Human autoantibodies to diacyl-phosphatidylethanolamine recognize a specific set of discrete cytoplasmic domains

The aim of this study was to characterize a novel human autoantibody-autoantigen system represented as cytoplasmic discrete speckles (CDS) in indirect immunofluorescence (IIF). A distinct CDS IIF pattern represented by 3-20 discrete speckles dispersed throughout the cytoplasm was identified among other cytoplasmic speckled IIF patterns. The cytoplasmic domains labelled by human anti-CDS-1 antibodies did not co-localize with endosome/lysosome markers EEA1 and LAMP-2, but showed partial co-localization with glycine-tryptophan bodies (GWB). CDS-1 sera did not react with several cellular extracts in immunoblotting and did not immunoprecipitate recombinant GW182 or EEA1 proteins. The typical CDS-1 IIF labelling pattern was abolished after delipidation of HEp-2 cells. Moreover, CDS-1 sera reacted strongly with a lipid component co-migrating with phosphatidylethanolamine (PE) in high performance thin-layer chromatography (HPTLC)-immunostaining of HEp-2 cell total lipid extracts. The CDS-1 major molecular targets were established by electrospray ionization-mass spectrometry (ESI-MS), HPTLC-immunostaining and chemiluminescent enzyme-linked immunosorbent assay as diacyl-PE species, containing preferentially a cis-C18 : 1 fatty acid chain at C-2 of the glycerol moiety, namely 1,2-cis-C18 : 1-PE and 1-C16 : 0-2-cis-C18 : 1-PE. The clinical association of CDS-1 sera included a variety of systemic and organ-specific autoimmune diseases but they were also observed in patients with no evidence of autoimmune disease.     

Endogenous ligands of PPAR-gamma reduce the liver injury in haemorrhagic shock

We demonstrate here for the first time that the novel, potent peroxisome proliferator-activated receptor (PPAR)-gamma antagonist GW9662 (2-chloro-5-nitrobenzanilide) augments the degree of liver injury associated with haemorrhagic (haemorrhage for 90 min and resuscitation for 4 h), but not endotoxic (6 mg/kg E. coli endotoxin i.v. for 6 h) shock in the anaesthetised rat. Thus, endogenous ligands for PPAR-gamma are released in haemorrhagic, but not endotoxic, shock in sufficient amounts to protect against injury.     

Cloning, tissue expression, and regulation of beagle dog CYP4A genes.

In addition to its function as a fatty acid hydroxylase, the peroxisome proliferator-activated receptor alpha (PPARalpha) target gene, CYP4A, has been shown to be important in the conversion of arachidonic acid to the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid, suggesting a role for this enzyme in mediating vascular tone. In the present study, the cDNA sequence of beagle dog CYP4A37, CYP4A38, and CYP4A39 from the liver was determined. Open reading frame analysis predicted that CYP4A37, CYP4A38, and CYP4A39 each comprised 510 amino acids with approximately 90% sequence identity to one another, and approximately 71 and 78% sequence identity to rat CYP4A1 and human CYP4A11, respectively. PCR analysis revealed that the three dog CYP4A isoforms are expressed in kidney > liver > lung > intestine > skeletal muscle > heart. Treatment of primary dog hepatocytes with the PPARalpha agonists GW7647X and clofibric acid resulted in an increase in CYP4A37, CYP4A38, and CYP4A39 mRNA expression (up to fourfold), whereas HMG-CoA synthase mRNA expression was increased to a greater extent (up to 10-fold). These results suggest that dog CYP4A37, CYP4A38, and CYP4A39 are expressed in a tissue-dependent manner and that beagle dog CYP4A is not highly inducible by PPARalpha agonists, similar to the human CYP4A11 gene.     

过氧化物酶体增殖物激活受体β亚型激动剂GW501516对胰岛素抵抗模型小鼠的胰岛素增敏作用(英文)

