Changes in chromogranin A-immunoreactive guinea-pig pulmonary neuroendocrine cells after sensitization and challenge with ovalbumin

Immunohistochemical study with antisera to chromogranin A and neuron-specific enolase, a general marker for nerves and endocrine cells, was used to quantify changes in bronchial neuroendocrine cells in guineapigs sensitized and challenged with ovalbumin. Actively sensitized animals were killed 2, 6, 24, 48, 72, and more than 144 hours after being challenged by an aerosolized solution of ovalbumin. The number of chromogranin A-immunoreactive cells was significantly greater in sensitized but unchallenged animals and in sensitized animals killed 2 and 6 h after challenge when compared to controls; it decreased significantly in animals killed more than 24 h after challenge when compared to sensitized, unchallenged animals. The number of neuron-specific-enolase-immunoreactive cells did not change. We conclude that the peptide content of bronchial neuroendocrine cells increases during sensitization and in the early phase of a hypersensitivity reaction, and that the cells release their granule contents in the late phase of such a reaction. They may therefore play a role in immunoallergic events in the lung.     

急性脑梗死患者血清同型半胱氨酸、D-二聚体和神经元特异性烯醇化酶水平的变化及其临床意义

目的：探讨急性脑梗死（acute cerebral infarction,ACI）患者血清同型半胱氨酸（homocysteine,Hcy）、D-二聚体（d—dimer,DD）和神经元特异性烯醇化酶（neuronspecific enolase,NSE）水平的变化及其临床意义,为ACI的诊治、预防和病情监测提供更多的实验依据。方法：选择我院神经内科2010年1月~2012年7月收治的急性脑梗死患者82例,及同期50例健康体检人群作为正常对照组,观察和比较不同病灶及不同神经功能缺损程度患者的血清Hcy、DD和NSE水平,同时对急性脑梗死患者急性期和恢复期血中Hcy、DD、NSE进行分析。结果：①急性脑梗死组Hcy、DD和NSE含量均显著高于正常对照组（P〈0．01）,急性脑梗死患者急性期血清Hcy、DD和NSE水平显著高于恢复期（P〈0．01）,但恢复期与对照组之间无显著性差异（P〉0．05）。②根据病灶大小,急性脑梗死各组之间血中Hcy、DD和NSE含量亦存在显著性差异（P〈0．05）。③根据神经功能缺损程度,脑梗死各组之间血中Hcy、DD和NSE含量亦存在显著性差异（P〈0．05）。结论：Hcy、DD和NSE与ACI的发生、发展密切相关,Hcy、DD和NSE的检测对ACI的诊断、治疗和预后均有重要意义。     

急性脑梗死患者血清同型半胱氨酸、D- 二聚体和神经元特异性 烯醇化酶水平的变化及其临床意义

目的：探讨急性脑梗死（acute cerebral infarction,ACI）患者血清同型半胱氨酸（homocysteine,Hcy）、D- 二聚体（d-dimer,DD） 和神经元特异性烯醇化酶（neuronspecific enolase,NSE）水平的变化及其临床意义,为ACI的诊治、预防和病情监测提供更多的实 验依据。方法：选择我院神经内科2010 年1月～2012 年7 月收治的急性脑梗死患者82 例,及同期50 例健康体检人群作为正常 对照组,观察和比较不同病灶及不同神经功能缺损程度患者的血清Hcy、DD和NSE 水平,同时对急性脑梗死患者急性期和恢复 期血中Hcy、DD、NSE 进行分析。结果：①急性脑梗死组Hcy、DD和NSE含量均显著高于正常对照组（P<0.01）,急性脑梗死患者 急性期血清Hcy、DD和NSE 水平显著高于恢复期（P<0.01）,但恢复期与对照组之间无显著性差异（P>0.05）。②根据病灶大小,急 性脑梗死各组之间血中Hcy、DD和NSE 含量亦存在显著性差异（P<0.05）。③根据神经功能缺损程度,脑梗死各组之间血中Hcy、 DD和NSE 含量亦存在显著性差异（P<0.05）。结论：Hcy、DD 和NSE 与ACI 的发生、发展密切相关,Hcy、DD 和NSE的检测对 ACI的诊断、治疗和预后均有重要意义。     

Neuropeptide changes and neuroactive amino acids in CSF from humans and sheep with neuronal ceroid lipofuscinoses (NCLs, Batten disease)

Anomalies in neuropeptides and neuroactive amino acids have been postulated to play a role in neurodegeneration in a variety of diseases including the inherited neuronal ceroid lipofuscinoses (NCLs, Batten disease). These are often indicated by concentration changes in cerebrospinal fluid (CSF). Here we compare CSF neuropeptide concentrations in patients with the classical juvenile CLN3 form of NCL and the classical late infantile CLN2 form with neuropeptide and neuroactive amino acid concentrations in CSF from sheep with the late infantile variant CLN6 form.A marked disease related increase in CSF concentrations of neuron specific enolase and tau protein was noted in the juvenile CLN3 patients but this was not observed in an advanced CLN2 patient nor CLN6 affected sheep. No changes were noted in S-100b, GFAP or MBP in patients or of S-100b, GFAP or IGF-1 in affected sheep. There were no disease related changes in CSF concentrations of the neuroactive amino acids, aspartate, glutamate, serine, glutamine, glycine, taurine and GABA in these sheep.The changes observed in the CLN3 patients may be progressive markers of neurodegeneration, or of underlying metabolic changes perhaps associated with CLN3 specific changes in neuroactive amino acids, as have been postulated. The lack of changes in the CLN2 and CLN6 subjects indicate that these changes are not shared by the CLN2 or CLN6 forms and changes in CSF concentrations of these compounds are unreliable as biomarkers of neurodegeneration in the NCLs in general.     

