一类灰色项时间序列模型及其应用

利用灰色系统理论与时间序列分析,提出了带灰色项的时间序列模型,对这类模型进行了分析,给出了建模与预报方法,并将其应用于我国农业产值问题的预报与研究之中,模型的正确性得到了检验．     

DARPin-Assisted Crystallography of the CC2-LZ Domain of NEMO Reveals a Coupling between Dimerization and Ubiquitin Binding

NEMO is an integral part of the IκB kinase complex and serves as a molecular switch by which the NF-κB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding site affect IκB kinase activation in the NF-κB signaling pathway.     

人MCM7 N-端融合蛋白的原核表达、纯化及其与AR蛋白相互作用的研究

目的：制备人MCM7 N-端的GST融合蛋白（GST-MCM7N）,研究其是否和雄激素受体（AR）蛋白存在直接的相互作用。方法：利用RT-PCR获得长度为744 bp的人mcm7 基因N-端cDNA碱基片段,把该片段构建到原核表达载体pGEX-5x-3中,转化大肠杆菌BL-21菌株。经IPTG诱导菌株基因表达产生了GST-MCM7N融合蛋白;使用Glutathione Sepharose 4B球珠分离纯化。利用GST pull-down 技术把纯化的融合蛋白分别和前列腺癌细胞LNCaP细胞裂解液及基因工程获得的AR蛋白孵育,检测MCM7蛋白的 N-端是否和AR蛋白直接相互作用。结果：酶切鉴定及基因测序表明,mcm7 基因cDNA的 N-端片段被构建到表达载体pGEX-5x-3中;SDS-PAGE及Western blotting结果分别显示,本研究获得了GST和GST-MCM7N融合蛋白;GST pull-down 结果证实GST-MCM7N和AR蛋白存在相互作用。结论：MCM7蛋白的N-端和AR蛋白至少在体外可以发生直接的相互作用。     

Paradoxical nature of estrogen agonist and antagonist binding in rat liver

Whereas Tamoxifen exerts potent antiestrogenic action in ER dependent breast cancer, it was largely without effect on rat liver gluconeogenesis which could be dramatically diminished by estrogens and androgens. Although estradiol was preferentially bound to an ER₄ component that coeluted with CBG from DE-52 columns, ³H-tamoxifen labelled the ER₃ moiety that was clearly distinct from transcortin. Similarly, testosterone was bound to the AR₄ entity but R-1881 was eluted in the AR₃ region. All these ER and AR populations were furthermore distinct from liver GR. These, for the first time, demonstrate polymorphic nature of AR and ER and suggest that agonist and antagonist actions may be expressed via separate populations of the receptor, contrary to the established, classical view that dictates competitive antagonism between them for the one and the same site.     

5-HT(1A) receptor mutant mice exhibit enhanced tonic, stress-induced and fluoxetine-induced serotonergic neurotransmission

Mutant mice that lack serotonin(1A) receptors exhibit enhanced anxiety-related behaviors, a phenotype that is hypothesized to result from impaired autoinhibitory control of midbrain serotonergic neuronal firing. Here we examined the impact of serotonin(1A) receptor deletion on forebrain serotonin neurotransmission using in vivo microdialysis in the frontal cortex and ventral hippocampus of serotonin(1A) receptor mutant and wild-type mice. Baseline dialysate serotonin levels were significantly elevated in mutant animals as compared with wild-types both in frontal cortex (mutant = 0.44 +/- 0.05 n M; wild-type = 0.28 +/- 0.03 n M) and hippocampus (mutant = 0.46 +/- 0.07 n M; wild-type = 0.27 +/- 0.04 n M). A stressor known to elicit enhanced anxiety-like behaviors in serotonin(1A) receptor mutants increased dialysate 5-HT levels in the frontal cortex of mutant mice by 144% while producing no alteration in cortical 5-HT in wild-type mice. There was no phenotypic difference in the effect of this stressor on serotonin levels in the hippocampus. Fluoxetine produced significantly greater increases in dialysate 5-HT content in serotonin(1A) receptor mutants as compared with wild-types, with two- and three-fold greater responses being observed in the hippocampus and frontal cortex, respectively. This phenotypic effect was mimicked in wild-types by pretreatment with the serotonin(1A) antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide (p-MPPI). These results indicate that deletion of central serotonin(1A) receptors results in a tonic disinhibition of central serotonin neurotransmission, with a greater dysregulation of serotonin release in the frontal cortex than ventral hippocampus under conditions of stress or increased interstitial serotonin levels.     

