Activity of anandamide (AEA) metabolic enzymes in rat placental bed

Endocannabinoids are endogenous lipid mediators, with anandamide (AEA) being the first member identified. It is now widely accepted that AEA influences early pregnancy events and its levels, which primarily depend on its synthesis by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and degradation by a fatty acid amide hydrolase (FAAH), must be tightly regulated. Previous studies demonstrated that AEA levels require in situ regulation of these respective metabolic enzymes, and thus, any disturbance in AEA levels may impact maternal remodeling processes occurring during placental development. In this study, the activities of the AEA-metabolic enzymes that result in the establishment of proper local AEA levels during rat gestation were examined. Here, we demonstrate that during placentation NAPE-PLD and FAAH activities change in a temporal manner. Our findings suggest that NAPE-PLD and FAAH create the appropriate AEA levels required for tissue remodeling in the placental bed, a process essential to pregnancy maintenance.     

Role of the basolateral amygdala in mediating the effects of the fatty acid amide hydrolase inhibitor URB597 on HPA axis response to stress

The endocannabinoid system is an important regulator of neuroendocrine and behavioral adaptation in stress related disorders thus representing a novel potential therapeutic target. The aim of this study was to determine the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on stress mediators of HPA axis and to study the role of the basolateral amygdala (BLA) in responses to forced swim stress.Systemic administration of URB597 (0.1 and 0.3 mg/kg) reduced the forced swim stress-induced activation of HPA axis. More specifically, URB597 decreased stress-induced corticotropin-releasing hormone (CRH) mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus, and pro-opiomelanocortin (POMC) mRNA expression dose-dependently in pituitary gland without affecting plasma corticosterone levels. URB597 treatment also attenuated stress-induced neuronal activation of the amygdala and PVN, and increased neuronal activation in the locus coeruleus (LC) and nucleus of solitary tract (NTS). Injection of the CB1 receptor antagonist AM251 (1 ng/side) in the BLA significantly attenuated URB597-mediated effects in the PVN and completely blocked those induced in the BLA.These results suggest that the BLA is a key structure involved in the anti-stress effects of URB597, and support the evidence that enhancement of endogenous cannabinoid signaling by inhibiting FAAH represents a potential therapeutic strategy for the management of stress-related disorders.     

外周疼痛模型大鼠脊髓背角环氧化酶表达时程的研究

目的 探讨环氧化酶参与疼痛过程的特点,从而为揭示疼痛的中枢机制提供线索.方法 选取雄性Sprague-Dawley大鼠50只,20只大鼠右侧后肢足掌皮下注射生理盐水100μl作为对照组.30只大鼠右侧后肢足掌皮下注射5%(体积分数)福尔马林液100μl作为实验组,注射后2、6 h和1、3、7、14天作为测试时间点,每个时间点随机选取5只大鼠,断头后取大鼠脊髓腰膨大部位1 cm(L4～5)组织并分离出背角部分,以免疫印迹法检测不司时间点环氧化酶1和环氧化酶2蛋白的表达.结果 福尔马林注射后大鼠脊髓环氧化酶1表达在注射后1 d开始增多,达到对照组的(2.44±0.43)倍,持续到14 d表达仍明显增多,达对照组的(3.30±0.39)倍.环氧化酶2的表达仪在注射后2 h表达明显增多,达对照组的(1.87±0.34)倍,注射后6 h即恢复到正常,观察到第14天仍然没有变化.结论 环氧化酶1和2可能都参与疼痛的中枢机制,环氧化酶2在疼痛的早期或发生中起作用,而环氧化酶1可能参与疼痛的维持.

Abstract：

Objective To compare the expression of two cyclooxygenase (COX) isoforms (COX-1 and COX-2) in the spinal cord of rats with inflammatory pain following peripheral formalin injection. Methods Experiments were performed on adult male Sprague-Dawley rats. 100μl of 5% formalin were injected subcutaneously into plantar hind paw of 30 rats to induce pain. The changes of COX-1 and COX-2 expression were evaluated by western blot in 30 rats, respectively at 2 hours, 6 hours, 1 day, 3 days, 7 days and 14 days after injection. The control group rats were injected with 100 μl of 0.9% saline instead of formalin in 20 rats. Results The expression of COX-1 significantly increased at 1day, 3 days, 7 days and 14 days, but the expression of COX-2 only transiently increased at 2 hours. Conclusions COX-1 and COX-2 are increasingly expressed in the spinal cord, furthermore,spinal COX-2 expression is induced early and transiently increased. COX-1 is up-regulated later and remains elevated for long time, which may explain the long-term hyperalgesia evoked by formalin injection.     

