分类: 生物学 >> 生物物理学 >> 神经科学 提交时间: 2016-06-06
摘要: Previous studies have shown that the Hippo pathway effector yes-associated protein (YAP) plays an important role in maintaining stem cell proliferation. However, the precise molecular mechanism of YAP in regulating murine embryonic neural stem cells (NSCs) remains largely unknown. Here, we show that bone morphogenetic protein-2 (BMP2) treatment inhibited the proliferation of mouse embryonic NSCs, that YAP was critical for mouse NSC proliferation, and that BMP2 treatment-induced inhibition of mouse NSC proliferation was abrogated by YAP knockdown, indicating that the YAP protein mediates the inhibitory effect of BMP2 signaling. Additionally, we found that BMP2 treatment reduced YAP nuclear translocation, YAP-TEAD interaction, and YAP-mediated transactivation. BMP2 treatment inhibited YAP/TEAD-mediated Cyclin D1 (ccnd1) expression, and knockdown of ccnd1 abrogated the BMP2-mediated inhibition of mouse NSC proliferation. Mechanistically, we found that Smad1/4, effectors of BMP2 signaling, competed with YAP for the interaction with TAED1 and inhibited YAP's cotranscriptional activity. Our data reveal mechanistic cross talk between BMP2 signaling and the Hippo-YAP pathway in murine NSC proliferation, which may be exploited as a therapeutic target in neurodegenerative diseases and aging.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-15
摘要: Insulin resistance and abdominal obesity are present in the majority of people with the metabolic syndrome. Antioxidant therapy might be a useful strategy for type 2 diabetes and other insulin-resistant states. The combination of vitamin C (Vc) and vitamin E has synthetic scavenging effect on free radicals and inhibition effect on lipid peroxidation. However, there are few studies about how to define the best combination of more than three anti-oxidants as it is difficult or impossible to test the anti-oxidant effect of the combination of every concentration of each ingredient experimentally. Here we present a math model, which is based on the classical Hill equation to determine the best combination, called Fixed Dose Combination (FDC), of several natural anti-oxidants, including Vc, green tea polyphenols (GTP) and grape seed extract proanthocyanidin (GSEP). Then we investigated the effects of FDC on oxidative stress, blood glucose and serum lipid levels in cultured 3T3-L1 adipocytes, high fat diet (HFD)-fed rats which serve as obesity model, and KK-ay mice as diabetic model. The level of serum malondialdehyde (MDA) in the treated rats was studied and Hematoxylin-Eosin (HE) staining or Oil red slices of liver and adipose tissue in the rats were examined as well. FDC shows excellent antioxidant and anti-glycation activity by attenuating lipid peroxidation. FDC determined in this investigation can become a potential solution to reduce obesity, to improve insulin sensitivity and be beneficial for the treatment of fat and diabetic patients. It is the first time to use the math model to determine the best ratio of three anti-oxidants, which can save much more time and chemical materials than traditional experimental method. This quantitative method represents a potentially new and useful strategy to screen all possible combinations of many natural anti-oxidants, therefore may help develop novel therapeutics with the potential to ameliorate the worldwide metabolic abnormalities. (C) 2015 The Authors. Published by Elsevier B.V.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-15
摘要: GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the function of GADD45A in autophagy remains unknown. In this report, we demonstrate that GADD45A plays an important role in regulating the process of autophagy. GADD45A is able to decrease LC3-II expression and numbers of autophagosomes in mouse tissues and different cancer cell lines. Using bafilomycin A(1) treatment, we have observed that GADD45A regulates autophagosome initiation. Likely, GADD45A inhibition of autophagy is through its influence on the interaction between BECN1 and PIK3C3. Immunoprecipitation and GST affinity isolation assays exhibit that GADD45A directly interacts with BECN1, and in turn dissociates the BECN1-PIK3C3 complex. Furthermore, we have mapped the 71 to 81 amino acids of the GADD45A protein that are necessary for the GADD45A interaction with BECN1. Knockdown of BECN1 can abolish autophagy alterations induced by GADD45A. Taken together, these findings provide the novel evidence that GADD45A inhibits autophagy via impairing the BECN1-PIK3C3 complex formation.
