OKN-007 decreases free radical levels in a preclinical F98 rat glioma model

Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immunospin-trapping (IST) methodologies. Free radicals are trapped with the spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immunospin trapping by an antibody against DMPO adducts. In this study, we combined mMRI with a biotin–Gd-DTPA–albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007-treated rat F98 gliomas. OKN-007 was found to significantly decrease (P < 0.05) free radical levels detected with an anti-DMPO probe in treated animals compared to untreated rats. Immunoelectron microscopy was used with gold-labeled antibiotin to detect the anti-DMPO probe within the plasma membrane of F98 tumor cells from rats administered anti-DMPO in vivo. OKN-007 was also found to decrease nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase, 3-nitrotyrosine, and malondialdehyde in ex vivo F98 glioma tissues via immunohistochemistry, as well as decrease 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells via ELISA. The results indicate that OKN-007 effectively decreases free radicals associated with glioma tumor growth. Furthermore, this method can potentially be applied toward other types of cancers for the in vivo detection of macromolecular free radicals and the assessment of antioxidants.     

β-Arrestin-1 protects against endoplasmic reticulum stress/p53-upregulated modulator of apoptosis-mediated apoptosis via repressing p-p65/inducible nitric oxide synthase in portal hypertensive gastropathy

Portal hypertensive gastropathy (PHG) is a serious cause of bleeding in patients, and is associated with portal hypertension. β-Arrestins (β-arrestin-1 and β-arrestin-2) are well-established mediators of endocytosis of G-protein-coupled receptors (GPCRs), ubiquitination, and G-protein-independent signaling. The role of β-arrestin-1 (β-arr1) in mucosal apoptosis in PHG remains unclear. The aim of this study was to investigate the involvement of β-arr1 in PHG via its regulation of endoplasmic reticulum (ER) stress/p53-upregulated modulator of apoptosis (PUMA) apoptotic signaling. Gastric mucosal injury and apoptosis were studied in PHG patients and in PHG mouse models. The induction of β-arr1 and the ER stress/PUMA signaling pathway were investigated, and the mechanisms of β-arr1-regulated gastric mucosal apoptosis were analyzed in vivo and in vitro experiments. β-arr1 and ER stress/PUMA signaling elements were markedly induced in the gastric mucosa of PHG patients and mouse models. Blockage of ER stress demonstrably attenuated the mucosal apoptosis of PHG, while targeted deletion of β-arr1 significantly aggravated the injury and ER stress/PUMA-mediated apoptosis. β-arr1 limited the activation of p65 to repress TNF-α-induced inducible nitric oxide synthase (iNOS) expression and NO release, which could regulate ER stress/PUMA-mediated mucosal apoptosis in PHG. In vivo and in vitro experiments further demonstrated that β-arr1 protected against mucosal apoptosis by repressing TNF-α-induced iNOS expression via inhibiting the activation of p65. These results indicated that β-arr1 regulated ER stress/PUMA-induced mucosal epithelial apoptosis through suppression of the TNF-α/p65/iNOS signaling pathway activation and that β-arr1 is a potential therapeutic target for PHG.     

水蕨(Ceratopteris thalictroides)查尔酮合成酶基因的克隆和表达

查尔酮合酶(chalcone synthase, CHS)是植物类黄酮化合物合成的关键酶,有关蕨类植物CHS基因的序列及功能信息尚不完善。本研究采用快速扩增cDNA末端(RACE)技术克隆获得了模式蕨类植物——水蕨(Ceratopteris thalictroides)CtCHS基因(GenBank登录号:JX027616.1),其cDNA序列全长为1616 bp,具有3个外显子和2个内含子,开放阅读框(ORF)为1215 bp,编码404个氨基酸。进化树分析表明,CtCHS与问荆(Equisetum arvense)、松叶蕨(Psilotum nudum)和3种薄囊蕨的查尔酮合成酶基因聚为一枝,说明这些蕨类植物亲缘关系较近且为单系起源。通过构建原核表达体系成功获得CtCHS蛋白的多克隆抗体并用于免疫印迹分析,结果表明CtCHS基因的表达明显受紫外光(UV)诱导。CtCHS基因的克隆与表达分析为进一步研究水蕨类黄酮化合物的合成及其调控机制提供了依据。     

Six week swimming followed by acute uptakes of ginsenoside Rg1 may affect aerobic capacity of SD rats

[Purpose]

The purpose of this study is to examine the effects of six-weeks of endurance swim training and short-term intake of Rg1 on the expression of related proteins as well as improvement of aerobic exercise capacity in 8-week-old male SD rats.

[Methods]

The groups were divided into placebo (NP, n=6), Rg1 (NRG, n=6), exercise+placebo (EP, n=7), and exercise+Rg1 (ERG, n=7). On completion of the 6-week swimming exercise, Rg1-intake groups were treated with acute uptakes (3 times within 24hrs) of Rg1. After the treatment, all groups were subjected to a swim to exhaustion test, and then the mass of muscle tissue, mRNA expression level and activity of citrate synthase (CS) were analyzed on plantaris.

