based on a homology model followed by a cell-based calcium mobilization assay, we found that sulfonamides are a new class of small organic antagonists for GPR40. One of the compounds, DC260126, dose-dependently inhibited GPR40-mediated Ca(2+) elevations stimulated by linoleic acid, oleic acid, palmitoleic acid and lauric acid (IC(50): 6.28+/-1.14, 5.96+/-1.12, 7.07+/-1.42, 4.58+/-1.14 microM, respectively), reduced GTP-loading and ERK1/2 phosphorylation stimulated by linoleic acid in GPR40-CHO cells, suppressed palmitic acid potentiated glucose-stimulated insulin secretion, and negatively regulated GPR40 mRNA expression induced by oleic acid in Min6 cells.

Bioactivities of ricin retained and its immunoreactivity to anti-ricin polyclonal antibodies alleviated through pegylation.

Ricin has long been employed to construct immunotoxins, whose efficacy was often undermined by immunogenicity. Pegylation (modification of proteins with polyethylene glycol, PEG) was one of those recently developed approaches to circumvent immunogenicity of legions of drugs. Herein, pegylation of ricin was found to have barely changed the RNA N-glycosidase activity and protein synthesis inhibiting activity of ricin, but remarkably altered the cytotoxicity of ricin on hepatoma cell line (BEL7404) or the immunoreactivity with polyclonal anti-ricin antibodies. This result suggested that the attached PEG or monomethyloxyl polyethylene glycol (mPEG) groups did not hinder ricin from hydrolyzing ribosomal RNA, but indeed covered some areas on the surface of ricin molecule, including those involved in the interaction with cellular receptors and epitopes recognized by polyclonal antibodies. Pegylation, masking certain epitopes of ricin, might contribute to alleviate the immunogenicity of the toxin. Approach in this work, if applied to thereby constructed immunotoxins, would help improve the prospective efficacy of these toxins.

Transcription Regulation of bcl-2 Gene.

Various extracelluar stimulation, including those by growth factors and cytokines, can induce the bcl-2 gene expression. Bcl-2 protein induced by this stimulation seems to be essential for cell survival. In order to understand the regulations of bcl-2 transcription, recent advances at transcriptional and post-transcriptional levels in this field will be described.

Interaction between Jak3 and Nucleosome Assembly Protein 1.

Tyrosine kinase Jak3 plays a critical role in the interleukin 2 IL-2 signaling because it not only participates the Jak-Stat pathway, but also interacts with unidentified signal transducers and regulates expression of some oncogenes such as c-fos and c-myc. Abundant evidence demonstrated that phosphorylated tyrosine was necessary for the interaction between two proteins. Therefore, in order to clarify the role of Jak3 in IL-2 signal transduction, the tyrosine-phosphorylation-involved yeast two-hybrid system was constructed and the N-terminal region JH3-JH7 of Jak3 was used as a bait to screen a peripheral blood cDNA library. About 50 double-positive colonies were obtained. Sequence analysis indicated that one of them was from nucleosome assembly protein 1 gene (Nap1), and encoded a protein of 392 amino acid residues. Two-hybrid system results demonstrated that interaction between Jak3 and Nap1 depended on the level of tyrosine phosphorylation. Furthermore, immunoprecipitation and Western blot experiments confirmed that Jak3 really interacted with Nap1 in murine pro-B lymphocyte BAF/BO3beta cells.

A Novel Type of Apoptosis Induced by Cadmium in BA/F3beta Cells.

Apoptosis is usually accompanied by DNA fragmentation and up-regulation of reactive oxygen species, and it can be inhibited by overexpression of Bcl-2. Here, cadmium was found to induce apoptosis in BA/F3beta cells. MTT assay, Hochest 33258 staining, and transmission electron microscopy analysis were used to detect the apoptosis, however, neither DNA fragmentation nor up-regulation of reactive oxygen species were observed in this type of apoptosis. Furthermore, Bcl-2 overexpression had no effect on this type of apoptosis. In conclusion, these data suggested that cadmium induced a novel type of apoptosis in BA/F3beta cells.