Androgen receptor transcriptional activity and chromatin modifications on the ABCB1/MDR gene are critical for taxol resistance in ovarian cancer cells

We report here that the androgen receptor (AR) and ABCB1 are upregulated in a model of acquired taxol resistance (txr) in ovarian carcinoma cells. AR silencing sensitizes txr cells to taxol threefold, whereas ectopic AR expression in AR-null HEK293 cells induces resistance to taxol by 1.7-fold. AR activation using the agonist dihydrotestosterone (DHT) or sublethal taxol treatment upregulates ABCB1 expression in both txr cells and AR-expressing HEK293 cells. In contrast, AR inactivation using the antagonist bicalutamide downregulates ABCB1 expression and enhances cytotoxicity to taxol. A functional ABCB1 promoter containing five predicted androgen-response elements (AREs) is cloned. Deletion assays reveal a taxol-responsive promoter segment which harbors ARE4. Notably, DHT- or taxol-activated AR potentiates binding of the AR to ARE4 as revealed by the chromatin immunoprecipitation. On the other hand, txr cells display an increase in chromatin remodeling. AR/H3K9ac and AR/H3K14ac complexes bind specifically to ARE4 in response to taxol. Furthermore, acetyltransferase protein levels (p300 and GCN5) are upregulated in txr cells. Silencing of p300 or GCN5 reduces chromatin modification and enhances cytotoxicity in both parental and txr SKOV3 cells. While the phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (AKT) pathway is significantly activated by taxol, taxol-induced ABCB1 expression, histone posttranslational modifications, and p300 binding to ARE4 are suppressed following inhibition of the PI3K/AKT cellular pathway. These results demonstrate that the AKT/p300/AR axis can be activated to target ABCB1 gene expression in response to taxol, thus revealing a new treatment target to counter taxol resistance.     

Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract

The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltrasferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.     

let‐7e replacement yields potent anti‐arrhythmic efficacy via targeting beta 1‐adrenergic receptor in rat heart

Beta‐adrenoceptor (β‐AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let‐7e in the up‐regulation of β₁‐AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. β₁‐AR expression was significantly up‐regulated and let‐7a, c, d, e and i were markedly down‐regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let‐7e suppressed β₁‐AR expression at the protein level, without affecting β₁‐AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let‐7e by let‐7e antisense inhibitor (AMO‐let‐7e) enhanced β₁‐AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let‐7e (len‐pre‐let‐7e) significantly inhibited β₁‐AR expression in rats, whereas len‐AMO‐let‐7e up‐regulated β₁‐AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β₁‐AR by endogenous let‐7e. Len‐negative control (len‐NC) did not produce significant influence on β₁‐AR expression. Len‐pre‐let‐7e also profoundly reduced the up‐regulation of β₁‐AR induced by AMI and this effect was abolished by len‐AMO‐let‐7e. Importantly, len‐pre‐let‐7e application significantly reduced arrhythmia incidence after AMI in rats and its anti‐arrhythmic effect was cancelled by len‐AMO‐let‐7e. Notably, anti‐arrhythmic efficacy of len‐pre‐let‐7e was similar to propranolol, a non‐selective β‐AR blocker and metoprolol, a selective β₁‐AR blocker. Down‐regulation of let‐7e contributes to the adverse increase in β₁‐AR expression in AMI and let‐7e supplement may be a new therapeutic approach for preventing adverse β₁‐AR up‐regulation and treating AMI‐induced arrhythmia.     

Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins

Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how post-translational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.