The subunit fine structure of isolated, purified Na,K-adenosine triphosphatase : Freeze-fracture study

The ultrastructural features of a purified fraction of Na⁺,K⁺-adenosine triphosphatase (ATPase) isolated from dog kidney medulla were compared with those of the initial crude microsomal fraction in the purification sequence. Although both fractions consist of vesicular structures, the purified fraction is more homogeneous with respect to overall size and intramembrane protein particle size and distribution. Polyacrylamide gel electrophoresis profiles of both fractions reveal multiple proteins in the microsomal fraction but only two in the final purified fraction. The membranes of the pure fraction comprised one class of particles roughly 95–120 Å in diameter which represent the in vitro configuration of Na⁺,K⁺-ATPase.     

RUS家族对植物生长发育的调控作用

叶绿体基因组编码许多参与光合作用和其他代谢过程的关键蛋白质,在叶绿体中合成的代谢物对于植物正常的生长发育至关重要。根对紫外线-B辐射敏感[Root-UVB (ultraviolet radiation B)-sensitive, RUS]蛋白属于叶绿体蛋白,由高度保守的DUF647结构域组成,在参与植物形态发生、物质运输和能量代谢等多种生命活动的调控中发挥作用。本文就近年来关于RUS家族在植物的胚胎发育、光形态建成、维生素B6稳态、生长素转运和花药发育等生长发育过程中的相关研究进行回顾和总结,为深入研究其在植物生长发育中的分子调控机制提供了参考。     

I-κBα depletion by transglutaminase 2 and μ-calpain occurs in parallel with the ubiquitin-proteasome pathway

Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-κB through the polymerization-mediated depletion of I-κBα without IKK activation. This NF-κB activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-κBα, which raises the question of how the polymerized I-κBα can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-κBα polymers in MCF7 cells transfected with TGase 2, and induced high levels of I-κBα polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-κBα polymers in HEK-293 cells in case of TGase 2 transfection either with I-κBα or I-κBα mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-κBα polymers and I-κBα, concurrent with an inhibition of NF-κB activity in MDA-MB-231 cells. This suggests that μ-calpain proteasome-dependent I-κBα polymer degradation may contribute to cancer progression through constitutive NF-κB activation.     

The affinity of human RANK binding to its ligand RANKL

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL play critical roles in bone re-modeling, immune function, vascular disease and mammary gland development. To study the interaction of RANK and RANKL, we have expressed both extracellular domain of RANK and ectodomain of RANKL using Escherichia coli expression system. RANK was expressed as an inclusion body first which properly refolded later, while RANKL was initially produced as a GST fusion protein, after which the GST was removed by enzyme digestion. Soluble RANK existed as a monomer while RANKL was seen as a trimer in solution, demonstrated by gel filtration chromatography and cross-linking experiment. The recombinant RANK and RANKL could bind to each other and the binding affinity of RANKL for RANK was measured with surface plasmon resonance technology and K_D value is about 1.09 × 10⁻¹⁰ M.     

Predicting RNA secondary structures with pseudoknots by MCMC sampling

The most probable secondary structure of an RNA molecule, given the nucleotide sequence, can be computed efficiently if a stochastic context-free grammar (SCFG) is used as the prior distribution of the secondary structure. The structures of some RNA molecules contain so-called pseudoknots. Allowing all possible configurations of pseudoknots is not compatible with context-free grammar models and makes the search for an optimal secondary structure NP-complete. We suggest a probabilistic model for RNA secondary structures with pseudoknots and present a Markov-chain Monte-Carlo Method for sampling RNA structures according to their posterior distribution for a given sequence. We favor Bayesian sampling over optimization methods in this context, because it makes the uncertainty of RNA structure predictions assessable. We demonstrate the benefit of our method in examples with tmRNA and also with simulated data. McQFold, an implementation of our method, is freely available from http://www.cs.uni-frankfurt.de/~metzler/McQFold.     

Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.     

Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis

Emerging studies indicate that intracellular eukaryotic ceramide species directly activate cathepsin B (CatB), a lysosomal‐cysteine‐protease, in the cytoplasm of osteoclast precursors (OCPs) leading to elevated RANKL‐mediated osteoclastogenesis and inflammatory osteolysis. However, the possible impact of CatB on osteoclastogenesis elevated by non‐eukaryotic ceramides is largely unknown. It was reported that a novel class of phosphoglycerol dihydroceramide (PGDHC), produced by the key periodontal pathogen Porphyromonas gingivalis upregulated RANKL‐mediated osteoclastogenesis in vitro and in vivo. Therefore, the aim of this study was to evaluate a crosstalk between host CatB and non‐eukaryotic PGDHC on the promotion of osteoclastogenesis. According to a pulldown assay, high affinity between PGDHC and CatB was observed in RANKL‐stimulated RAW264.7 cells in vitro. It was also demonstrated that PGDHC promotes enzymatic activity of recombinant CatB protein ex vivo and in RANKL‐stimulated osteoclast precursors in vitro. Furthermore, no or little effect of PGDHC on the RANKL‐primed osteoclastogenesis was observed in male and female CatB‐knock out mice compared with their wild type counterparts. Altogether, these findings demonstrate that bacterial dihydroceramides produced by P. gingivalis elevate RANKL‐primed osteoclastogenesis via direct activation of intracellular CatB in OCPs.     

