牛小脑肌醇磷脂激酶PI(4)K高产率纯化与特征

对牛小脑膜区肌醇磷脂激酶进行了11 500倍纯化,过程包括：TritonX-100抽提,硫酸铵沉淀,阳离子交换层析(phosphocellulose),亲和层析(Heparin Sepharose CL-6B)和阴离子交换层析(DEAE10,FPLC)等.纯化程度可达95％以上,对SDS-PAGE电泳结果进行扫描分析测其分子质量为56 ku.纯化的肌醇磷脂激酶的特异活性为450 nmol/mg·min, 动力学性质表现为ATP的表观K_m值为7.9×10^-7 mol/L,PI的表观K_m值为6.6×10^-7 mol/L. 腺嘌呤核苷是该酶的有效抑制剂,3.5×10^-7 mol/L腺嘌呤核苷可使该酶活力降低约50％,而TritonX-100对该酶活力具有刺激作用,0.5% TritonX-100可使该酶表现为最高活力.     

绿色木霉MJ1产内切β-葡聚糖苷酶的分离纯化及酶学性质

利用KTAUPC-900快速蛋白液相色谱系统(FPLC)从绿色木霉MJ1固体发酵产物中分离纯化出内切β-葡聚糖苷酶。分离纯化后酶的比活力提高了28.6倍,回收率为19.7%。SDS-PAGE后经BIO-RAD凝胶成像系统分析该内切酶的分子量为64.7kD。酶学试验研究表明:该酶的最适反应温度53℃,最适pH为4.2,Lineweaver-Burk法求得动力学参数,K_m和V_max分别为1.230×10^{-2相似文献}   

细菌3-脱氧葡糖醛酮代谢酶的纯化及性质研究

细菌Bacillus sp.2粗酶液通过(NH₄)₂SO₄分级分离、Q Sepharose FF、Sephadex G-100(Ⅰ)、Hydroxyapatite和Sephadex G-100(Ⅱ)柱层析分离,纯化了一种以NADPH为辅酶的3-脱氧葡糖醛酮(3-DG)代谢酶,定性为2-羰基醛还原酶.纯化酶的比活力为63.75 U/mg,在SDS-聚丙烯酰胺凝胶上显示一条蛋白质带.该酶分子质量约为32 ku,酶反应最适pH约为6.2, 在pH 5～8, 温度25～30℃之间酶保持稳定;该酶对3-DG的K_m为2.3 mmol/L.添加适量的EDTA、巯基乙醇或二硫苏糖醇能明显提高酶的活性;而碘乙酸、N-乙基顺丁烯二酰亚胺抑制酶的活性.     

恶臭假单胞菌SJTE-1中转化17β-雌二醇的3-酰基-ACP还原酶的鉴定与功能研究

[目的]假单胞菌SJTE-1可高效转化17β-雌二醇,但是催化该转化的酶尚不清楚。本文鉴定了该菌株的一个新的3-酮酰基-ACP还原酶（ANI01589.1）,并对其进行了功能研究。[方法]首先,我们克隆了该3-酰基-ACP还原酶的编码基因,在大肠杆菌BL21（DE3）菌株中进行了异源表达;利用金属离子亲和层析法,纯化获得了重组蛋白。体外检测了重组蛋白的活性与酶学性质,并利用高效液相色谱法（HPLC）测定了该酶的催化产物。[结果]3-酮酰基-ACP还原酶可被17β-雌二醇诱导表达,重组蛋白纯化量可达19.6 mg/L。蛋白序列比对结果表明,该蛋白包含短链脱氢酶/还原酶（SDR）的2个共有区域和多个保守残基。该酶以NAD⁺为辅助因子,将17β-雌二醇转化为雌酮;其K_m值为0.071 mmol/L,k_cat值为2.4±0.06/s^-1,5 min内可转化超过95.8%的雌二醇。该酶的最佳反应温度为42℃,最佳pH为8.0。不同二价离子对该酶的活性影响不同,Mg²⁺和Mn²⁺可增强其酶活性。[结论]这一假单胞菌SJTE-1来源的3-酮酰基-ACP还原酶可高效催化17β-雌二醇的转化,该酶可能在该菌株的雌激素代谢过程中起到重要作用。     