Long-term inhibition of UCHL1 decreases hypertension and retinopathy in spontaneously hypertensive rats

ObjectiveTo investigate the role of the deubiquitinase ubiquitin C-terminal hydrolase L1 (UCHL1) in hypertension and retinopathy in the spontaneously hypertensive rat (SHR).MethodsWistar–Kyoto (WKY) rats and SHRs were administered the UCHL1 inhibitor LDN57444 (20 μg/kg/day) for 4 months. Pathological changes were detected with hematoxylin and eosin, immunofluorescence, and dihydroethidium staining. The mRNA and protein expression of UCHL1 were examined by real-time PCR and immunoblotting analysis.ResultsAt 6 months of age, SHRs showed significantly increased mRNA and protein levels of UCHL1 in the retina compared with WKY rats. Moreover, SHRs exhibited significantly increased central retinal thickness, inflammation, and reactive oxygen species production compared with WKY rats, and these effects were markedly attenuated by systemic administration of the UCHL1 inhibitor LDN57444. The beneficial effects of LDN57444 were possibly associated with reduced blood pressure and the inactivation of several signaling pathways.ConclusionUCHL1 is involved in hypertension and retinopathy in SHRs, suggesting that UCHL1 may be used as a potential therapeutic target for treating hypertensive retinopathy.     

泛素羧基末端水解酶L1在年龄相关性白内障晶状体上皮细胞中的表达及其抗氧化作用

背景 氧化损伤是年龄相关性白内障发生的主要原因,而泛素蛋白酶系统参与晶状体的分化发育,研究发现其关键酶泛素羧基末端水解酶L1( UCHL1)参与帕金森病和阿尔茨海默病等年龄相关性疾病的发生发展,且与氧化应激有关. 目的 研究UCHL1在年龄相关性白内障发病过程中的作用.方法 收集24例单纯年龄相关性白内障患者术后获得的晶状体囊膜(皮质性白内障12例、核性白内障12例)、5例正常人晶状体前囊膜上皮和人晶状体上皮细胞(LECs)系SRA01/04细胞,采用免疫荧光法检测UCHL1在各组人晶状体前囊膜上皮细胞中的表达情况.构建UCHL1真核表达质粒,鉴定后采用脂质体转染法转染SRA01/04细胞作为UCHL1过表达组,同时采用绿色荧光蛋白(GFP)真核表达质粒转染SRA01/04细胞作为GFP过表达组,使用梯度过氧化氢叔丁醇(TBHB)处理24h后,采用MTT法检测各组人LECs的活性变化.结果 免疫荧光检测表明,UCHL1在各组人LECs中均有表达,但在正常晶状体囊膜、皮质性白内障以及核性白内障晶状体囊膜上皮细胞表达量的总体差异有统计学意义(F=13.441,P=0.000).皮质性白内障组以及核性白内障组晶状体囊膜上皮细胞中UCHL1的表达量均低于正常晶状体组(P=0.000、0.000),但皮质性白内障组和核性白内障组之间UCHL1的表达量差异无统计学意义(P=0.164).Western blot鉴定结果表明,UCHL1真核表达质粒转染后可见SRA01/04细胞中UCHL1的强表达.MTT检测结果显示,0.3 mol/L TBHB处理24 h后,UCHL1过表达组细胞活性吸光度(A570/630)值与GFP过表达组比较有增高的趋势,而0.2、0.4、0.5 mol/LTBHP均导致SRA01/04细胞的耐受或者大量凋亡. 结论 UCHL1具有抗氧化作用,且可能在年龄相关性白内障的发生发展过程中起抑制作用.     

