Selective modulation of the CAP/Cbl pathway in the adipose tissue of high fat diet treated rats

A high-fat diet (HFD) is associated with reduced glucose uptake in muscle, but not in adipose tissue. In the present study, we investigated whether a HFD can modulate glucose uptake in adipose tissue by increasing signal transduction through the CAP/Cbl pathway, independently of the PI3-K/Akt pathway. Our results suggest that, in HFD, the differential regulation of insulin-induced glucose uptake between skeletal muscle and adipose tissue may, in part, be a consequence of the CAP/Cbl/C3G pathway, since the expression of CAP and Cbl, and also the activation of this pathway were increased in adipose tissue but not in muscle.     

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.     

Interactions among challenges of hydromineral balance, angiotensin-converting enzyme, and cystine aminopeptidase

Enzymatic cleavage of some peptides could be included among the mechanisms of water-electrolyte homeostasis. To test this hypothesis, the angiotensin-converting activity (ACE) of plasma and the L-cystine-di-β-naphthylamidase activity (CAP) of plasma and of soluble and particulate fractions from different areas of the central nervous system (CNS) were investigated in rats submitted to treatments eliciting hydromineral imbalance. CAP in the CNS was unchanged by hydromineral challenges. The correlations observed between plasma osmolality and CAP, and plasma CAP and ACE suggested a contribution of these activities to the restoration of basal water-electrolyte and blood pressure conditions through the hydrolysis of vasopressin, oxytocin, angiotensin I and bradykinin.     

Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter

The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

HIV-1 CAP2NC蛋白的表达及体外自组装

构建并表达HIV-1 CAP2NC蛋白,探索其体外自组装条件。通过PCR技术扩增HIV-1(NL4-3毒株)CAP2NC基因片段,并将其连接到原核表达载体pTO-T7,获得重组质粒pTO-T7-CAP2NC,然后转化至大肠杆菌BL21(DE3)菌株,经疏水层析纯化后获得重组蛋白CAP2NC。SDS-PAGE结果表明,重组蛋白CAP2NC可在大肠杆菌可溶高效表达,经纯化后纯度约为95%。ELISA检测表明重组蛋白CAP2NC可被HIV-1衣壳蛋白特异性单克隆抗体识别,具有较好反应活性。重组蛋白透析后在非原性SDS-PAGE中呈现为多种聚体形式。分子筛排阻层析分析CAP2NC蛋白透析后可进行组装,负染电镜进一步观察显示CAP2NC蛋白在RNA存在条件下,可形成空心管状颗粒,其形态结构与HIV-1病毒衣壳体外自组装形成的类似。上述结果表明HIV-1 CAP2NC蛋白具有体外自组装的性质,为进一步在体外研究非成熟病毒样颗粒结构奠定基础。     

Troglitazone activates TRPV1 and causes deacetylation of PPARγ in 3T3-L1 cells

Published research suggests that activation of transient receptor potential vanilloid subfamily 1 (TRPV1) enhances the expression and deacetylation of peroxisome proliferator-activated receptor gamma (PPARγ) to cause browning of white adipose tissue. Here, we show that TRPV1 activation by capsaicin significantly prevents high fat diet-induced obesity in mice. This is associated with an increase in the expression and deacetylation of PPARγ in the epididymal fat of these mice. Consistent with the TRPV1 activation in vivo, overexpression of TRPV1 enhanced the PPARγ and other thermogenic genes in cultured 3T3-L1 preadipocytes. To determine the interaction between TRPV1 and PPARγ signaling, we analyzed the effect of Troglitazone (Trog; a thiazolidinedione derivative and an agonist of PAARγ) treatment on cultured 3T3-L1 cells. Trog enhanced the expression of TRPV1, PPARγ and thermogenic proteins in undifferentiated 3T3-L1 cells but not in differentiated cells. Acute application of Trog stimulated a robust Ca²⁺ influx into 3T3-L1 cells and TRPV1 inhibition by capsazepine prevented this. More interestingly, Trog or capsaicin treatment caused the deacetylation of PPARγ in 3T3-L1 cells and inhibition of TRPV1 or Sirtuin 1 - prevented this. Our data suggest a novel effect of Trog to induce PPARγ deacetylation by activating TRPV1. This research has a significant implication on the role of TRPV1 and PPARγ signaling in the browning of white adipose tissue.     

Antibacterial cathelicidin peptide CAP11 suppresses the anandamide production from lipopolysaccharide-stimulated mononuclear phagocytes

The action of antibacterial cathelicidin peptide CAP11 on the anandamide production from mononuclear phagocytes was examined. Lipopolysaccharide (LPS)-stimulation induced the anandamide production from macrophage-like RAW264.7, accompanied with the enhanced anandamide-synthesizing enzyme activity; however, the anandamide-degrading enzyme activity was not changed by LPS-stimulation. Importantly, CAP11 suppressed the LPS-induced anandamide production and the increase of anandamide-synthesizing enzyme activity. Furthermore, CAP11 abrogated the LPS-binding to CD14-positive RAW264.7. These observations indicate that CAP11 inhibits the binding of LPS to CD14-positive mononuclear phagocytes, thereby suppressing the anandamide synthesizing enzyme activity and the anandamide production from the cells.     

α7烟碱型乙酰胆碱受体在胆碱能抗炎通路中的作用机制及应用现状

胆碱能抗炎通路是一条神经-免疫调节通路,以中枢神经系统的迷走神经为起点,以神经递质乙酰胆碱为基础,以免疫细胞膜表面的α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor, α7 nAChR)为关键作用位点。其中,电信号与化学信号相互转化,激活JAK-STAT3、PI3K-Akt等信号通路,抑制NF-κB的核转位,进而抑制促炎因子的释放,促进抑炎因子的释放,调节和控制机体的炎症,具体的作用机制正在研究中。研究表明,可以通过使用药物激活α7 nAChR和电刺激迷走神经这2种方法,激活胆碱能抗炎通路。其中,激活α7 nAChR具有操作简便、创伤小和效果显著等优势。常用药物有选择性激动剂,例如PNU282987和GTS-21等,非选择性激动剂例如烟碱。多项研究显示,该方法在脓毒症、缺血再灌注、胃肠炎、骨关节炎和自身免疫病等外周器官炎性疾病治疗中都具有一定的效果。α7 nAChR作为胆碱能抗炎通路的关键作用位点,成为许多炎性疾病的潜在治疗靶点,本文对胆碱能抗炎通路中的α7 nAChR所涉及的抗炎机制、通路激活方式,以及其近年来在炎性疾病中的应用进行了综述,为未来研究其具体作用机制和新的治疗靶点提供参考。