Differential Metabotypes in Synovial Fibroblasts and Synovial Fluid in Hip Osteoarthritis Patients Support Inflammatory Responses

Changes in cellular metabolism have been implicated in mediating the activated fibroblast phenotype in a number of chronic inflammatory disorders, including pulmonary fibrosis, renal disease and rheumatoid arthritis. The aim of this study was therefore to characterise the metabolic profile of synovial joint fluid and synovial fibroblasts under both basal and inflammatory conditions in a cohort of obese and normal-weight hip OA patients. Furthermore, we sought to ascertain whether modulation of a metabolic pathway in OA synovial fibroblasts could alter their inflammatory activity. Synovium and synovial fluid was obtained from hip OA patients, who were either of normal-weight or obese and were undergoing elective joint replacement surgery. The synovial fluid metabolome was determined by 1H NMR spectroscopy. The metabolic profile of isolated synovial fibroblasts in vitro was characterised by lactate secretion, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF Analyser. The effects of a small molecule pharmacological inhibitor and siRNA targeted at glutaminase-1 (GLS1) were assessed to probe the role of glutamine metabolism in OA synovial fibroblast function. Obese OA patient synovial fluid (n = 5) exhibited a different metabotype, compared to normal-weight patient fluid (n = 6), with significantly increased levels of 1, 3-dimethylurate, N-Nitrosodimethylamine, succinate, tyrosine, pyruvate, glucose, glycine and lactate, and enrichment of the glutamine–glutamate metabolic pathway, which correlated with increasing adiposity. In vitro, isolated obese OA fibroblasts exhibited greater basal lactate secretion and aerobic glycolysis, and increased mitochondrial respiration when stimulated with pro-inflammatory cytokine TNFα, compared to fibroblasts from normal-weight patients. Inhibition of GLS1 attenuated the TNFα-induced expression and secretion of IL-6 in OA synovial fibroblasts. These findings suggest that altered cellular metabolism underpins the inflammatory phenotype of OA fibroblasts, and that targeted inhibition of glutamine–glutamate metabolism may provide a route to reducing the pathological effects of joint inflammation in OA patients who are obese.     

Loss of SDHB Induces a Metabolic Switch in the hPheo1 Cell Line toward Enhanced OXPHOS

Background: Enzymes of tricarboxylic acid (TCA) have recently been recognized as tumor suppressors. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) cause pheochromocytomas and paragangliomas (PCCs/PGLs) and predispose patients to malignant disease with poor prognosis. Methods: Using the human pheochromocytoma cell line (hPheo1), we knocked down SDHB gene expression using CRISPR-cas9 technology. Results: Microarray gene expression analysis showed that >500 differentially expressed gene targets, about 54%, were upregulated in response to SDHB knock down. Notably, genes involved in glycolysis, hypoxia, cell proliferation, and cell differentiation were up regulated, whereas genes involved in oxidative phosphorylation (OXPHOS) were downregulated. In vitro studies show that hPheo1 proliferation is not affected negatively and the cells that survive by shifting their metabolism to the use of glutamine as an alternative energy source and promote OXPHOS activity. Knock down of SDHB expression results in a significant increase in GLUD1 expression in hPheo1 cells cultured as monolayer or as 3D culture. Analysis of TCGA data confirms the enhancement of GLUD1 in SDHB mutated/low expressed PCCs/PGLs. Conclusions: Our data suggest that the downregulation of SDHB in PCCs/PGLs results in increased GLUD1 expression and may represent a potential biomarker and therapeutic target in SDHB mutated tumors and SDHB loss of activity-dependent diseases.     

Identification and Synthesis of Volicitin and Related Components from Beet Armyworm Oral Secretions

Oral secretion of beet armyworm caterpillars (BAW), when applied to damaged tissues of corn seedlings, induces the seedlings to emit volatile compounds that attract the natural enemies of the caterpillars. The key elicitor present in BAW oral secretions is N-[17-hydroxylinolenoyl]-L-glutamine (volicitin). Analysis of the oral secretion showed that it also contained N-[17-hydroxyolinoleoyl]-L-glutamine, free 17-hydroxylinolenic, and 17-hydroxylinoleic acid, the glutamine conjugates of linolenic and linoleic acid as well as free linolenic and linoleic acid. Here we present the identification and synthesis of the hydroxy acids and of glutamine conjugates.     