目的在长期(4个月)高脂高糖饮食诱导的胰岛素抵抗小鼠模型上,评价过氧化物酶体增殖物激活受体β(PPARβ)亚型激动剂GW501516对胰岛素抵抗的改善作用,并对可能的相关机制进行探讨。方法C57BL/6J小鼠采用高脂高糖饮食(35%脂肪,30%麦芽糖)诱导4个月,待产生明显的糖脂代谢紊乱。实验分为正常对照、饮食导致的肥胖(DIO)模型与DIO模型+GW501516(10mg·kg-1·d-1)给药组。隔天监测体重与进食量情况,以葡萄糖氧化酶法检测血糖,并进行口服葡萄糖耐量试验和血脂(甘油三酯、总胆固醇和高密度脂蛋白)含量的检测。以组织学方法检测肝脏异位脂积聚及病理变化情况。为确证其相关作用机制,采用RT-PCR方法检测骨骼肌内PPARβ下游糖脂代谢靶基因的表达。结果GW501516有效改善模型小鼠的胰岛素抵抗,显著降低口服糖耐量曲线下面积〔DIO模型组,(32.4±4.6)mmol·h·L-1, DIO +GW501516组,(23.4±2.5)mmol·h·L-1,n=7 ~8, P<0.05〕,降低空腹血糖,增加血清高密度脂蛋白含量,减轻模型小鼠的肝脂肪变性。此外,RT-PCR结果表明,骨骼肌卡尼汀(肉碱)软脂酰转移酶1b,解偶联蛋白(UCP)2,UCP3明显上调,同时葡萄糖转运蛋白也明显上调。结论GW501516显著改善模型小鼠的胰岛素抵抗,恢复其空腹血糖值,降低肝脏内异位脂积聚,其治疗作用机制可能与①促进骨骼肌内脂肪酸氧化和能量的解偶联,②促进骨骼肌内的糖摄取有关,提示PPARβ可能是胰岛素抵抗及代谢综合征的有效治疗靶标。     

吡格列酮对局灶性脑缺血再灌注损伤大鼠核转录因子及干扰素-γ的影响

目的探讨局灶性脑缺血再灌注损伤大鼠脑组织中NF—KB及IFN-γ的变化及毗格列酮干预对上述指标的影响。方法64只sD雄性大鼠随机分为4组：①假手术组（s组,n=16）;②模型组（M组,n=16）;③吡格列酮组（P组,n=16）;@GW9662＋吡格列酮组（GP组,n=16）。除s组外,其余各组均通过线栓法建立大鼠大脑中动脉阻塞（MCAO）再灌注损伤模型。于术前30分钟分别给予相应处理。缺血120分钟再灌注24小时后,对大鼠进行神经功能评分;干湿质量法检测脑含水量;EUsA法检测NF—KB、IFN-γ含量水平。结果①与S组比较,M组神经功能评分、脑含水量明显增加（P〈0．05）;与M组比较,P组神经功能评分、脑含水量明显降低（P〈0.05）。②与S组比较,M组NF—KB、IFN-γ含量水平明显增加（P〈O．05）,P组较M组NF—KB、IFN-γ含量水平显著降低（P〈0．05）,且P组中两者呈显著正相关（P〈0．001）。结论NF—KB及IFN-γ的改变与脑缺血再灌注损伤的炎症反应存在相关性,而吡格列酮可能通过抑制炎症反应,减轻细胞水肿,从而对神经细胞发挥保护作用。     

Biochemical isolation of Argonaute protein complexes by Ago-APP

During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.To repress gene expression, microRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target sites on mRNAs (1, 2). Ago proteins contain four different domains with distinct functions. The N domain is important for small RNA loading, the PAZ domain binds the 3′ and the MID domain the 5′ end of the small RNA. The PIWI domain is structurally similar to RNase H, and indeed some Ago proteins can function as small RNA-guided endonucleases. In humans, only Ago2 is catalytically active whereas Ago1, Ago3, and Ago4 are inactive (3, 4). Recent structural and biochemical experiments identified small structural elements that affect the endonucleolytic activity of Ago proteins (5–9). For miRNA-guided repression independently of Ago-mediated RNA cleavage, Ago proteins recruit a member of the TNRC6/GW182 protein family (also referred to as GW proteins and TNRC6A-C in human), which coordinate all downstream silencing events including binding to the poly(A)-binding proteins on the poly(A) tail of the mRNA, recruiting deadenylase complexes such as PAN2–PAN3 or the CCR4–NOT complex and translational repression (10–13). GW (glycine-tryptophan) proteins are characterized by an unstructured, tryptophan (Trp)-rich N-terminal half that serves as an Ago-binding domain (Fig. 1A). In particular, two Trp of a GW protein bind into two specific pockets on the Ago surface (10, 14, 15). We have recently identified a short TNRC6B-derived peptide that efficiently interacts with Ago proteins (Fig. 1A, T6B peptide) (15, 16). We hypothesized that such a peptide might be a powerful tool for the isolation of endogenous Ago proteins from cell or tissue lysates. We find that this peptide precipitates all four endogenous human Ago proteins efficiently, and we refer to this method as “Ago protein Affinity Purification by Peptides” (Ago-APP). Furthermore, it also allows for an accurate quantification of Ago protein levels in different primary tissues. Strikingly, Ago-APP binds Ago proteins that are involved in miRNA-guided gene silencing from any animal lysate. Furthermore, in plants, where GW proteins are not conserved, the T6B peptide mimics interactions with components of the RNA-guided DNA methylation pathway, and indeed we have efficiently isolated Ago-associated small RNAs within a length frame indicative for this pathway. Finally, transfection of the T6B peptide leads to a strong repression of endogenous miRNA pathways. Taken together, we have developed and characterized a novel highly efficient tool to study small RNA pathways in many different cell types, tissues, and species.Open in a separate windowFig. 1.Precipitation of Ago complexes by Ago-APP. (A) Schematic representation of the TNRC6B domain organization and its Ago-interacting regions. The position and amino acid sequence of the peptide used for Ago-APP are shown. (B) Ago-APP from HEK293 cells overexpressing FH-Ago1–4 (lanes 1–6) and FH-tagged human PIWI proteins (lanes 7–9). (C and D) Comparison of Ago2-immunoprecipitation (IP) and Ago-APP in terms of Ago enrichment (C) and coprecipitation of miRNAs (D). A monoclonal antibody against human Ago2 and an unrelated antibody were used for IPs. Recombinant GST-T6B and GST alone were used for Ago-APPs. Asterisks indicate unspecific bands. (E) Schematic representation of an Ago protein and miRNA codepletion experiment using Ago-APP. (F) Codepletion of Ago proteins and associated miRNAs. Supernatants of Ago-APPs were repeatedly incubated with T6B-coupled beads as shown in E. Ago1 and Ago2 protein levels were analyzed by Western blotting. The levels of the highly abundant let-7a and the weakly expressed miRNA-30a were analyzed by Northern blotting. A probe against U6 snRNA was used to control for equal RNA loading. (G) Quantification of Western and Northern blot signals shown in F. Background signals of the blots were deduced to obtain the protein and miRNA signal intensities of input and supernatant samples.     