分类: 生物学 >> 生物物理学 >> 肿瘤学 提交时间: 2016-05-12
摘要: The signal transduction pathway initiated by vascular endothelial growth factor-vascular endothelial growth factor receptor 2 (VEGF-VEGFR2) plays an important role in the angiogenesis of tumors. The effective antagonists of VEGFR2 would behave as potent drugs for the treatment of malignant cancers. In our study, specific binding peptides with high affinity to VEGFR2 were obtained through bacterial display technology. Conserved motif (FF/YEXWGVK) among those peptide sequences was discovered. One of the selected peptides, VRBP1 (YDGNSFYEMWGVKPASES) was identified by screening the biased bacterial peptide library and its physiochemical feature was further characterized. The results of surface plasmon resonance (SPR) assay indicated that the dissociation constant (K-D) value of VRBP1 was 228.3 nM and this peptide competed with VEGF binding to VEGFR2. Particles conjugated with VRBP1 could recognize the human umbilical vein endothelial cells (HUVEC) which express VEGFR2 on the surface. Further therapeutic effect of VRBP1 was examined by in vivo experiments. VRBP1 could result in a significant decrease in tumor size of H460 xenografts. The results from the immunohistochemical assay showed that CD31 positive signals in VRBP1-treated group were fewer than those in the control ones. These data highlighted the potential of VEGFR2-binding peptides as effective molecules for cancer diagnosis and therapy.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5' end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-12
摘要: The Hippo signaling pathway restricts organ size by inactivating the Yorkie (Yki)/Yes-associated protein (YAP) family proteins. The oncogenic Yki/YAP transcriptional coactivator family promotes tissue growth by activating target gene transcription, but the regulation of Yki/YAP activation remains elusive. In mammalian cells, we identified Brg1, a major subunit of chromatin-remodeling SWI/SNF family proteins, which interacts with YAP. This finding led us to investigate the in vivo functional interaction of Yki and Brahma (Brm), the Drosophila homolog of Brg1. We found that Brm functions at the downstream of Hippo pathway and interacts with Yki and Scalloped (Sd) to promotes Yki-dependent transcription and tissue growth. Furthermore, we demonstrated that Brm is required for the Crumbs (Crb) dysregulation-induced Yki activation. Interestingly, we also found that crb is a downstream target of Yki-Brm complex. Brm physically binds to the promoter of crb and regulates its transcription through Yki. Together, we showed that Brm functions as a critical regulator of Hippo signaling during tissue growth and plays an important role in the feedback loop between Crb and Yki. (C) 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.
分类: 生物学 >> 生物物理学 >> 细胞生物学 提交时间: 2016-05-11
摘要: Membrane receptors at the surface of target cells are key host factors for virion entry; however, it is unknown whether trafficking and secretion of progeny virus requires host intracellular receptors. In this study, we demonstrate that dengue virus (DENV) interacts with KDEL receptors (KDELR), which cycle between the ER and Golgi apparatus, for vesicular transport from ER to Golgi. Depletion of KDELR by siRNA reduced egress of both DENV progeny and recombinant subviral particles (RSPs). Coimmunoprecipitation of KDELR with dengue structural protein prM required three positively charged residues at the N terminus, whose mutation disrupted protein interaction and inhibited RSP transport from the ER to the Golgi. Finally, siRNA depletion of class II Arfs, which results in KDELR accumulation in the Golgi, phenocopied results obtained with mutagenized prME and KDELR knockdown. Our results have uncovered a function for KDELR as an internal receptor involved in DENV trafficking.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Cerebral cavernous malformation 2 (CCM2) functions as an adaptor protein implicated in various biological processes. By interacting with the mitogen-activated protein kinase MEKK3, CCM2 either mediates the activation of MEKK3 signaling in response to osmotic stress or negatively regulates MEKK3 signaling, which is important for normal cardiovascular development. However, the molecular basis governing CCM2-MEKK3 interaction is largely unknown. Here we report the crystal structure of the CCM2 C-terminal part (CCM2ct) containing both the five-helix domain (CCM2ct(s)) and the following C-terminal tail. The end of the C-terminal tail forms an isolated helix, which interacts intramolecularly with CCM2ct(s). By biochemical studies we identified the N-terminal amphiphilic helix of MEKK3 (MEKK3-n(helix)) as the essential structural element for CCM2ct binding. We further determined the crystal structure of CCM2ct(s)-MEKK3-n(helix) complex, in which MEKK3-n(helix) binds to the same site of CCM2ct(s) for CCM2ct intramolecular interaction. These findings build a structural framework for understanding CCM2ct-MEKK3 molecular recognition.
