The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

MyD88抑制乙型肝炎病毒复制依赖活化NF-κB信号通路

目的 研究髓样细胞分化蛋白(MyD88)抗乙型肝炎病毒(HBV)效应的作用机制。方法 构建MyD88的截短突变体,获得核因子kappa B(NF-κB)超抑制剂IkBa-SR或者NF-κB信号通路激活剂IKKα/IKKβ的表达质粒,分别与HBV复制型质粒瞬时转染Huh7细胞,检测细胞上清液中HBeAg,HBsAg的表达以及胞质中HBV复制中间体DNA的含量,并以NF-κB依赖的荧光素酶报道系统检测它们活化NF-κB的程度。结果 MyD88全长蛋白和2个截短突变体M(1-151)、M(151-296)活化NF-κB的程度与其抑制HBV蛋白以及复制中间体DNA合成的能力相一致。与空载相比,表达NF-κB信号通路激活剂IKKα/IKKβ的质粒共同瞬转细胞后,转染MyD88和HBV表达质粒的细胞中NF-κB的通路明显活化,同时HBV core蛋白的合成显著降低;而NF-κB的超抑制剂IκBα-SR共同瞬转的细胞中core蛋白的表达量显著增加,检测细胞培养上清液中HBeAg和HBsAg及胞质中HBV复制中间体DNA的合成,得到相似结果。结论 NF-κB信号通路的活化在MyD88抑制HBV复制中发挥了关键作用     

赤眼鳟MyD88基因cDNA克隆与表达特性研究

实验使用RACE-PCR技术获得了赤眼鳟(Squaliobarbus curriculus)髓样分化因子88 (Myeloid differentiationfactor 88, MyD88)的cDNA全长, 命名为ScMyD88。ScMyD88的cDNA全长为1779 bp, 其中开放阅读框855 bp, 共编码284个氨基酸残基, 推导的蛋白质分子量为33.053 kD, 理论等电点为5.66。赤眼鳟MyD88具有典型的MyD88结构特征, 包括死亡结构域和TIR结构域(Toll-IL-1 receptor domain, TIR), 其氨基酸序列和鲤科鱼类具有高度保守性, 相似性达到了90%以上, 特别是和武昌鱼相比, 相似性达到了98%。在检测的9个赤眼鳟组织和器官中均有MyD88表达, 其中肝脏、头肾和体肾中表达水平最高, 在脑中表达量最低。在草鱼呼肠弧病毒(Grass carp reovirus, GCRV)感染初期(12h内), MyD88在赤眼鳟免疫组织中表达水平急剧上升, 特别是在脾脏和体肾中尤为明显, 随后恢复正常水平。研究表明, MyD88在赤眼鳟抵抗GCRV入侵的免疫应答反应初期发挥了重要作用。     

Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets

The protective effects of egg-yolk antibodies obtained from hens immunized with fimbrial antigens from a local strain (Escherichia coli K88+ MB, Manitoba, Canada) of K88+ piliated enterotoxigenic E. coli (ETEC) were evaluated in 3- and 21-day-old piglets in which ETEC diarrhea was induced and also in early-weaned piglets in a commercial farm. The results demonstrated that the E. coli K88+ MB-induced diarrhea in 3-day-old piglets was cured 24 h after treating with egg-yolk antibodies while those treated with egg-yolk powder from conventional hens continued to have diarrhea and 62.5% of them died of severe diarrhea. For 21-day-old weaned piglets, those fed egg-yolk antibodies had transient diarrhea, positive body weight gains and 100% survival during the period of the experiment, whereas control piglets that were treated with placebo had severe diarrhea and dehydration and some died within 48 h after infection. In the field trial, the incidence and severity of diarrhea of 14-18-day-old weaned piglets fed egg-yolk antibodies were much lower than in those fed a commercial diet containing an antibiotic. These results indicate that the neonatal and early-weaned piglets that received the egg-yolk antibodies were protected against ETEC infection.     

吉粳88高效再生体系的建立

以吉粳88为材料,通过对不同培养基和激素的筛选,确定最佳的再生体系。结果表明:NMB培养基为吉粳88最适的愈伤组织诱导培养基。通过L₉(3³)正交实验研究最适激素浓度配比,筛选出吉粳88愈伤组织诱导的最佳激素配比为2.0 mg/L 2,4-D+0.2 mg/L 6-BA+0.10 mg/L NAA。在添加2.0 mg/L 6-BA+0.5 mg/L NAA的NMB基本培养基条件下,吉粳88的分化率最高,成培苗率也最高。     

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology^1,2, behavior³, and memory and learning^4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access.     

Potentiation of antibiotic activity by Passiflora cincinnata Mast. front of strains Staphylococcus aureus and Escherichia coli

The development of new drugs from plants is an interesting alternative approach to overcoming microbial resistance. Passiflora cincinnata shows resistance to diseases and pests and a higher concentration of chemical components that may be useful in the pharmaceutical industry. We investigated the potential antimicrobial and antibiotic-modifying activity of hydroalcoholic extracts of leaves, stems, bark, pulp and seeds of P. cincinnata. The extracts were prepared by homogenization of material in 50% ethanol. Minimum inhibitory concentration (MIC) was determined by the broth dilution method, and the bacterial strains tested were Staphylococcus aureus and Escherichia coli. Antibiotic-modifying activity was evaluated against the strains S. aureus 03 and E. coli 08, using a subinhibitory concentration of extract. The antibiotics tested were: amikacin, gentamicin, ampicillin, potassium benzylpenicillin and oxacillin. The extracts did not show antimicrobial activity of clinical relevance, where the MIC was equal to or greater than 1024 μg/mL. S. aureus showed 13 events, while E. coli showed only 4 events. Among these events, 14 involved synergistic activity, potentiating the effect of the antibiotics, and only 3 events demonstrated antagonistic activity toward ampicillin. Hydroalcoholic extracts are potential antimicrobial agents when combined with conventional drugs little utilized in in vivo treatment.     

How Mitochondrial Metabolism Contributes to Macrophage Phenotype and Functions

Metabolic reprogramming of cells from the innate immune system is one of the most noteworthy topics in immunological research nowadays. Upon infection or tissue damage, innate immune cells, such as macrophages, mobilize various immune and metabolic signals to mount a response best suited to eradicate the threat. Current data indicate that both the immune and metabolic responses are closely interconnected. On account of its peculiar position in regulating both of these processes, the mitochondrion has emerged as a critical organelle that orchestrates the coordinated metabolic and immune adaptations in macrophages. Significant effort is now underway to understand how metabolic features of differentiated macrophages regulate their immune specificities with the eventual goal to manipulate cellular metabolism to control immunity. In this review, we highlight some of the recent work that place cellular and mitochondrial metabolism in a central position in the macrophage differentiation program.     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane