Developmental expression and evolution of hexamerin and haemocyanin from Folsomia candida (Collembola)

Haemocyanins constitute a group of copper‐containing respiratory proteins, and hexamerins were derived from hexapod haemocyanin but lost the ability to transport oxygen and serve as storage proteins. Although hexamerins have been reported in most insect species, none of them has been identified in Collembola, one of the most primitive hexapod lineages, thereby preventing us from exploring relevant evolutionary scenarios regarding the origin and evolution of hexamerins in hexapods. Here we report on collembolan hexamerins for the first time, and investigated the temporal expression profiles of hexamerin and haemocyanin in the collembolan Folsomia candida. Haemocyanin was expressed over the entire life cycle, with higher expression at the embryonic stage than at other stages, whereas hexamerin expression was restricted to embryos, unlike insect hexamerins, which are generally expressed from larval to adult stages. A phylogenetic analysis and molecular clock estimation suggested that all investigated hexapod hexamerins have a single and ancient origin (~423 Ma), coincident with the rise of atmospheric oxygen levels in the Silurian–Devonian period, indicating a physiological link between molecular evolution and Palaeozoic oxygen changes.     

Mosquito hexamerins: characterization during larval development

We report here the first examination of hexamerins expressed during mosquito larval development. Haemolymph proteins from fourth-instar larvae of six species representing the two major subfamilies of mosquitoes were characterized by immunoblotting using antisera to calliphorin, the major hexamerin of the blowfly, Calliphora vicina , or to LSP1 or LSP2, the two distinct hexamerins of Drosophila melanogaster . In each mosquito species the antisera demonstrated the presence of multiple abundant hexamerin polypeptides of 66–85 kDa in molecular weight. According to the subunit composition of native proteins, the larval hexamerins from both Aedes aegypti and Anopheles gambiae form heterohexamers. Furthermore, the two major Aedes hexamerin subunits (AaHex1 and AaHex2) are neither rich in aromatic amino acids nor methionine. cDNA clones encoding AaHex1 and AaHex2 were isolated and used to show that hexamerin mRNA is uniquely expressed in fourth-instar larvae of both A. aegypti and A. gambiae and disappears rapidly at the onset of pupal development.     

A putative hexamerin from a Campodea sp. suggests an independent origin of haemocyanin-related storage proteins in Hexapoda

Haemocyanins are copper-containing respiratory proteins in the arthropod haemolymph. In hexapods, haemocyanins gave rise to hexamerins, which have lost the ability to bind copper and thus oxygen. Hexamerins are thought to act mainly as storage proteins in nonfeeding periods. So far, hexamerins have only been identified in ectognathan hexapods, but not in Entognatha. Here we report the identification of a putative hexamerin from Campodea sp. (Diplura). The full-length cDNA of Campodea sp. hexamerin 1 (CspHex1) measures 2188 bp and translates into a native polypeptide of 667 amino acids. As in other hexamerins, the six copper-coordinating histidines are not conserved. However, sequence comparison and phylogenetic analyses demonstrated that CspHex1 is not closely related to other hexapod hexamerins, which derive from hexapod type 1 haemocyanin subunits in the ectognathan lineage, but rather resembles a derivative of hexapod type 2 haemocyanin subunits. Hence, haemocyanin-related storage proteins emerged at least two times independently in Hexapoda.     

Sequence and evolution of a hexamerin from the ant Camponotus festinatus

In the ant Camponotus festinatus, two different hexamerins accumulate stage-specifically during the late larval period and at various times in adults. These hexamerins serve as storage proteins and play important roles in brood nourishment and colony founding. We report an analysis of the cDNA sequence of C. festinatus hexamerin 2 (CfeHex2). The native protein contains 732 amino acids, which are moderately enriched in aromatic amino acids, aspartate and asparagine. Phylogenetic analyses show a close relationship of CfeHex2 to a putative toxin of the braconid wasp, Bracon hebetor. The divergence of Formicidae and Braconidae hexamerins was calculated to have begun 187 MYA, an estimate consistent with currently accepted phylogeny of insect orders.     