大豆液泡膜H+-ATPase功能与构象关系的初步研究

大豆液泡膜V型H⁺-ATPase是ATPases中的一种,它在植物细胞的生长发育中有重要的作用.利用竹红菌乙素(HB）和KI这两种分别猝灭蛋白质疏水区域内源荧光和亲水区域内源荧光的荧光猝灭剂,在不同pH值、温度条件下对纯化的大豆液泡膜V型ATPase进行荧光猝灭实验,初步探讨了V型H⁺-ATPase的水解活性同其蛋白质折叠状态间的关系.研究表明,通过比较不同pH值、温度条件下蛋白质疏水区域和亲水区域内源荧光的荧光猝灭常数（K_SV）,发现当环境pH值、温度偏离酶的最适pH值和温度时,蛋白质的内源荧光强度降低且疏水区域和亲水区域内源荧光的荧光猝灭常数（K_SV）降低,说明伴随着酶的水解活性降低,蛋白质的折叠状态发生了变化.我们认为蛋白质在膜内的折叠状态变化是酶失活机制的一个重要方面,为植物的抗冻和抗盐研究提供了一定的参考.     

谷氨酸棒状杆菌尿素传感器的研究

基于腺酶催化尿素分解产生氨,以氨气敏电极为基础电极,用含脲酶丰富的谷氨酸棒状杆菌研制成测定尿素的微生物传感器.在30℃、pH8.0、0.1mol/L磷酸盐缓冲液中,该传感器的线性范围为1.1×10^-4～1.4×10^-2mol/L,斜率为51.2mV/decade,检测下限为1.0×10^-5mol/L,寿命可达45d.考察了传感器响应初速和底物浓度之间的关系,测定了微生物膜中脲酶的表观米氏常数K_m及最大响应初速v_m.     

人白三烯A4水解酶的提取和纯化

人白细胞匀浆中的LTA₄H经硫酸铵分段盐析,DEAE-纤维素柱层析,Sephadex柱层析和HpLCmono-Q阳离子交挟柱层析,再经羟基磷灰石柱纯化,得到其纯度可达90—95％的产物。酶活性为212LTB₂nmol·mg^-1·min^-1。较纯化前活性提高350倍。SDS聚丙烯酰胺凝胶电泳显示酶的分子量为68000—70000的单体蛋白质。与以前报道人红细胞LTA₄H不同。     

壳聚糖固定化半纤维素酶的研究

从青霉菌m8提取出半纤维素酶,将其固定在用戊二醛交联的壳聚糖载体上.0.5 g壳聚糖与4%的戊二醛结合固定2.5 mg蛋白质,酶活回收率为45.6%. 原酶的最适pH为4.6,固定化酶为pH 3.6.原酶的最适温度为55℃,固定化酶在60～75℃都具有较高活性.固定化酶的耐热性优于原酶. 以半纤维素为底物,固定化酶的表观K_m值略低于原酶,前者为5.0×10^－2 g/L,后者为3.58×10^－2 g/L.     

用Blue Sepharose CL-6B快速纯化天花粉蛋白

差光谱显示染料cibacron blue F3GA与天花粉蛋白(TCS)有特异性结合,复合物在可见光部分的最大吸收波长在690 nm,摩尔消光系数ε=2.6×10^-3(mol/L)^-1·cm^-1,解离常数K_d=1.8 μmol/L,0.5 mol/L NaCl可使复合物解离.根据这一特点,用Blue-Sepharose CL-6B凝胶从栝篓块茎中亲和纯化了TCS.此法快速、简便、高效,易于大量制备.     

重组刺桐胰蛋白酶抑制剂a的制备及其在tPA突变体纯化中的应用

将基因工程菌株E.coliBL21(DE3) pET22b-mETIa高密度发酵,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导,重组刺桐胰蛋白酶抑制剂a(rETIa)蛋白在E.coli中得到较高水平表达,表达量占菌体总蛋白的40%以上.经菌体破碎、包涵体变性、复性,二步柱层析纯化得到电泳纯的rETIa蛋白.测得rETIa对t-PA突变体(NTA)的抑制平衡常数K_i为8.72×10^－8 mol/L.据此利用纯化的rETIa蛋白制备rETIa-Sepharose 4B亲和层析柱.直接一步纯化NTA复性液,纯化的NTA纯度达90 %以上,收率为96.2 %,纯化倍数为13.2,比活为(565.7±71.3) U/μg.     

