Biogenesis of glutaminyl-mt tRNAGln in human mitochondria

Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA^Gln is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA^Gln to form Glu-tRNA^Gln. In addition, we have identified a human homolog of the Glu-tRNA^Gln amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA^Gln accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA^Gln formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA^Gln has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA^Gln is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.     

Mutation Screening and Association Study of the Folylpolyglutamate Synthetase (FPGS) Gene with Susceptibility to Childhood Acute Lymphoblastic Leukemia

Background: Folylpolyglutamate synthetase (FPGS), an important enzyme in the folate metabolic pathway,plays a central role in intracellular accumulation of folate and antifolate in several mammalian cell types. Loss ofFPGS activity results in decreased cellular levels of antifolates and consequently to polyglutamatable antifolatesin acute lymphoblastic leukemia (ALL). Materials and Methods: During May 1997 and December 2003, 134children diagnosed with ALL were recruited from one hospital in Thailand. We performed a mutation analysis inthe coding regions of the FPGS gene and the association between single nucleotide polymorphisms (SNPs) withinFPGS in a case-control sample of childhood ALL patients. Mutation screening was conducted by polymerasechain reaction-single strand conformation polymorphism (PCR-SSCP) and subsequently with direct sequencing(n=72). Association analysis between common FPGS variants and ALL risk was done in 98 childhood ALL casesand 95 healthy volunteers recruited as controls. Results: Seven SNPs in the FPGS coding region were identifiedby mutation analysis, 3 of which (IVS13+55C>T, g.1297T>G, and g.1508C>T) were recognized as novel SNPs.Association analysis revealed 3 of 6 SNPs to confer significant increase in ALL risk these being rs7039798 (p=0.014, OR=2.14), rs1544105 (p=0.010, OR= 2.24), and rs10106 (p=0.026, OR= 1.99). Conclusions: These findingssuggested that common genetic polymorphisms in the FPGS coding region including rs7039789, rs1544105, andrs10106 are significantly associated with increased ALL risk in Thai children.     

Roles of Long-chain Acyl Coenzyme A Synthetase in Absorption and Transport of Fatty Acid

Abstract Long-chain acyl coenzyme A synthetase (ACSL) is a member of the synthetase family encoded by a multigene family; it plays an important role in the absorption and transport of fatty acid. Here we review the roles of ACSL in the regulating absorption and transport of fatty acid, as well as the connection between ACSL and some metabolic diseases.     

谷氨酰胺体外对大鼠晶状体上皮细胞HSP70和NF-κB表达的影响

目的:探讨谷氨酰胺(Gln)体外对晶状体上皮细胞HSP70和NF-κB的作用。方法:采用ELISA法检测在MEM培养液里培养3,12和24h晶状体上皮细胞HSP70和NF-κB的表达水平。结果:谷氨酰胺注射各组HSP70蛋白的表达量均显著高于对照组(P<0.05),谷氨酰胺注射各组NF-κB的表达较对照组显著降低(P<0.05)。结论:谷氨酰胺能诱导晶状体上皮HSP70蛋白的表达,抑制NF-κB的表达。     

Glucosamine attenuates increases of intraabdominal fat,serum leptin levels,and insulin resistance induced by a high-fat diet in rats

The levels of circulating nonesterified fatty acids increase during obesity and contribute to insulin resistance by inhibiting insulin-stimulated glucose transport and phosphorylation in human muscles. In cells, glucose-6-phosphate is primarily used in glycogenesis and glycolysis; only 1% to 3% is converted to glucosamine-6-phosphate, which enters the hexosamine-biosynthesis pathway. The major end product of this pathway, uridine-5′-diphosphate-N-acetyl-glucosamine, which is increased by exogenous glucosamine (GlcN) administration, mediates insulin resistance. We hypothesized that the administration of GlcN to rats receiving a high-fat (HF) diet may potentiate the effects of an HF diet on glucose tolerance and other metabolic variables. To evaluate this relationship, 2 groups of rats were fed with a control or HF diet; and another 2 groups received glucosamine hydrochloride at a dose of 500 mg/kg dissolved in drinking water for 21 weeks. Metabolic variables related to insulin resistance were then measured. The levels of blood glucose and serum insulin were higher in a glucose tolerance test in the HF group as compared with the control group. Rats receiving GlcN had reduced liver glycogen and only slightly worsened glucose tolerance as compared with control rats, although this did not induce insulin resistance as evaluated by the homeostasis model assessment. Glucosamine administration was able to partially or completely inhibit some effects of the HF diet by reducing fat depot weight and serum leptin levels, thus resulting in a smaller increase in the insulinemic response to a glucose injection and lower postabsorptive glycemia.     