Molecular dynamics simulation of interactions in glycolytic enzymes

Two glycolytic enzymes, phosphoglycerate mutase (PGM) and enolase from Saccharomyces cerevisiae, have been chosen to detect complex formation and possible channeling, using molecular dynamics simulation. The enzymes were separated by 10 angstroms distance and placed in a water-filled box of size 173 x 173 x 173 angstroms. Three different orientations have been investigated. The two initial 3-phosphoglycerate substrate molecules near the active centers of the initial structure of PGM have been replaced with final product (2-phosphoglycerate) molecules, and 150 mM NaCl together with three Mg2+ ions have been added to the system to observe post-catalytic activity under near-physiological conditions. Analysis of interaction energies and conformation changes for 3 nsec simulation indicates that PGM and enolase do show binding affinity between their near active regions, which is necessary for channeling to occur. Interaction of the C-terminal residues Ala239 and Val240 of PGM (which partially "cap" the 2-phosphoglycerate) with enolase also favors the existence of channeling.     

Structure and conformational stability of a tetrameric thermostable N‐succinylamino acid racemase

The N‐succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co²⁺ by using hydrodynamic and spectroscopic techniques. The Co²⁺, among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by α‐helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten‐globule‐like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co²⁺ did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 757–772, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

瑞芬太尼联合异丙酚对颅内动脉瘤夹闭术患者麻醉效果及血清S100β、NSE水平的影响

目的:探讨瑞芬太尼联合异丙酚对全麻下颅内动脉瘤夹闭术患者麻醉效果及血清中枢神经特异蛋白(S100β)、神经元特异性烯醇化酶(NSE)的影响。方法:选取2017年2月～2019年1月期间于我院行全麻下颅内动脉瘤夹闭术的患者103例,根据随机数字表法将患者分为对照组(n=51)和研究组(n=52),对照组给予异氟醚联合异丙酚麻醉,研究组给予瑞芬太尼联合异丙酚麻醉,比较两组患者麻醉效果、再出血率及血清S100β、NSE水平。结果:研究组硬脑膜切开前(T2)～动脉瘤夹闭即刻(T4)时间点心率(HR)、中心静脉压(MAP)均低于对照组(P0.05)。研究组自主呼吸恢复时间、定向力恢复时间、呼吸后睁眼时间、气管拔管时间、离开手术室时间均短于对照组(P0.05)。研究组T2～手术结束时(T5)时间点血清S100β、NSE水平低于对照组(P0.05)。两组再出血率比较差异无统计学意义(P0.05)。结论:瑞芬太尼联合异丙酚可改善全麻下颅内动脉瘤夹闭术患者的术后指标,维持血流动力学平稳,减轻脑损害。     

Structure of galactarate dehydratase,a new fold in an enolase involved in bacterial fitness after antibiotic treatment

Galactarate dehydratase (GarD) is the first enzyme in the galactarate/glucarate pathway and catalyzes the dehydration of galactarate to 3‐keto‐5‐dehydroxygalactarate. This protein is known to increase colonization fitness of intestinal pathogens in antibiotic‐treated mice and to promote bacterial survival during stress. The galactarate/glucarate pathway is widespread in bacteria, but not in humans, and thus could be a target to develop new inhibitors for use in combination therapy to combat antibiotic resistance. The structure of almost all the enzymes of the galactarate/glucarate pathway were solved previously, except for GarD, for which only the structure of the N‐terminal domain was determined previously. Herein, we report the first crystal structure of full‐length GarD solved using a seleno‐methoionine derivative revealing a new protein fold. The protein consists of three domains, each presenting a novel twist as compared to their distant homologs. GarD in the crystal structure forms dimers and each monomer consists of three domains. The N‐terminal domain is comprised of a β‐clip fold, connected to the second domain by a long unstructured linker. The second domain serves as a dimerization interface between two monomers. The C‐terminal domain forms an unusual variant of a Rossmann fold with a crossover and is built around a seven‐stranded parallel β‐sheet supported by nine α‐helices. A metal binding site in the C‐terminal domain is occupied by Ca²⁺. The activity of GarD was corroborated by the production of 5‐keto‐4‐deoxy‐D‐glucarate under reducing conditions and in the presence of iron. Thus, GarD is an unusual enolase with a novel protein fold never previously seen in this class of enzymes.     

Characterization and expression analyses of two plastidic enolase genes in rice

To verify the presence of enolase related to the chloroplastic glycolysis in rice, database search was carried out and identified seven putative enolase genes in the rice genome. Among them, OsEno1 and OsEno3 encode long proteins with N-terminal extensions. GFP protein fusions of these N-terminal extensions were both targeted to plastids of onion epidermal cell. Promoter::GUS analysis showed that OsEno3 was highly expressed in young developing leaves, but its expression was drastically decreased during leaf development and greening. On the other hand, the expression of OsEno1 was low and detected in limited portions such as leaf sheath at the tiller base. Recombinant OsEno1 protein showed enolase activity with a pH optimum at pH 8.0, whereas OsEno3 did not exhibit detectable activity. Although it remains obscure if OsEno3 encodes a functional enolase in vivo, our results demonstrate that the entire glycolytic pathway does not operate in rice chloroplasts.