Unmasking of LPA1 receptor-mediated migration response to lysophosphatidic acid by interleukin-1β-induced attenuation of Rho signaling pathways in rat astrocytes

Action mechanism of lipopolysaccharide (LPS), interleukin-1β (IL-1β), and lysophosphatidic acid (LPA) to regulate motility, an important process of astrogliosis, was investigated in rat astrocytes. While LPA exerted no significant effect on the cell migration, the prior treatment of the cells with LPS or IL-1β resulted in the appearance of migration activity in response to LPA. The LPS induction of the migration response to LPA was associated with the production of IL-1β precursor protein and inhibited by the IL-1 receptor antagonist. The IL-1β treatment also allowed LPA to activate Rac1. The LPA-induced Rac1 activation and migration were inhibited by pertussis toxin, a small interfering RNA specific to LPA(1) receptors, and LPA(1) receptor antagonists, including Ki16425. However, the IL-1β treatment had no appreciable effect on LPA(1) receptor mRNA expression and LPA-induced activation of ERK, Akt, and proliferation. The induction of the migration response to LPA by IL-1β was inhibited by a constitutively active RhoA. Moreover, LPA significantly activated RhoA through the LPA(1) receptor in the control cells but not in the IL-1β-treated cells. These results suggest that IL-1β inhibits the LPA(1) receptor-mediated Rho signaling through the IL-1 receptor, thereby disclosing the LPA(1) receptor-mediated G(i) protein/Rac/migration pathway.     

TNFα-induced up-regulation of miR-155 inhibits adipogenesis by down-regulating early adipogenic transcription factors

Tumor necrosis factor α (TNFα) is known to inhibit adipogenesis, but the molecular mechanism of this inhibition remains elusive. In the present study, we found that TNFα-induced inhibition of adipogenesis mainly occurs when 3T3-L1 preadipocytes are treated with TNFα within 2 h induction of adipogenesis. We revealed that TNFα treatment results in the up-regulation of miR-155 through the NFκB pathway in 3T3-L1 cells. This overexpression of miR-155 may suppress the expression of C/EBPβ and CREB by directly targeting their 3′ untranslated regions (3′ UTRs). Importantly, anti-miR-155 reduces the TNFα-induced inhibition of adipogenesis, whereas exogenous expression of mir-155 inhibits adipogenesis. Taken together, these findings reveal a novel role for TNFα in the regulation of anti-adipogenic miRNAs.     

Old and new generation lipid mediators in acute inflammation and resolution

Originally regarded as just membrane constituents and energy storing molecules, lipids are now recognised as potent signalling molecules that regulate a multitude of cellular responses via receptor-mediated pathways, including cell growth and death, and inflammation/infection. Derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), each lipid displays unique properties, thus making their role in inflammation distinct from that of other lipids derived from the same PUFA. The diversity of their actions arises because such metabolites are synthesised via discrete enzymatic pathways and because they elicit their response via different receptors. This review will collate the bioactive lipid research to date and summarise the findings in terms of the major pathways involved in their biosynthesis and their role in inflammation and its resolution. It will include lipids derived from AA (prostanoids, leukotrienes, 5-oxo-6,8,11,14-eicosatetraenoic acid, lipoxins and epoxyeicosatrienoic acids), EPA (E-series resolvins), and DHA (D-series resolvins, protectins and maresins).     

Gastrophysa polygoni herbivory on Rumex confertus: single leaf VOC induction and dose dependent herbivore attraction/repellence to individual compounds

We report large induction (>65_fold increases) of volatile organic compounds (VOCs) emitted from a single leaf of the invasive weed mossy sorrel, Rumex confertus Willd. (Polygonaceae), by herbivory of the dock leaf beetle, Gastrophysa polygoni L. (Coleoptera: Chrysomelidae). The R. confertus VOC blend induced by G. polygoni herbivory included two green leaf volatiles ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate) and three terpenes (linalool, ß-caryophyllene, (E)-ß-farnesene). Uninjured leaves produced small constitutive amounts of the GLVs and barely detectable amounts of the terpenes. A Y-tube olfactometer bioassay revealed that both sexes of adult G. polygoni were attracted to (Z)-3-hexenal and (Z)-3-hexen-1-yl acetate at a concentration of 300 ng h⁻¹. No significant G. polygoni attraction or repellence was detected for any VOC at other concentrations (60 and 1500 ng h⁻¹). Yet, G. polygoni males and females were significantly repelled by (or avoided) at the highest test concentration (7500 ng h⁻¹) of both GLVs and (E)-ß-farnesene. Mated male and female G. polygoni might be attracted to injured R. confertus leaves, but might avoid R. confertus when VOC concentrations (especially the terpene (E)-ß-farnesene) suggest high overall plant injury from conspecifics, G. viridula, or high infestations of other herbivores that release (E)-ß-farnesene (e.g., aphids). Tests in the future will need to examine G. polygoni responses to VOCs emitted directly from uninjured (constitutive) and injured (induced) R. confertus, and examine whether R. confertus VOC induction concentrations increase with greater tissue removal on a single leaf and/or the number of leaves with feeding injury.     