PPAR-γ在多囊卵巢综合征大鼠卵巢组织中的表达及意义

目的:探讨过氧化物酶增殖物激活受体-γ(PPAR-γ)在多囊卵巢综合征(PCOS)大鼠卵巢的表达与其性激素变化及胰岛素抵抗(IR)的关系。方法:应用脱氢表雄酮(DHEA)皮下注射23日龄SD雌性大鼠20d,观察卵巢巢重量及光镜(HE染色)形态学改变,应用ELISA(酶联免疫法)测定T、E2、LH、FSH及血清胰岛素、糖耐量试验,应用荧光定时定量聚合酶链反应(real-timePCR)检测PPAR-γmRNA的表达。结果:实验组卵巢重量显著高于对照组(P<0.05),实验组卵巢呈多囊样改变而黄体形体比例减少;实验组血清T、E2、FSH、空腹血糖水平显著高于对照组(P<0.05),空腹胰岛素及胰岛素抵抗指数(HOMA-IR)显著高于对照(P<0.05);在PCOS大鼠卵巢中PPAR-γ相对含量显著高于对照组(P<0.05)。结论:DHEA诱导的PCOS大鼠动物模型与PCOS患者相似,PPAR-γ在PCOS大鼠卵巢发育中起部分调节作用,并与PCOS的IR密切相关。     

Detection and localization of cytokine immunoreactivity in retro-ocular connective tissue in Graves' opthalmopathy

Abstract. Paracrine interactions between fibroblasts residing in the retro-ocular space and infiltrating lymphocytes/macrophages are thought to be of central importance in the pathogenesis of Graves' opthalmopathy (GO). Although various roles have been suggested for interferon-γ (IFNγ), tumour necrosis factor-αTNFα) and interleukin-lα (IL-lα) in GO, their actual presence in Graves' retro-ocular connective tissue has not been demonstrated. We examined surgical specimens obtained during orbital decompression from patients with severe GO (n= 6), and from normal individuals (n= 5), for the presence of IFNγ, TNFα and IL-1α. We used immunohistochemical methods on frozen tissue sections and primary fibroblast cultures, and sodium dodecylsulfate polyacryl-amide-gel electrophoresis of tissue extracts and tissue culture supernatants. In addition, immunohistochemical staining of tissues for characterization of the mononuclear cell infiltrates was performed. Aggregates of mononuclear cells in retro-ocular connective and fatty tissue were found in five of six GO tissue specimens, but in none of the control specimens. We detected immunoreactivity for the three cytokines (IFNγ, TNFα and IL-lα) in the five GO tissue specimens that contained mononuclear cell aggregates. In addition, IL-la immunoreactivity was demonstrable in primary and subsequent GO fibroblast cultures and in their supernatants. In contrast, no immunoreactivity for any of these cytokines was detected in tissue specimens, primary cultures or culture supernatants derived from normal individuals. The presence of mononuclear cell infiltrates and associated immunoreactivity for IFNγ, TNFα, IL-1α in retro-ocular connective tissue derived from patients with GO suggests that the previously demonstrated in vitro functions of these cytokines may indeed be operative in vivo. Our results further support the concept that IFNγ, TNFα and IL-lα may play a role in the pathogenesis of GO.     