No evidence of maternal cell colonization in reverted liver nodules of tyrosinemia type I patients

BACKGROUND AND AIMS: Hereditary tyrosinemia type I (HTI) is a recessively inherited disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine catabolic pathway. The mosaic pattern of FAH expression observed in the livers of >85% of studied patients was shown to result from the correction of the mutation in one of the FAH alleles. Bilateral cell trafficking can occur between mother and fetus and such an event could be responsible for the chimerism observed in some diseases. It also has been reported that the liver repopulation observed in a HTI murine model by serial transplantation of bone marrow-derived cells was caused by a fusion of these cells to host hepatocytes. These observations led us to test the possibility that the transfer of nucleated heterozygous maternal cells in the fetal circulation could be responsible for the mosaic liver expression of FAH in HTI patients. METHODS: We used polymorphic markers of short cytosine-adenine DNA repeats to compare DNA from corrected liver sections of 4 HTI patients with DNA from their parents' blood. RESULTS: Genotyping showed that only one maternal allele is present in DNA isolated from FAH-expressing liver nodules of each proband for at least 1 marker. CONCLUSIONS: The corrected liver nodules in HTI patients are not of maternal origin and do not support cell trafficking and cell fusion as mechanisms of correction of the gene defect in hepatocytes of tyrosinemia patients.     

Association between polymorphisms of microsomal epoxide hydrolase and COPD: results from meta-analyses

Background and objective: COPD is a complex polygenic disease in which gene–environment interactions are very important. The gene encoding microsomal epoxide hydrolase (EPHX1) is one of several candidate loci for COPD pathogenesis and is highly polymorphic. Based χ on the polymorphisms of EPHX1 gene (tyrosine/histidine 113, histidine/arginine 139), the population can be classified into four groups of putative EPHX1 phenotypes (fast, normal, slow and very slow). A number of studies have investigated the association between the genotypes and phenotypes of EPHX1 and COPD susceptibility in different populations, with inconsistent results. A systematic review and meta‐analysis of the published data was performed to gain a clearer understanding of this association. Methods: The MEDLINE database was searched for case–control studies published from 1966 to August 2007. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Results: Sixteen eligible studies, comprising 1847 patients with COPD and 2455 controls, were included in the meta‐analysis. The pooled result showed that the EPHX1 113 mutant homozygote was significantly associated with an increased risk of COPD (OR 1.59, 95% CI: 1.14–2.21). Subgroup analysis supported the result in the Asian population, but not in the Caucasian population. When the analysis was limited to only the larger‐sample‐size studies, studies in which controls were in Hardy–Weinberg equilibrium and studies in which controls were smokers/ex‐smokers, the pooled results supported the conclusion. The EPHX1 139 heterozygote protected against the development of COPD in the Asian population, but not in the Caucasian population. The other gene types of EPHX1 113 and EPHX1 139 were not associated with an increased risk of COPD. The slow activity phenotype of EPHX1 was associated with an increased risk of COPD. The fast activity phenotype of EPHX1 was a protective factor for developing COPD in the Asian population, but not in the Caucasian population. However, the very slow activity phenotype of EPHX1 was a risk for developing COPD in the Caucasian population, but not in the Asian population. Conclusions: The polymorphisms of EPHX1 113 and EPHX1 139 are genetic contributors to COPD susceptibility in Asian populations. The phenotypes of EPHX1 were contributors to overall COPD susceptibility.     

Functional analysis of an α-1,2-mannosidase from <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Identification of enzymes that are expressed during host colonization and characterization of their biochemical properties are prerequisite to understanding their role in the pathogen–host interaction. Nine α-1,2-mannosidase homologs were identified in the analysis of the Magnaporthe oryzae genome. Endoplasmic reticulum localized α-1,2-mannosidases play an important role in protein glycosylation. However, several members of the α-1,2-mannosidase gene family are predicted to be secreted. The biological role of such extracellular enzymes in host colonization has not been defined. Here, we characterized a secreted α-1,2-mannosidase of M. oryzae, MGG_00994.6, and found that the mature polypeptide is a glycoprotein capable of hydrolyzing α-1,2 linked mannobiose. The gene is expressed during growth in vitro and during colonization on rice plants, however, deletion of the gene did not affect pathogenicity. Five other members of the α-1,2-mannosidase of M. oryzae were expressed with a pattern similar to MGG_00994.6, suggesting the potential for functional redundancy. These results form the basis for additional studies on the role of this gene family in the rice blast fungus and its interaction with rice. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.