[Results]

There were no differences in the effect of 6-week swimming exercise and short-term intake of Rg1 on body weight and muscle mass between groups. Although the CS mRNA expression was elevated in the exercise group and combined treatment group, there was no significant difference in CS activity. Acute uptakes of Rg1 did not affect swimming time to exhaustion, but it was increased by 235% and 314% by the 6-weeks of exercise and combined treatment of exercise and Rg1, respectively, which suggests that the combined treatment increased the effect on the capacity of aerobic exercise.

[Conclusion]

Based on these results, it was confirmed that even a short-term treatment of Rg1 can give an additive effect for improvement of exercise function, and additional studies are needed for the mechanisms and modes of its working.     

Identification of chalcone synthase genes and their expression patterns reveal pollen abortion in cotton

Chalcone synthase (CHS) is a key enzyme and producing flavonoid derivatives as well play a vital roles in sustaining plant growth and development. However, the systematic and comprehensive analysis of CHS genes in island cotton (G. barbadense) has not been reported yet especially response to cytoplasmic male sterility (CMS). To fill this knowledge gap, a genome-wide investigation of CHS genes were studied in island cotton. A total of 20 GbCHS genes were identified and grouped into five GbCHSs. The gene structure analysis revealed that most of GbCHS genes consisted of two exons and one intron, and 20 motifs were identified. Twenty five pairs duplicated events (12 GbCHS genes) were identified including 23 segmental duplication pairs and two tandem duplication events, representing that GbCHS gene family amplification mainly owned to segmental duplication events and evolving slowly. Gene expression analysis exhibited that the GbCHS family genes presented a diversity expression patterns in various organs of cotton. Coupled with functional predictions and gene expression, the abnormal expression of GbCHS06, 10, 16 and 19 might be associated with pollen abortion of CMS line in island cotton. Conclusively, GbCHS genes exhibited diversity and conservation in many aspects, which will help to better understand functional studies and a reference for CHS research in island cotton and other plants.     

Asparagine-84, a regulatory allosteric site residue,helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.     

Continuous administration of polyphenols from aqueous rooibos (Aspalathus linearis) extract ameliorates dietary-induced metabolic disturbances in hyperlipidemic mice

The incidence of obesity and related metabolic diseases is increasing globally. Current medical treatments often fail to halt the progress of such disturbances, and plant-derived polyphenols are increasingly being investigated as a possible way to provide safe and effective complementary therapy. Rooibos (Aspalathus linearis) is a rich source of polyphenols without caloric and/or stimulant components. We have tentatively characterized 25 phenolic compounds in rooibos extract and studied the effects of continuous aqueous rooibos extract consumption in mice. The effects of this extract, which contained 25% w/w of total polyphenol content, were negligible in animals with no metabolic disturbance but were significant in hyperlipemic mice, especially in those in which energy intake was increased via a Western-type diet that increased the risk of developing metabolic complications. In these mice, we found hypolipemiant activity when given rooibos extract, with significant reductions in serum cholesterol, triglyceride and free fatty acid concentrations. Additionally, we found changes in adipocyte size and number as well as complete prevention of dietary-induced hepatic steatosis. These effects were not related to changes in insulin resistance. Among other possible mechanisms, we present data indicating that the activation of AMP-activated protein kinase (AMPK) and the resulting regulation of cellular energy homeostasis may play a significant role in these effects of rooibos extract. Our findings suggest that adding polyphenols to the daily diet is likely to help in the overall management of metabolic diseases.     

NF-κB介导非对称性二甲基精氨酸上调大鼠主动脉诱导型一氧化氮合酶的表达

目的探讨非对称性二甲基精氨酸(ADMA)上调大鼠主动脉诱导型一氧化氮合酶(iNOS)的表达是否是通过激活NF-κB实现的。方法 Wistar大鼠50只随机分为四组:①对照组(n=10):标准饲料喂养。②H组(n=12):高脂饲料喂养。③A+H组(n=14):予ADMA[0.2mg/kgd]灌胃,高脂饲料喂养。④P+A+H组(n=14):予吡咯烷二硫代氨基甲酸盐(pyrrolidine dithiocarbamate,PDTC)[40mg/kgd]腹腔注射、ADMA[0.2mg/kgd]灌胃、高脂饲料喂养。对照组、H组予等体积的生理盐水灌胃及腹腔注射、A+H组给予等体积的生理盐水腹腔注射。18周后麻醉大鼠、取主动脉。以实时荧光定量PCR和Westen blotting分别检测iNOS mRNA和蛋白表达,以电泳迁移率变动分析(EMSA)和增强化学发光法(ECL)检测NF-κB活性。结果①A+H组iNOS mRNA和蛋白表达量较对照组和H组增加(P<0.05),P+A+H组较A+H组iNOSmRNA和蛋白表达量降低(P<0.05)。②A+H组NF-κB活性较对照组和H组显著升高(P<0.05),P+A+H组NF-κB活性较A+H组明显降低(P<0.05)③相关分析:NF-κB活性与iNOS mRNA和蛋白表达量呈正相关(相关系数r分别为0.854、0.876,P<0.05)。结论 ADMA可能通过激活NF-κB途径上调iNOS表达,从而促进动脉粥样硬化的发生发展。