Anti-atherosclerotic potential of baicalin mediated by promoting cholesterol efflux from macrophages via the PPARγ-LXRα-ABCA1/ABCG1 pathway

BackgroundAtherosclerosis (AS) is associated with severe cardiovascular disease. The anti-inflammatory, anti-oxidation, and lipid regulating properties of baicalin suggest potential as an anti-atherosclerotic agent. We therefore investigated whether baicalin can protect against the development of atherosclerosis in an AS rabbit model and explored the underling mechanisms in THP-1 macrophages.Methods and resultsIn vivo, treatment with baicalin markedly decreased atherosclerotic lesion sizes and lipid accumulation in AS rabbit carotid arteries. Western blotting revealed that the protein expression levels of both peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha (LXRα) were up-regulated in the baicalin group compared with the model group. In vitro, baicalin restricted oxidized-low density lipoprotein (ox-LDL)-induced intracellular lipid accumulation and foam cell formation in THP-1 macrophages. Molecular data showed that baicalin significantly increased the expression levels of PPARγ, LXRα, ATP binding cassette transporters (ABC) A1 and ABCG1. Cell transfection experiments (including PPARγ and LXRα siRNAs) suggested that the effects of baicalin are mediated by the PPARγ-LXRα signalling pathway, which stimulates the expression of ABCA1 and ABCG1.ConclusionThese results suggest that baicalin potentially exerts anti-atherosclerosis effects, possibly through the PPARγ-LXRα-ABCA1/ABCG1 pathway, by promoting efflux of cholesterol from macrophages and delaying the formation of foam cells.     

Plasma cytokine levels and risks of abdominal aortic aneurysms: A population-based prospective cohort study

Abstract

Background. Abdominal aortic aneurysm (AAA) is characterized by inflammatory cell accumulation in AAA lesions that produce inflammatory cytokines and advance its pathogenesis. Peripheral cytokines may predict the degree or risk of AAA.

Methods and results. ELISA determined plasma interleukin-6 (IL6), IL10, IL17A, IFN-γ, and C-reactive protein (CRP) from 476 AAA patients and 200 controls. AAA patients had lower IL6, IFN-γ, IL10, IL17A, and higher CRP than controls. IL10 correlated positively with IFN-γ, IL17A, or IL6, but not CRP in control or AAA populations. IL10 associated negatively with systolic blood pressure, whereas CRP associated positively with diastolic blood pressure and body mass index. CRP was an independent AAA risk factor and correlated positively with aortic diameters before and after adjustments for other risk factors. IFN-γ, IL17A, and CRP correlated positively with cross-sectional AAA area after adjustment. IL10 correlated positively with AAA growth rate before and after adjustment. The risk of death doubled in AAA patients with CRP levels above the median.

Conclusions. Reduced IFN-γ, IL10, and IL17A in AAA patients, positive correlations of IFN-γ and IL17A with cross-sectional AAA area, IL10 with AAA growth rate, and IL10 with IFN-γ and IL17A suggest combined Th1, Th2, and Th17 immune responses in human AAAs.     

Integrin α2β1 induces phosphorylation-dependent and phosphorylation-independent activation of phospholipase Cγ2 in platelets: role of Src kinase and Rac GTPase

Summary. Background: Platelet adhesion promoted by integrin  α₂β₁ induces integrin  α_IIbβ₃ activation through the phospholipase C (PLC)‐dependent stimulation of the small GTPase Rap1b. Objective: To analyze the mechanism of PLC activation downstream of α₂β₁ that is required for regulation of Rap1b and α_IIbβ₃. Methods: Human and murine platelets were allowed to adhere to immobilized type I monomeric collagen through α₂β₁. Tyrosine phosphorylation of PLCγ2, PLC activation, accumulation of GTP‐bound Rap1b and fibrinogen binding were measured and compared. Results: Integrin  α₂β₁ recruitment induced an evident PLC activation that was concomitant with robust tyrosine phosphorylation of PLCγ2, and was suppressed in platelets from PLCγ2‐knockout mice. Moreover, PLCγ2^?/? platelets were unable to accumulate active Rap1b and to activate α_IIbβ₃ upon adhesion through α₂β₁. Inhibition of Src kinases completely prevented tyrosine phosphorylation of PLCγ2 in adherent platelets, but did not affect its activation, and both Rap1b and α_IIbβ₃ stimulation occurred normally. Importantly, α_IIbβ₃‐induced phosphorylation and activation of PLCγ2, as well as accumulation of active Rap1b, were totally suppressed by Src inhibition. Integrin  α₂β₁ recruitment triggered the Src kinase‐independent activation of the small GTPase Rac1, and activation of Rac1 was not required for PLCγ2 phosphorylation. However, when phosphorylation of PLCγ2 was blocked by the Src kinase inhibitor PP2, prevention of Rac1 activation significantly reduced PLCγ2 activation, GTP‐Rap1b accumulation, and α_IIbβ₃ stimulation. Conclusions: Src kinases and the Rac GTPases mediate independent pathways for PLCγ2 activation downstream of α₂β₁.     