定点突变的谷氨酰胺合成酶的表达条件和在酶法合成谷氨酰胺中的应用

针对酶法生产谷氨酰胺中高浓度铵盐条件下的谷氨酰胺合成酶（GS）腺苷酰化问题,从谷氨酸棒杆菌Corynebacterium glutamicum ATCC 14067调取编码GS的基因glnA,将GS的腺苷酰化位点Tyr405定点突变为Phe405,并在大肠杆菌中表达突变后的GS,优化产酶条件。用突变的GS在摇瓶中进行酶催化过程,通过补加酶催化底物谷氨酸钠和氯化铵,可以提高定点突变的GS生产谷氨酰胺的能力,谷氨酰胺产量达到16.8 g·L^-1;在5 L反应器规模的酶催化生产谷氨酰胺过程中,通过调控底物补加方案和反应条件,谷氨酰胺的产量达到34.2 g·L^-1,谷氨酰胺对谷氨酸的摩尔转化率为96.3%。     

水解面筋蛋白高效制备谷氨酰胺结合肽的研究

采用食品级胃蛋白酶、胰蛋白酶、Protamex复合酶双酶组合对面筋蛋白进行水解制备Gln结合肽。先确定三种单酶各自对面筋蛋白适宜的初始pH、温度、酶浓度和底物浓度。并以双酶进行两两组合水解。在进行水解度控制实验时取样,分析水解液中氨基氮的含量,计算平均肽链长度,并用高效液相色谱分析水解液中Gln的得率。实验结果表明胰蛋白酶和胃蛋白酶联合水解的效果最好,条件为:胰蛋白酶在pH8.0,50℃,S%=12.8%(W/V),E%=9%(W/W)水解6h,再用胃蛋白酶pH2.0,40℃,E%=5%(W/W)条件下水解5h,可得到平均肽链长度为2.20个氨基酸残基的蛋白水解液,Gln得率最高,为60.43%。认为胰蛋白酶和胃蛋白酶组合是面筋蛋白中高效制备谷氨酰胺结合肽的最佳组合。     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

谷氨酰胺提取的研究

对强酸性阳离子交换树脂提取分离发酵液中L-谷氨酰胺（L-Gln）的工艺条件进行了研究。确立了提取的最佳条件为：强酸性阳离子交换树脂的上柱液pH为3．5，氨水的洗脱浓度为0．03mol／L，洗脱速度为1．0mL／min，提取得率为70％左右。     

适用于辐射加工的谷氨酰胺晶溶发光剂量计及其BPLL型测量装置

本文介绍了我们实验室研制的BPLL型晶溶发光剂量计的测量装置。本装置采用直流电流积分法。选用GDB-52型光电倍增管做光探测器,使用NWF-02A型微电流放大器和电压—频率转换器。装置的灵敏度可进行调节,对于剂量测量,八小时的稳定性是令人满意的。48个测量数据的平均值的标准偏差为±0.4％。在此装置上对适用于辐射加工的谷氨酰胺晶溶发光剂量计的剂量学特性进行了研究。谷氨酰胶的剂量响应线性范围为10~2～10~4Gy。论述了溶剂的温度、样品的质量对光产额的影响以及射线照射后的存贮效应。     

谷氨酰胺对小儿心内直视手术的神经元烯醇化酶的影响

目的探讨谷氨酰胺（Gln）对先天性心脏病心内直视手术患儿的血清特异性神经元烯醇化酶（NSE）的影响和可能脑保护机制。方法将择期行体外循环（CPB）下先天性心脏病（VSD/ASD）心内直视手术患儿20例随机分为2组,小儿氨基酸＋Gln组（G组）和小儿氨基酸＋生理盐水组（P组）,每组10例。2组患儿统一进行术前用药、麻醉诱导、麻醉维持、CPB以及术后管理。于术前30 min（T0）、主动脉开放后10 min（T1）以及CPB结束后1 h（T2）、6 h（T3）、24 h（T4）分别采集颈内静脉球部静脉血检测一氧化氮合酶（NOS）、乳酸（LD）和NSE水平变化。结果 2组血清学指标在T0时差异无显著性意义（P〉0.05）;T2-T4时间点NOS活性G组与P组比较有所上升、LD有所下降（P〈0.05或P〈0.01）;NSE值G组在T3时点与P组比较在有所下降（P〈0.05）。结论外源性补充Gln能够安全有效地调节先天性室间隔缺损/房间隔缺损心内直视手术患儿血清NOS活性,降低LD和NSE水平,具有一定的脑保护作用。