Sequences in the proximal 5′ flanking region of the rat neuron-specific enolase (NSE) gene are sufficient for cell type-specific reporter gene expression

We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic mouse studies have shown that a 1.8-kb segment of the ratNSE gene 5′ flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a reporter gene in parallel with endogenousNSE. These data suggest thatcis-acting elements responsible for the spatial and temporal pattern ofNSE gene expression are located within the proximal 1.8 kb of the 5′ flanking sequence. To further investigate this region, we joined the 1.8-kb regulatory cassette to thecat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5′ end. These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity. We found that as little as 255 bp of 5′ flanking sequence was able to confer cell type-specificity on the reporter gene. Further truncation to 120 bp of 5′ sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating thatcis-acting elements controlling the regulation ofNSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2 site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific peripherin gene. Reduction to 95 bp of 5′ sequence resulted in a slight downregulation of CAT activity in all cell lines tested, and further truncation to 65 bp of 5′ sequence caused a universal reduction to background levels of CAT activity, concomitant with the disruption of the basalNSE promoter. Our results show that the 5′ flanking region of theNSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible for this are located within the proximal 255 bp.     

Enolase 1 of Candida albicans binds human CD4+ T cells and modulates naïve and memory responses

To obtain a better understanding of the biology behind life-threatening fungal infections caused by Candida albicans, we recently conducted an in silico screening for fungal and host protein interaction partners. We report here that the extracellular domain of human CD4 binds to the moonlighting protein enolase 1 (Eno1) of C. albicans as predicted bioinformatically. By using different anti-CD4 monoclonal antibodies, we determined that C. albicans Eno1 (CaEno1) primarily binds to the extracellular domain 3 of CD4. Functionally, we observed that CaEno1 binding to CD4 activated lymphocyte-specific protein tyrosine kinase (LCK), which was also the case for anti-CD4 monoclonal antibodies tested in parallel. CaEno1 binding to naïve human CD4⁺ T cells skewed cytokine secretion toward a Th2 profile indicative of poor fungal control. Moreover, CaEno1 inhibited human memory CD4⁺ T-cell recall responses. Therapeutically, CD4⁺ T cells transduced with a p41/Crf1-specific T-cell receptor developed for adoptive T-cell therapy were not inhibited by CaEno1 in vitro. Together, the interaction of human CD4⁺ T cells with CaEno1 modulated host CD4⁺ T-cell responses in favor of the fungus. Thus, CaEno1 mediates not only immune evasion through its interference with complement regulators but also through the direct modulation of CD4⁺ T-cell responses.     

肺癌癌组织中M2-PK、和ENO1的表达水平及对其生存期的预测价值

目的研究肺癌组织中肿瘤型丙酮酸激酶(M2-PK)和烯醇化酶-α(ENO1)的表达对患者生存期的预测价值。方法采用免疫组织化学染色法分别对182例肺癌患者和68例肺良性病变组织切片进行检测,观察M2-PK和ENO1在肺癌组织和良性病变组织中的表达情况,分析M2-PK和ENO1表达情况与肺癌临床病理特征及患者生存期的关系。结果肺癌组织中M2-PK和ENO1阳性表达率分别为78.02%、67.58%,高于良性病变组织的32.36%和29.41%,差异具有统计学意义(P<0.001),其表达与肺癌浸润分期(P=0.027,P=0.019)、分化程度(P=0.041,P=0.037)、淋巴转移(P=0.009,P=0.001)、TNM分期均显著相关(P=0.012,P=0.015);肺癌组织中M2-PK与ENO1表达呈正相关(r=0.769,P<0.05);肺癌组织中M2-PK和ENO1的高表达与患者5存活率分别为37.35%和32.31%,低于低表达的62.62%和52.99%,差异具有统计学意义(P<0.05);M2-PK与ENO1共高表达患者5年存活率为28.84%,低于共低表达的59.32%(P<0.05)。结论肺癌组织中M2-PK和ENO1的高表达与肺癌的发生、发展及预测患者的生存期有一定的相关性,可能存在临床应用价值。     