结核性脑膜炎患者脑脊液中细胞因子水平的诊断价值

目的:探讨分泌性靶抗原-6(ESAT-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)和干扰素-γ(IFN-γ)对于早期诊断结核性脑膜炎(TBM)的临床应用价值。方法:选取48例TBM患者为研究组,29例非结核性中枢神经系统感染患者为对照组,35例普通头痛患者为正常组。采用ELISA法分别检测所有患者脑脊液中的ESAT-6、IL-10、TNF-α和IFN-γ水平。结果:研究组的ESAT-6、TNF-α、IFN-γ水平与对照组相比增高,研究组与对照组的ESAT-6、IL-10、TNF-α、IFN-γ水平均高于正常组,差异均有统计学意义(P<0.05)。研究组的ESAT-6与IFN-γ水平呈正相关(r=0.96,P<0.05)。4项指标联合检测的灵敏度明显高于使用ESAT-6、IL-10、TNF-α、IFN-γ单一指标,差异均具有统计学意义(χ2=10.936,18.104,12.647,13.978;P<0.05)。结论:ESAT-6、TNF-α、IFN-γ和IL-10在TBM患者脑脊液中的表达水平显著升高,对早期诊断TBM具有重要的临床价值,而且4项联合检测较单一检测的诊断效果更佳。     

Association between biosynthesis of nitric oxide and changes in immunological and vascular parameters in patients treated with interleukin-2

Abstract Hypotension is a dose-limiting side effect of interleukin-2 (IL-2) therapy. This may be due to increased biosynthesis of the potent vasodilator nitric oxide (NO) induced by cytokines such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), which are known to be generated during IL-2 therapy. We describe the relationship between NO biosynthesis and changes in immunological and vascular parameters during IL-2 therapy in 13 patients with metastatic cancer. Plasma concentrations of neopterin and nitrite plus nitrate (NO_x) were higher in cancer patients prior to treatment compared with normal subjects (neopterin; 10·8±1·4 vs. 2·0±0·4 ng ml^-1, P<0·001: NO_x; 45±6 vs. 28±2 μM, P<0·005). Pretreatment TNF-α and IFN-γ plasma concentrations were not significantly different in cancer patients from those in controls. During infusion of IL-2 (18 times 10⁶ international units m^-2 per day for 5 days) these parameters increased, reaching maximal concentrations at day 3 for IFN-γ and day 5 for TNF-α, neopterin and NO_x. The maximal induced NO_x correlated with maximal TNF-α (r = 0·60, P<0·04), IFN-γ (r = 0·63, P<0·02) and neopterin (r = 0·66, P<0·01). As plasma NO_x concentrations increased, systolic blood pressure fell, reaching a minimum at day 3 despite a continued rise in NO_x concentrations. These changes were accompanied by a continuous increase in pulse rate throughout the infusion period. These findings indicate that induction of NO biosynthesis contributes to hypotension induced during IL-2 therapy. Inhibitors of NO synthase may be useful in limiting toxicity, thus allowing administration of higher and possibly more efficacious doses of this cytokine in the treatment of cancer.     

