Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.     

Mammalian Prion protein expression in yeast; a model for transmembrane insertion

The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian microsomes, 50% being found as transmembrane (TM) proteins with the central TM1 segment spanning the membrane. TM1 hydrophobicity is marginal for lateral membrane insertion, which is primarily driven by hydrophobic interaction between the ER translocon and substrates in transit. Most inserted TM1 has its N-terminus in the ER lumen (Ntm orientation), as expected for arrest of normal secretion. However, 20% is found in inverted Ctm orientation. These are minor species in vivo, presumably a consequence of efficient quality control. PrP mutations that increase TM1 hydrophobicity result in increased Ctm insertion, both in vitro and in mouse brain, and a strong correlation is found between CtmPrP insertion and neuropathology in transgenic mice; a copper-dependent pathogenicity mechanism is suggested. PrP fusions with a C-terminal epitope tag, when expressed in yeast cells at moderate levels, appear to interact efficiently with the translocon, providing a useful model for testing the effects of PrP mutations on TM insertion and orientation. However, secretion of PrP by the mammalian translocon requires the TRAP complex, absent in yeast, where essentially all PrP ends up as TM species, 85–90% Ntm and 10–15% Ctm. Although yeast is, therefore, an incomplete mimic of mammalian PrP trafficking, effects on Ctm insertion of mutations increasing TM1 hydrophobicity closely reflect those seen in vitro. Electrostatic substrate-translocon interactions are a major determinant of TM protein insertion orientation and the yeast model was used to investigate the role of the large negative charge difference across TM1, a likely cause of translocation delay that would favor TM insertion and Ctm orientation. An increase in ΔCh from −5 to −7 caused a marked increase in Ctm insertion, while a decrease to −3 or −1 allowed 35 and about 65% secretion, respectively. Utility of the yeast model and the role of this charge difference in driving PrP membrane insertion are confirmed.     

Kinetic analysis of the interaction between HIV-1 protease and inhibitors using optical biosensor technology

The interaction between HIV-1 protease and reversible inhibitors was studied by surface plasmon resonance biosensor technology. The steady-state binding level and the time course of association and dissociation could be observed by measuring the binding of inhibitors injected in a continuous flow of buffer to the immobilized enzyme. Fourteen low molecular weight inhibitors (500-700 Da), including the four clinically used HIV-1 protease inhibitors (indinavir, nelfinavir, ritonavir, and saquinavir), were analyzed. Affinities were estimated as B(50) values from a series of sensorgrams at different concentrations of inhibitors. These values were found to be correlated with inhibition constants (K(i)) determined by an enzyme inhibition assay (r(2) = 0.84, logarithmic values). Dissociation rates were estimated at a single saturating concentration of the inhibitors as t(1/2,obs), but these values did not correlate with K(i) (r(2) = 0.26, logarithmic values). Indinavir had the highest affinity (B(50) = 11 nM) and the fastest dissociation (t(1/2,obs) = 500 s) among the clinically used inhibitors while saquinavir had a lower affinity (B(50) = 25 nM) and the slowest dissociation rate (t(1/2,obs) = 6500 s). Since these two inhibitors have similar K(i) values, the differences in dissociation rates reveal important characteristics in the interaction that cannot be obtained by the inhibition studies. The biosensor data are expected to be of greater in vivo relevance since the experiments were performed in a buffer more similar to physiological conditions.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02-0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

情前期与间情期大鼠内侧视前区-下丘脑前区神经元的电活动

在氯化筒箭毒制动的情前期及间情期大鼠上进行实验。用玻璃微电极记录内侧视前区-下丘脑前区(mPOA-AHA)的单位放电。mPOA-AHA 的大多数单位表现连续性或周期性自发放电。情前期大鼠周期性放电单位所占的百分比显著地多于间情期大鼠(P<0.01)。刺激子宫颈部可以使单位放电发生两种反应。一种单位对宫颈刺激发生放电频率增加的反应(宫颈兴奋神经元,CE 神经元),另一种单位则发生放电频率降低的反应(宫颈抑制神经元,CI神经元)。情前期大鼠 CE 与 CI 神经元所占的百分比与间情期大鼠没有明显的差异(P>0.05)。但情前期大鼠的 CE和 CI 神经元多呈现周期性放电,而间情期大鼠的 CE 和 CI 神经元则多呈现连续性放电。这说明 mPOA-AHA 神经元的电活动随生殖周期发生明显的变化。脑室注射去甲肾上腺素(NE)能使 mPOA-AHA 内大多数 CE 神经元对宫颈刺激的兴奋反应暂时受到抑制,而注射 NE 却使大多数 CI 神经元发生抑制解除。这些结果提示 NE 可能有抑制 CE 神经元和刺激 CI 神经元的作用。     

The Plant Cell Surface

Multicellular organization and tissue construction has evolved along essentially different lines in plants and animals. Since plants do not run away, but are anchored in the soil, their tissues are more or less firm and stiff. This strength stems     

玉米黑粉菌DNA载体的构建和它的原生质球的转化和再生

我们用玉米黑粉菌(Ustilago maydis)热休克基因(Heat shock gene,hsp 70)的启动子和终止子与新霉素磷酸转移酶基因相连,构建成了有效的双功能质粒pDL1(在E。coli中用Amp作为选择标记,在玉米黑粉菌中用新霉素作为选择标记)。以分离的一株新霉素敏感的玉米黑粉菌作为受体菌,用构建的pDL1质粒作为供体DNA,对影响受体菌原生质球的形成条件(培养基、菌龄、各种酶、酶的浓度、作用时间和渗透压稳定剂)、再生条件和DNA转化条件进行了初步研究。对数前中期收集的菌体,以5mg/ml真菌溶壁酶处理30分钟左右,90%都形成原生质球,其再生率为60—80%,转化率为300—1000转化子/μg DNA。随机选出25个转化子,DNA杂交都为阳性。分析dot-blot和Southern blot DNA杂交结果,发现质粒在细胞中以整合形式存在,一个细胞可以有多拷贝的整合质粒,质粒可能以非完全同源性的重组形式,参入性地整合进染色体中。