脑出血继发癫痫患者血清NSE和炎症细胞因子浓度测定对病情的预测价值

目的:探讨脑出血急性期继发癫痫患者血清NSE,炎症细胞因子的变化及对病情的预测价值。方法:检测脑出血继发癫痫组,单纯脑出血组和健康对照组各31例对象的血清肿瘤坏死因子(TNF-α),白介素-β(IL-1β),白介素-6(IL-6),C-反应蛋白(CRP)与神经元特异性烯醇化酶(NSE)的含量,并同步采用美国卫生研究院卒中量表(NIHSS)进行评分。结果:组间脑出血继发癫痫组和单纯脑出血组患者各指标水平均高于健康对照组,(P<0.001),有非常显著差异。多元回归分析显示炎症细胞因子的变化与NSE及NIHSS有线性关系,NSE是NIHSS的独立预测因子。结论:脑出血急性期继发癫痫的病理机制是炎症细胞因子进一步表达导致神经元再次受损,NSE升高,检测血清中NSE浓度对患者的病情预测有重要意义。     

Elevated concentrations of γ-enolase in renal cell tumors in rats: similarity to renal cell carcinoma in man

Concentrations of enolase isozymes in normal kidney and renal cell tumors in rats were determined using a highly sensitive enzyme immunoassay, and the isozymes were immunohistochemically localized in tissue sections. Levels of -enolase in renal cell turnors were significantly lower than in normal kidney, whereas those of -enolase were significantly elevated (mean ±SD:211±129 ng/mg protein, n=15, as compared to 27.1±2.9 ng/mg protein, n=7). The proportion of -enolase in the total enolases in the tumor tissues (1.6±0.5%) was significantly higher than in normal kidney (0.15±0.005). Immunohistochemistry revealed epithelial cells of all nephron segments to be positive for the -isozyme, whereas -enolase staining was strongly positive only in the loops of Henle, being faint in the distal tubules and absent in the proximal tubules. Both - and -enolases demonstrated positive immunostaining in all of the seven renal cell tumors studied. These findings indicate that an isozyme switch from - to -enolase occurs during rat kidney carcinogenesis, taking into account the derivation from proximal tubules, consistent with the findings for renal cell carcinomas in man.     

高压氧治疗对脑梗死患者血清神经元特异性烯醇化酶的影响

目的 为观察脑梗死后血清神经元特异性烯醇化酶的动态变化，以及高压氧治疗后血清NSE的变化，判断高压氧治疗脑梗死的疗效。方法 将脑梗死患者84例，随机分为高压氧治疗组42例，常规治疗组42例，分别测定发病第1、2、3、12天的血清NSE水平，采用50例健康老年人的血清NSE作为正常对照组。结果 脑梗死患者血清NES水平明显高于正常组，P〈0．01，HBO治疗组第2、3、12天血清NSE水平与常规治疗组相比有显著差异，（第2天P〈0．05，第3天，第12天P均〈0．01）。结论 NSE可以作为反映脑梗塞患者脑细胞损伤程度的生化指标，早期高压氧治疗有助于减轻脑梗死病情。     

Proteomic characterisation of neuronal sphingolipid-cholesterol microdomains: role in plasminogen activation

Sorting of certain membrane proteins requires a mechanism involving rafts, protein-lipid complexes enriched in glycosphingolipids and cholesterol. These microdomains remain at the plasma membrane of different cell types and play a role in signal transduction. Although recent reports have begun to describe molecules associated with rafts, their protein composition remains largely unknown, especially in neuronal cells. To address this question, we have purified detergent-insoluble raft fractions (DRMs) from primary cultures of hippocampal neurons. Bidimensional gel analysis and pharmacological raft lipid manipulation allowed the identification of neuronal raft proteins and their characterisation by MALDI-TOF analysis. Enolases were found among the proteins identified and functional studies demonstrate their participation in plasminogen binding. We also show the specific enrichment in rafts of several other plasminogen binding molecules and the exclusive activation of plasminogen to the protease plasmin in these microdomains. These observations suggest that neuronal rafts may play, in addition to intracellular signaling, a role in extracellular/membrane protein proteolysis.     