分类: 生物学 >> 生物物理学 >> 神经科学 提交时间: 2016-05-05
摘要: Postoperative cognitive dysfunction (POCD) is an important complication following major surgery and general anesthesia in older patients. However, the etiology of POCD remains largely to be determined. It is unknown how surgical stress and psychological stress affect the postoperative learning and memory function in geriatric patients. We therefore established a pre-clinical model in aged C57BL/6 mice and aimed to investigate the effects of surgical stress and psychological stress on learning and memory function and the possible roles of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway. The surgical stress was induced by abdominal surgery under local anesthesia, and the psychological stress was induced by a communication box. Cognitive functions and markers of the AKT/mTOR pathway were assessed at 1, 3 and 7 days following the stress. The impairments of learning and memory function existed for up to 7 days following surgical stress and surgical stress plus psychological stress, whereas the psychological stress did not affect the cognitive function alone or combined with surgical stress. Analysis of brain tissue revealed a significant involvement of the AKT/mTOR pathway in the impairment of cognition. These data suggested that surgical stress could induce cognitive impairment in aged mice and perioperative psychological stress is not a constitutive factor of POCD. The AKT/mTOR pathway is likely involved as one of the underlying mechanisms of the development of POCD. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-05
摘要: Oxidative stress is a major cause of sporadic Parkinson's disease (PD). Here, we demonstrated that c-Abl plays an important role in oxidative stress-induced neuronal cell death. C-Abl, a nonreceptor tyrosine kinase, was activated in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced acute PD model. Conditional knockout of c-Abl in neurons or treatment of mice with STI571, a c-Abl family kinase inhibitor, reduced the loss of dopaminergic neurons and ameliorated the locomotive defects induced by short-term MPTP treatment. By combining the SILAC (stable isotope labeling with amino acids in cell culture) technique with other biochemical methods, we identified p38 alpha as a major substrate of c-Abl both in vitro and in vivo and c-Abl-mediated phosphorylation is critical for the dimerization of p38 alpha. Furthermore, p38 alpha inhibition mitigated the MPTP-induced loss of dopaminergic neurons. Taken together, these data suggested that c-Abl-p38 alpha signaling may represent a therapeutic target for PD.
分类: 生物学 >> 生物物理学 >> 免疫学 提交时间: 2016-05-05
摘要: Background: Programmed cell death ligand-1 (PD-L1) with its receptor PD-1 pathway is overactivated in many tumors. Inhibiting the interaction of PD-L1 and PD-1 is an attractive strategy to restore tumor-specific T cell immunity for tumor therapy. Methods: A fully human anti-PD-L1 monoclonal antibody (mAb) B60-55 was identified by yeast surface display. The affinity, specificity, activity, and efficacy of mAb B60-55 were investigated in vitro or in vivo. Results: mAb B60-55 (purity >99%) could bind to PD-L1 that is expressed on HEK293 cells with a dissociation constant of 02 nM, and specifically bind to human or cynomolgus macaque PD-L1 without a cross-reaction with murine PD-L1. Moreover, mAb B60-55 is an antagonistic antibody, which can block PD-L1 binding to its receptors, including PD-1 (PDCD1) and B7.1 (CD80). In vitro assays demonstrated the ability of mAb B60-55 to enhance T cell responses and cytokine production in the mixed lymphocyte reaction. In vivo studies showed that administration of mAb B60-55 exhibited a potent antitumor activity toward tumor cell carcinoma xenograft, with a mean half-life of 177.9 h in cynomolgus monkeys. Conclusion: mAb B60-55 is a potential candidate for clinical development in cancer treatment. (C) 2016 Elsevier B.V. All rights reserved.
分类: 生物学 >> 植物学 >> 植物生物化学、植物生物物理学 提交时间: 2016-05-04
摘要: Burley tobaccos (Nicotiana tabacum) display a nitrogen-use-deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco-specific nitrosamines (TSNAs). Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen-assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field-grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well-documented animal carcinogens found in tobacco products.