A Nuclear Receptor Ligand Down-Regulates Cytosolic Phospholipase A<Subscript><Emphasis Type="BoldItalic">2</Emphasis></Subscript> Expression to Reduce Bile Acid–Induced Cyclooxygenase 2 Activity in Cholangiocytes: Implications of Anticarcinogenic Action of Farnesoid X Receptor Agonists

Bile acids are considered to be involved in the development of biliary tract carcinoma, although the underlying mechanisms are yet to be established. The aims of this study were (1) to investigate the carcinogenic role of bile acids in the biliary system based on the arachidonate–prostanoid pathway and (2) to clarify the therapeutic role of a farnesoid X receptor (FXR) ligand that modifies bile acid metabolism. Immortalized mouse cholangiocytes were incubated with glycochenodeoxycholate (GCDC), taurocholate, taurochenodeoxycholate, taurodeoxycholate, and tauroursodeoxycholate. GCDC induced cyclooxygenase 2 (COX-2) expression (Western blotting, 1.7-fold; RT-PCR, 2.3-fold) and prostaglandin (PG) production (PGE₂, 6.3-fold; PGF₂, 8.5-fold), whereas cytosolic phospholipase A₂ (cPLA₂) expression and activity were reduced. In contrast, no marked changes were induced by the other bile acids. When the same experiment was performed in the presence of a synthetic FXR ligand (GW4064), cPLA₂ expression and activity were reduced, although COX-2 expression was unchanged. GW4064 also suppressed PG generation by 40%. In conclusion, the present findings suggest a carcinogenic potential of GCDC. A synthetic FXR ligand (GW4064) inhibited the induction of COX-2 activity (detected as PG production) by GCDC, suggesting its anticarcinogenic potential. This effect seemed to be due to down-regulation of cPLA₂. FXR ligands may have therapeutic potential against biliary carcinogenesis, but a delivery system for these agents is still to be developed.     

Quantitative comparison of the inhibitory effects of GW5638 and tamoxifen on angiogenesis in the cornea pocket assay

GW5638 is a novel tissue-selective estrogen receptor (ER) modulator. Structurally, it is a derivative of tamoxifen that is known for its inhibitory effects on angiogenesis in an ER-independent manner. Therefore, it is possible that GW5638 has the same effects as tamoxifen on angiogenesis. To test this hypothesis, we used the rat cornea pocket assay and developed a new method that could precisely determine the total projected area of microvessels induced by basic fibroblast growth factor (bFGF) in the cornea. Animals in the study were treated with corn oil (control group), tamoxifen, or GW5638. After treatment, we observed that both GW5638 and tamoxifen could inhibit angiogenesis in the cornea (P<0.05) and that the inhibitory effects were not mediated by blocking functions of estrogen. Meanwhile, GW5638 had minimal effects on the body weight of animals whereas tamoxifen significantly reduced the body weight. Based on these observations, we concluded that GW5638 was as effective as tamoxifen in antiangiogenic treatment but less toxic than tamoxifen.