The antimicrobial peptide, tilapia hepcidin 2-3, and PMA differentially regulate the protein kinase C isoforms, TNF-α and COX-2, in mouse RAW264.7 macrophages

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, were previously studied. Herein, we report the differential modulation of protein kinase C (PKC)-associated proteins by TH2-3, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), in RAW264.7 macrophages. Treatment with TH2-3 at 40 or 80 μg/ml did not affect the cell morphology, but TH2-3 at 120 μg/ml produced morphological changes similar to those after treatment with PMA in RAW264.7 cells. The coexistence of the PKC inhibitor, Ro-31-8220, prevented morphological changes induced by either PMA or 120 μg/ml TH2-3 in RAW264.7 cells. Since PMA is known to induce expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, activation of the TNF-α promoter in response to TH2-3 and PMA treatments in lipopolysaccharide (LPS)-stimulated cells was compared. In LPS-stimulated RAW264.7 cells, TNF-α promoter activity was significantly suppressed by TH2-3, but not by PMA. In addition, PMA activated prostaglandin synthase-associated cyclooxygenase (COX)-2 proteins on the cell surface, while the presence of TH2-3 inhibited its expression. Western blotting demonstrated that the expressions of PKC-μ, phosphorylated (p)-PKCμ at serine (S)-744, and p-PKCδ were activated by PMA, but were suppressed by TH2-3. In addition, p-PKC at S-916 was activated by TH2-3 and inhibited by PMA. In conclusion, the differential regulation of PKC isoforms by PMA and TH2-3 may influence morphological changes and regulation of TNF-α in RAW264.7 cells.     

4-Aryl-5-carbamoyl-3-isoxazolols as competitive antagonists of insect GABA receptors: Synthesis,biological activity,and molecular docking studies

Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC₅₀ values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC₅₀ values of 2.0 and 0.9?μM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100?μg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action.     

N-cadherin enhances APP dimerization at the extracellular domain and modulates Aβ production

Sequential processing of amyloid precursor protein (APP) by β- and γ-secretase leads to the generation of amyloid-β (Aβ) peptides, which plays a central role in Alzheimer's disease pathogenesis. APP is capable of forming a homodimer through its extracellular domain as well as transmembrane GXXXG motifs. A number of reports have shown that dimerization of APP modulates Aβ production. On the other hand, we have previously reported that N-cadherin-based synaptic contact is tightly linked to Aβ production. In the present report, we investigated the effect of N-cadherin expression on APP dimerization and metabolism. Here, we demonstrate that N-cadherin expression facilitates cis-dimerization of APP. Moreover, N-cadherin expression led to increased production of Aβ as well as soluble APPβ, indicating that β-secretase-mediated cleavage of APP is enhanced. Interestingly, N-cadherin expression affected neither dimerization of C99 nor Aβ production from C99, suggesting that the effect of N-cadherin on APP metabolism is mediated through APP extracellular domain. We confirmed that N-cadherin enhances APP dimerization by a novel luciferase-complementation assay, which could be a platform for drug screening on a high-throughput basis. Taken together, our results suggest that modulation of APP dimerization state could be one of mechanisms, which links synaptic contact and Aβ production.     

Phosphorylation of Serine 1137/1138 of Mouse Insulin Receptor Substrate (IRS) 2 Regulates cAMP-dependent Binding to 14-3-3 Proteins and IRS2 Protein Degradation

Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R_p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N⁶-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.     

三角帆蚌Hc-GSK3β基因鉴定及内壳色性状相关SNP筛选

为了探究三角帆蚌(Hyriopsis cumingii)糖原合成激酶-3β(GSK3β)基因对壳色的影响,研究采用RACE技术获得Hc-GSK3β基因cDNA全长1867 bp,其中包含1261 bp的ORF区编码420个氨基酸, ORF中含有一个S＿TKc结构域,该结构域序列高度保守。组织差异表达分析发现Hc-GSK3β基因在紫色蚌鳃、斧足、内脏团和边缘膜组织中表达量高于白色蚌的表达量(P<0.05),且在斧足和边缘膜表达差异水平达到极显著(P<0.01),而在紫色蚌闭壳肌组织中表达量显著低于白色蚌(P<0.05)。原位杂交(ISH)实验结果显示在三角帆蚌外套膜的外褶、中褶、內褶、背膜区和腹膜区均有阳性信号产生,且在外褶的信号表达较强烈。该基因经重测序比较,共鉴定出6个SNP位点,其中在C+185A位点的CA基因型在紫色蚌的分布频率显著高于白色三角帆蚌(P<0.05);在紫色蚌中, T+341G位点TT基因型三角帆蚌内壳颜色参数b值显著低于TG基因型(P<0.05)。研究表明, Hc-GSK3β基因参与了三角帆蚌壳色形成,筛选的SNP标记可用于三角帆蚌壳...