Priming of neutrophils with tumor necrosis factor-α measured as Fcγ receptor-mediated respiratory burst correlates with increased complement receptor 3 membrane density

Hyperactive or primed neutrophils which damage tissue via cytokines and membrane receptors may be implicated in the pathogenesis of inflammatory conditions. The purpose of this study was to elucidate the priming mechanism in neutrophils by assessing changes in membrane receptors and the FcγR-mediated respiratory burst, measured as chemiluminescence. Purified neutrophilic granulocytes from healthy volunteers were preincubated with recombinant human tumor necrosis factor-α. This had a priming effect, inqreasing both the Fcγ receptor-mediated luminol-enhanced chemiluminescence and the membrane expression of the C3bi receptor (CRB) (r=0.843). The membrane densities of FcγRII, FcγRIII, and CR1 were unaffected by tumor necrosis factor-α. The mechanism of increased chemiluminescence may involve redistribution of the Fcγ receptors and cooperation with upregulated CR3, facilitating crosslinking of the receptors. The experiments were performed in a buffer without divalent cations, since these increased the background activity and abolished the priming effect of tumor necrosis factor-α. In conclusion, a simultaneous increase in the FcγR-mediated respiratory burst and CR3 density after priming with tumor necrosis factor-α indicates a cooperation between FcγR and CR3.     

结核病合并慢性乙肝患者TNF—α、IFN-γ、IL-12变化及意义

【目的】研究结核病合并慢性乙肝患者血清肿瘤坏死因子-α（TNF-α）、γ干扰素（IFN-γ）及白细胞介素-12（IL-12）水平的变化及意义。【方法】肺结核并HBsAg阳性患者21例（A组）,单纯肺结核患者30例（B组）,健康对照组30例（C组）。应用酶联免疫吸附法（ELISA）测定三组血清中TNF-α、IFN-γ、IL-12浓度水平。【结果】A组和B组TNF-α水平均高于C组（P〈0．01）;但A组T组和B组之间TNF-α水平差异无显著性（P〉0．05）;B组IFN-γ、IL-12水平均低于C组（P〈0．05）;而A组IFN-γ、IL-12水平均低于B组（P〈0．05）。【结论】在结核病合并慢性乙肝患者的体内,可能存在着显著的Th1细胞免疫应答减弱。     

ITAM receptor‐mediated generation of reactive oxygen species in human platelets occurs via Syk‐dependent and Syk‐independent pathways

Background: Ligation of the platelet‐specific collagen receptor, GPVI/FcRγ, causes rapid, transient disulfide‐dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4. Objectives: The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcRγ or a second immunoreceptor tyrosine‐based activation motif (ITAM)‐containing receptor on platelets, FcγRIIa. Methods and Results: Using an H₂DCF‐DA‐based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen‐related peptide (CRP) or convulxin (Cvx), or the FcγRIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet‐rich plasma from 14 healthy donors displayed little inter‐individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15–20 min. The Syk inhibitor BAY61‐3606, which blocks ITAM‐dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase. Conclusions: Together, these results show for the first time that ROS generation downstream of GPVI or FcγRIIa consists of two distinct phases: an initial Syk‐independent burst followed by additional Syk‐dependent generation.     

Gqα‐linked phospholipase Cβ1 and phospholipase Cγ are essential components of the pheromone biosynthesis activating neuropeptide (PBAN) signal transduction cascade

Sex pheromone production for most moths is regulated by pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori, PBAN binding triggers the opening of store‐operated Ca²⁺ channels, suggesting the involvement of a receptor‐activated phospholipase C (PLC). In this study, we found that PLC inhibitors U73122 and compound 48/80 reduced sex pheromone production and that intracellular levels of ³H‐inositol phosphate species increased following PBAN stimulation. In addition, we amplified cDNAs from pheromone glands corresponding to PLCβ1, PLCβ4, PLCγ and two G protein α subunits, Go and Gq. In vivo RNA interference‐mediated knockdown analyses revealed that BmPLCβ1, BmGq1, and unexpectedly, BmPLCγ, are part of the PBAN signal transduction cascade.