Mitochondrial associated metabolic proteins are selectively oxidized in A30P alpha-synuclein transgenic mice--a model of familial Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra compacta. alpha-Synuclein is strongly implicated in the pathophysiology of PD because aggregated alpha-synuclein accumulates in the brains of subjects with PD, mutations in alpha-synuclein cause familial PD, and overexpressing mutant human alpha-synuclein (A30P or A53T) causes degenerative disease in mice or drosophila. The pathophysiology of PD is poorly understood, but increasing evidence implicates mitochondrial dysfunction and oxidative stress. To understand how mutations in alpha-synuclein contribute to the pathophysiology of PD, we undertook a proteomic analysis of transgenic mice overexpressing A30P alpha-synuclein to investigate which proteins are oxidized. We observed more than twofold selective increases in specific carbonyl levels of three metabolic proteins in brains of symptomatic A30P alpha-synuclein mice: carbonic anhydrase 2 (Car2), alpha-enolase (Eno1), and lactate dehydrogenase 2 (Ldh2). Analysis of the activities of these proteins demonstrates decreased functions of these oxidatively modified proteins in brains from the A30P compared to control mice. Our findings suggest that proteins associated with impaired energy metabolism and mitochondria are particularly prone to oxidative stress associated with A30P-mutant alpha-synuclein.     

Strict versus moderate glucose control after resuscitation from ventricular fibrillation

OBJECTIVE: Elevated blood glucose is associated with poor outcome in patients resuscitated from out-of-hospital cardiac arrest (OHCA). Our aim was to determine whether strict glucose control with intensive insulin treatment improves outcome of OHCA patients. DESIGN: A randomized, controlled trial. SETTING: Two university hospital intensive care units. PATIENTS: Ninety patients resuscitated from OHCA with ventricular fibrillation detected as the initial rhythm were treated with therapeutic hypothermia. INTERVENTIONS: Patients were randomized into two treatment groups: a strict glucose control group (SGC group), with a blood glucose target of 4-6 mmol/l, or a moderate glucose control group (MGC group), with a blood glucose target of 6-8 mmol/l. Both groups were treated with insulin infusion for 48 h, because a control group with no treatment was considered unethical. MEASUREMENTS AND RESULTS: Baseline data were similar in both groups. In the SGC group 71% of the glucose measurements were within the target range compared with 41% in the MGC group. Median glucose was 5.0 mmol/l in the SGC group and 6.4 mmol/l in the MGC group. The occurrence of moderate hypoglycemic episodes was 18% in the SGC group and 2% in the MGC group (p = 0.008). No episodes of severe hypoglycemia occurred. Mortality by day 30 was 33% in the SGC group and 35% in the MGC group (p = 0.846); the difference was 2% (95% CI -18% to +22%). CONCLUSIONS: We found no additional survival benefit from strict glucose control compared with moderate glucose control with a target between 6 and 8 mmol/l in OHCA patients.     

Identification and characterization of a novel protective antigen,Enolase of Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is a porcine and human pathogen with adhesive and invasive properties. The absence of suitable vaccine or virulent marker can be the bottleneck to control SS2 infection. In the present study, a novel immunogenic Enolase identified in the previous study was inducibly overexpressed in Escherichia coli, and the purified recombinant protein could elicit a significant humoral antibody response and confer efficient immunity against challenge with lethal dose of SS2 or SS7 infection in mouse model. The roles Enolase plays in pathogenicity of SS2 were also explored as reasons for which Enolase could be a protective antigen. The Enolase was an in vivo-induced antigen confirmed by the real-time PCR and could adhere to the Hep-2 cells by the indirect immunofluorescent assay and the inhibition assay. These suggested that Enolase could play important roles in pathogenicity and may serve as a novel vaccine candidate against SS2 infection.