N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification

Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification.     

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     

Differential changes in cell morphology,macromolecular composition and membrane protein profiles of neurons and astrocytes in chronic ethanol treated rats

Cellular morphology, macromolecular composition, (DNA, RNA and Protein content) marker enzyme activities for neurons [neuron specific enolase (NSE)] and astrocytes [glutamine synthetase (GS)] and plasma membrane protein profiles in the bulk isolated neurons and astrocytes from control and ethanol treated rats were studied. One month aged Wistar rats were given ethanol as sole drinking fluid for 10 weeks. Scanning electron microscopy revealed a characteristic cell surface smoothening in astrocytes due to ethanol treatment. DNA levels were unaltered, while RNA and Protein contents were decreased in astrocytes and neurons. Further,³H-leucine incorporation into proteins was decreased in neurons and astrocytes derived from ethanol treated rats indicating reduced protein synthesis in neurons and astrocytes. GS activity was affected severely suggesting impairment in astrocytic functions. Plasma membrane protein composition was analyzed by 2-D electrophoresis. The analysis indicated several protein defects in the plasma membranes of neurons and astrocytes, which might be involved in membrane disorder during ethanol challenge.¹²⁵I-Wheat Germ agglutinin binding studies showed three prominent proteins (160, 116 and 97 kDa) in astrocyte membrane fraction suggesting the possible involvement of N-terminal glycoproteins in altered astrocyte morphology during ethanol ingestion. Impairment in the astrocyte cell functions, protein changes in plasma membrane and cellular morphology studies suggest that astrocytes may be more vulnerable than neurons for ethanol effects.     

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.     

胚胎大鼠背根神经节细胞的分离培养、纯化及生物学特性的研究

本实验取E15 SD胎鼠的背根神经节,用胰蛋白酶消化分离成单细胞,在NBl培养基中培养,并通过差速贴壁法进行背根神经节神经元(DRGn)的分离纯化,用神经元特异性的烯醇化酶(NSE)鉴定培养的神经元。结果发现DRGn在体外合适条件下可存活3-4周,DRGn纯化培养的纯度达91％左右。DRGn在体外能存活较长时间,可作为神经科学研究的细胞模型。     

Fluoride inhibition of yeast enolase: Crystal structure of the enolase–Mg2+–F−–Pi complex at 2.6 Å resolution

Enolase in the presence of its physiological cofactor Mg²⁺ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase–Mg²⁺–F^?–P_i has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/σ(F) ≥ 3 to 2.6 Å resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-lo9, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate–enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg²⁺ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg²⁺. The synergism of inhibition by F^? and P_i most probably is due to the requirement of P_i, for the closed conformation. It is possible that other Mg²⁺-dependent enzymes that have OH^? ions bound to the metalion in the transition state also will be inhibited by fluoride ions. © Wiley-Liss, Inc.     

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。     

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.     

Ⅲ期非小细胞肺癌患者精确放疗前后血清CEA、SCC、NSE水平变化及与放疗疗效的关系研究

目的:研究Ⅲ期非小细胞肺癌(NSCLC)患者精确放疗前后血清癌胚抗原(CEA)、鳞状细胞癌相关抗原(SCC)、神经元特异性烯醇化酶(NSE)水平变化及与放疗疗效的关系。方法:选择2014年1月到2016年12月在亳州市人民医院肿瘤科就诊的60例Ⅲ期NSCLC患者纳入此次研究,其中鳞癌14例,腺癌26例,腺鳞癌20例。所有患者均实施4周的精确放疗,放疗后肿瘤标记物水平降低43例,升高17例。根据放疗疗效将患者分为有效组39例,无效组21例。对比不同病理类型的Ⅲ期NSCLC患者CEA、SCC、NSE水平,不同疗效组放疗前后CEA、SCC、NSE水平,并分析患者的肿瘤标记物水平变化与放疗疗效的关系。结果:腺癌Ⅲ期NSCLC患者的CEA、NSE水平高于鳞癌及腺鳞癌者,且腺鳞癌者又高于鳞癌者;SCC水平低于鳞癌及腺鳞癌者,且腺鳞癌者又低于鳞癌者(P0.05)。放疗后有效组CEA、SCC、NSE水平均低于放疗前和无效组,而无效组CEA、SCC、NSE水平高于放疗前(P0.05)。肿瘤标记物水平降低者的有效率高于升高者,差异有统计学意义(P0.05)。结论:在实施精确放疗后治疗有效的Ⅲ期NSCLC患者,其血清CEA、SCC、NSE水平均呈现出明显的下降趋势,且与病理类型密切相关,临床上可重点关注上述指标水平,有助于患者的诊疗过程。