谷氨酰胺强化肠内营养支持对全胃切除术后营养及免疫功能影响的研究

目的 探讨经胃十二指肠管谷氨酰胺强化肠内营养支持对全胃切除术后营养及免疫功能的影响。 方法 收集2006年10月至2009年2月上海交通大学医学院附属仁济医院普外科接受全胃切除术的72例进展期胃癌病人,随机分为谷氨酰胺强化肠内营养（EN+Gln）组、肠内营养（EN）组和对照组。在术后48h内待一般情况稳定后即开始给予肠内营养。观察肛门排气恢复时间、术后并发症发生率及住院时间,分别于术前1d、术后3d和12d检测总蛋白、白蛋白、前白蛋白、转铁蛋白,术后7d检测外周血NK细胞、CD4+T细胞、CD8+ T细胞和免疫球蛋白IgM、IgG。 结果 EN+Gln组与EN组术后并发症发生率及住院时间均低于对照组。术后第3天3组病人总蛋白、白蛋白、前白蛋白及转铁蛋白均较术前明显下降,且对照组总蛋白、白蛋白及转铁蛋白下降幅度更大（P<0.05）,术后第14天时EN+Gln组与EN组的总蛋白、白蛋白、前白蛋白及转铁蛋白水平明显高于对照组（P<0.05）。术后第7天EN+Gln组CD4+T细胞、NK细胞百分比和IgM、IgG较术前明显升高,且较EN组和对照组同期显著升高（P<0.05）。 结论 全胃切除术病人围手术期谷氨酰胺强化肠内营养支持有助于改善营养和免疫状态、促进术后恢复,减少并发症发生率和缩短住院时间。     

脓毒症与免疫营养治疗

脓毒症病人常因能量需求增加及某些特殊营养素缺乏,从而导致器官功能障碍。营养支持对脓毒症病人非常重要。某些特殊营养素包括谷氨酰胺、精氨酸、ω-3脂肪酸、核苷酸、维生素及微量元素除了能够发挥营养支持的作用之外,还能够发挥免疫调节作用以改善机体防御能力并促进恢复。谷氨酰胺能够抑制促炎因子反应并维持肠道黏膜屏障及细胞防御功能。精氨酸通过一氧化氮依赖性及非依赖性途径发挥代谢性、免疫调节性及血流动力学作用。ω-3脂肪酸能够减轻炎性反应。维生素和微量元素能够发挥抗氧化作用。但是,脓毒症病人免疫营养治疗仍存在理论与实践上的差异,目前并不能常规应用于脓毒症病人的治疗。临床医生应根据脓毒症病人的具体情况及相关影响因素仔细选择营养制剂的成分,合理应用免疫营养治疗。     

不同潮气量机械通气对大鼠肺组织γ-谷氨酰半胱氨酸合成酶和转化生长因子β1的影响

目的 观察不同潮气量机械通气大鼠肺组织γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase,γ-GCS)和转化牛长因子β_1(transforming growth factor-β_1,TGF-β_1)mRNA及其蛋白表达水平,探讨氧化/抗氧化失衡在呼吸机相关性肺损伤(ventilator associated lung injury,Vau)中的作用.方法 24只雄性Wistar大鼠随机(随机数字法)分为对照组、小潮气量组和大潮气量组(各组8只),测定其肺组织和血浆中MDA含量,并分别采用原位分子杂交技术和免疫组织化学染色法检测肺组织γ-GCS,TGF-β_1,mRNA及其蛋白表达水平,并进行相关性分析.组间差异比较采用方差分析法,各组均数间两两比较采用SNK-q检验,以P＜0.05表示差异有统计学意义,相关性分析采用Pearson直线相关分析法.结果 与对照组和小潮气量组比较,大潮气量组大鼠肺组织和血浆MDA含量、肺组织TGF-β_1mRNA及其蛋白表达水平明显升高(P＜0.01),而γ-GCS mRNA及其蛋白表达水平则明显低于对照组和小潮气量组(P＜0.01);小潮气量组与对照组各项指标比较,差异无统计学意义(P＞0.05).相关分析结果表明大潮气量组大鼠肺气道卜皮细胞γ-GCS和TGF-β_1 mRNA及其蛋白表达均呈负相关(r值分别为-0.96,-0.85,P＜0.01).结论 大潮气量机械通气町诱导肺组织TGF-β_1 mRNA及其蛋白高表达,使γ-GCS表达降低,谷胱甘肽合成减少,导致局部肺组织氧化/抗氧化系统失衡,是VALI发生的重要因素之一.     

NMDA-receptor activation and nitroxidative regulation of the glutamatergic pathway during nociceptive processing

The role of peroxynitrite (PN) as a mediator of nociceptive signaling is emerging. We recently reported that the development of central sensitization that follows the intraplantar injection of carrageenan in rats is associated with spinal PN synthesis. We now demonstrate that a significant pathway through which spinal PN modulates central sensitization is post-translational tyrosine nitration of key proteins involved in the glutamatergic pathway, namely glutamate transporter GLT-1 and glutamine synthetase (GS). We also reveal that spinal activation of the N-methyl-d-aspartate (NMDA) receptor provides a source of PN in this setting. Intraplantar injection of carrageenan led to the development of thermal hyperalgesia as well as nitration of GLT-1 and GS in dorsal horn tissues. Pretreatment with the PN decomposition catalyst FeTM-4-PyP⁵⁺ [Fe(III)5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin] or the NMDA receptor antagonist MK-801 blocked the development of hyperalgesia. Carrageenan-induced hyperalgesia was also associated with nitration and inactivation of spinal mitochondrial superoxide dismutase (MnSOD) known to provide a critical source of PN during central sensitization. Nitration of GLT-1 and GS contributes to central sensitization by enhancing glutamatergic neurotransmission. Our results support the critical role of nitroxidative stress in the development of hyperalgesia and suggest that post-translational nitration of enzymes and transporters linked to glutamatergic neurotransmission represent a novel mechanism of central sensitization.