妊娠合并乙肝病毒感染患者血清DNMT1、TIM-3与HBV-DNA病毒载量和妊娠结局的关系分析

摘要 目的：探讨妊娠合并乙肝病毒（HBV）感染患者血清脱氧核糖核酸甲基转移酶1（DNMT1）、T细胞免疫球蛋白粘蛋白-3（TIM-3）与乙型肝炎病毒-脱氧核糖核酸（HBV-DNA）病毒载量和妊娠结局的关系。方法：选取2021年1月～2022年1月南京医科大学第一附属医院收治的186例妊娠合并HBV感染患者为HBV感染组,根据HBV-DNA病毒载量分为阳性组56例和阴性组130例,根据妊娠结局分为结局不良组和结局良好组,另选取同期于南京医科大学第一附属医院进行孕检的150名健康孕妇为对照组。采用酶联免疫吸附法检测血清DNMT1、TIM-3水平。比较HBV感染组与对照组、阳性组与阴性组血清DNMT1、TIM-3水平。采用单因素及多因素Logistic回归分析妊娠合并HBV感染患者妊娠结局不良的影响因素,受试者工作特征（ROC）曲线分析血清DNMT1、TIM-3水平对妊娠合并HBV感染患者妊娠结局不良的预测价值。结果：与对照组比较,HBV感染组血清DNMT1、TIM-3水平升高（P＜0.05）。与阴性组比较,阳性组血清DNMT1、TIM-3水平升高（P＜0.05）。186例妊娠合并HBV感染患者妊娠结局不良发生率为55.38%（103/186）。单因素分析显示,妊娠结局不良与HBV感染孕周、HBV-DNA病毒载量、谷草转氨酶（AST）、谷丙转氨酶（ALT）、DNMT1、TIM-3有关（P＜0.05）。多因素Logistic回归分析显示,HBV-DNA病毒载量阳性和DNMT1＞34.94 ng/mL、TIM-3＞18.96 pg/mL为妊娠合并HBV感染患者妊娠结局不良的独立危险因素（P＜0.05）。ROC曲线分析显示,血清DNMT1、TIM-3水平单独和联合检测预测妊娠合并HBV感染患者妊娠结局不良的曲线下面积分别为0.798、0.791、0.870。结论：妊娠合并HBV感染患者血清DNMT1、TIM-3水平升高与HBV-DNA病毒载量阳性和妊娠结局不良密切相关,血清DNMT1、TIM-3水平联合对妊娠合并HBV感染患者妊娠结局预测价值良好。     

Dopamine depletion and subsequent treatment with L-DOPA, but not the long-lived dopamine agonist pergolide, enhances activity of the Akt pathway in the rat striatum

Dysregulation of signaling pathways is believed to contribute to Parkinson's disease pathology and l-DOPA-induced motor complications. Long-lived dopamine (DA) agonists are less likely to cause motor complications by virtue of continuous stimulation of DA receptors. In this study, we compared the effects of the unilateral 6-hydroxydopamine lesion and subsequent treatment with l-DOPA and DA agonist pergolide on signaling pathways in rats. Pergolide caused less pronounced behavioral sensitization than l-DOPA (25 mg/kg, i.p., 10 days), particularly at lower dose (0.5 and 0.25 mg/kg, i.p.). Pergolide, but not l-DOPA, reversed lesion-induced up-regulation of preproenkephalin and did not up-regulate preprodynorphine or DA D3 receptor in the lesioned hemisphere. Pergolide was as effective as l-DOPA in reversing the lesion-induced elevation of ERK2 phosphorylation in response to acute apomorphine administration (0.05 mg/kg, s.c.). Chronic l-DOPA significantly elevated the level of Akt phosphorylation at both Thr(308) and Ser(473) and concentration of phosphorylated GSK3alpha, whereas pergolide suppressed the lesion- and/or challenge-induced supersensitive Akt responses. The data indicate that l-DOPA, unlike pergolide, exacerbates imbalances in the Akt pathway caused by the loss of DA. The results support the hypothesis that the Akt pathway is involved in long-term actions of l-DOPA and may be linked to l-DOPA-induced dyskinesia.     

Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals

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