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1.
Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP.  相似文献   
2.
Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction.  相似文献   
3.
ObjectiveTo study the protective effect of total flavonoid in rabdosia rubescens on BIT model by brain ischemic tolerance (hereinafter BIT) model of mice.MethodBIT model is used to block bilateral common carotid arteries and to copy BIT model of mice. After 10 min of transient ischemia for rats in preconditioning group, the mice in the nimodipine group and naoluotong capsule group were given the total flavonoid in rabdosia rubescens (300 mg/kg, 150 mg/kg, 75 mg/kg) for gavage, sham operation group, ischemia/reperfusion injury (hereinafter IRI) group and BIT group were fed with the same volume of 0.5% sodium carboxymethyl cellulose (CMC) once a day for 5 days. After administration for 1 h on day 5 (120 h), the rats in the other groups except for the sham operation group were treated with blood flow block for 30 min and reperfusion for 22 h. The serum NSE level were measured and the brain NO content and NOS activity changes was measured to observe the histopathological changes of brain tissue.ResultsBIT models of mice and in rats were both successfully replicated. The total flavonoid in rabdosia rubescens can decrease the mortality of mice, decrease serum NSE level, increase the content of NO and the activity of NOS in the brain tissue of mice, and improve the pathological damage of cortex and hippocampus of mice.ConclusionThe total flavonoid in rabdosia rubescens can stimulate an endogenous protective mechanism by inducing the release of low levels of cytokines NO and NOS, which reduces the release of serum NSE, relieves the brain tissue ischemia-reperfusion injury, and further improves the protection effect of ischemic preconditioning on brain injury. The damage of brain tissue ischemia and reperfusion, and further improve the ischemia Protective effect of preconditioning on brain injury.  相似文献   
4.
To increase the menaquinone (MK) content of an Elizabethkingia meningoseptica, site-directed mutagenesis was generated to suppress 4-hydroxybenzoate octaprenyl transferase (UbiA) activity and subsequently blocked the ubiquinone (UQ) biosynthesis pathway. Fourteen conserved residues except L174 and G211 were mutated to analyze the effect of site-directed mutagenesis. The expression of UbiA in twelve mutants was decreased in both mRNA and protein levels, which resulted in the decrease of UQ concentration. Based on MenA expression level, 12 mutants were divided into two groups. Second group such as N72A, D76A, K81A, L139A, and D198A enhanced the expression of MenA, which increased MK production by 127.1%, 87.9%, 96.2%, 109.7% and 130.0% in wt-EmUbiA, respectively. In general, blocking UQ synthesis pathway for by site-directed mutagenesis of the active site of UbiA in E. meningoseptica was a promising strategy to increase MK production in E. meningoseptica.  相似文献   
5.
Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.  相似文献   
6.
High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.  相似文献   
7.
Natural killer (NK) cells are a sub-population of cytotoxic lymphocytes that can kill tumor or infected cells without prior exposure, by secreting the contents of preformed cytotoxic vesicles, containing perforin and granzymes, at the immune synapse. Cytohesin-associated scaffolding protein (CASP) is an adaptor molecule uniquely expressed in lymphocytes that forms complexes with both vesicle-initiating and sorting proteins, and has roles in NK cell migration, cytotoxicity, and cytokine secretion. In this study, we show that CASP contains a conserved granzyme B cleavage site that could modify its intracellular localization and interaction with sorting nexin 27. We also provide evidence that CASP is modified by ubiquitination. Both of these post-translational modifications could rapidly modify CASP function and highlight the dynamic regulatory mechanisms that direct its role in NK cell functions.  相似文献   
8.
Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.  相似文献   
9.
Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4+ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.  相似文献   
10.
Mutations in CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early-onset neurodegenerative disorder that is characterized by the accumulation of ceroid lipofuscin within lysosomes. The function of the CLN3 protein remains unclear and is presumed to be related to Endoplasmic reticulum (ER) stress. To investigate the function of CLN3 in the ER stress signaling pathway, we measured proliferation and apoptosis in cells transfected with normal and mutant CLN3 after treatment with the ER stress inducer tunicamycin (TM). We found that overexpression of CLN3 was sufficient in conferring increased resistance to ER stress. Wild-type CLN3 protected cells from TM-induced apoptosis and increased cell proliferation. Overexpression of wild-type CLN3 enhanced expression of the ER chaperone protein, glucose-regulated protein 78 (GRP78), and reduced expression of the proapoptotic protein CCAAT/-enhancer-binding protein homologous protein (CHOP). In contrast, overexpression of mutant CLN3 or siRNA knockdown of CLN3 produced the opposite effect. Together, our data suggest that the lack of CLN3 function in cells leads to a failure of management in the response to ER stress and this may be the key deficit in JNCL that causes neuronal degeneration.  相似文献   
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