Pattern recognition of neuron specific enolase and carcinoembryonic antigen in whole blood samples

New tools and methods for pattern recognition of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) were proposed for the screening of whole blood samples. The new tools were based on stochastic sensors designed using nanoporous gold microspheres, graphite, graphene, diamond paste as well as α‐CDs, and 5,10,15,20‐tetraphenyl‐21H,23H‐porphyrin. The best sensor for the assay of CEA was the one based on P/graphite (the limit of determination was 16 fg/ml and sensitivity was 2.32 × 10⁷ s mg^?1 ml), while for the assay of NSE the, best sensor was the one based on P/graphene (the limit of determination was 7.45 pg/ml and sensitivity was 2.49 × 10⁸ s mg^?1 ml). The sensor of choice for simultaneous detection of NSE and CEA is the one based on P/graphene because we need high sensitivity and low limit of determination for NSE. To our knowledge, this is the only one screening test for early detection of lung cancer, by identification of NSE and CEA in whole blood samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of a novel transgenic mouse line expressing Cre recombinase under the control of the Cdx2 neural specific enhancer

Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal‐related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R‐YFP reporter mice have indicated that the Cdx2NSE‐Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double‐transgenic embryos has revealed that this novel Cre‐driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso‐ventral axis (including pre‐migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non‐cranial neural crest derivatives of double‐transgenic embryos. Therefore, we believe that the Cdx2NSE‐Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems. genesis 51:777–784. © 2013 Wiley Periodicals, Inc.     

Elevated S100B and Neuron Specific Enolase Levels in Patients with Migraine-without Aura: Evidence for Neurodegeneration?

Although migraine has mainly been considered as a benign disease, there is cumulative evidence of silent changes in the brain, brainstem, or cerebellum and subtle subclinical cerebellar dysfunction. In this study, in order to investigate a possible neuronal and/or glial damage at the cellular level in migraine, we measured and compared serum levels of S100B which is a protein marker of glial damage or activation, and neuron specific enolase (NSE) which is a marker of neuronal damage, in migraine patients and control subjects. Serum levels of S100B and NSE were measured in blood samples from 41 patients with migraine-without aura taken during a migraine attack (ictal) and in the attack-free period between migraine attacks (interictal) and 35 age- and sex-matched controls. Patients with migraine-without aura had significantly higher ictal serum levels of S100B and NSE (P < 0.05, for both) than control subjects; whereas in the interictal phase, there was a significant increment only in S100B levels (P < 0.05) compared to controls. On the other hand, serum levels of S100B and NSE in ictal and interictal blood samples did not differ significantly. The findings of increased ictal serum S100B and NSE levels together with increased interictal levels of S100B suggested that migraine might be associated with glial and/or neuronal damage in the brain and a prolonged disruption of blood–brain barrier. Increased interictal serum levels of S100B might point out to an insidious and slow damaging process in migraine patients.     

Tau and GSK3β Dephosphorylations are Required for Regulating Pin1 Phosphorylation

Pin1 binds mitotically phosphorylated Thr231–Pro232 and Thr212–Pro213 sites on tau, and a Pin1 deficiency in mice leads to tau hyperphosphorylation. The aim of this study was to determine if the dephosphorylation or inhibition of tau and GSK3β phosphorylation induces the Pin1 phosphorylation. To test this, human SK-N-MC cells were stably transfected with a fusion gene containing neuron-specific enolase (NSE)-controlled APPsw gene(NSE/APPsw), to induce Aβ-42. The stable transfectants were then transiently transfected with NSE/Splice, lacking human tau (NSE/Splice), or NSE/hTau, containing human tau, into the cells. The NSE/Splice- and NSE/hTau-cells were then treated with lithium. We concluded that (i) there was more C99-β APP accumulation than C83-βAPP in APPsw-tansfectant and thereby promoted Aβ-42 production in transfectants. (ii) the inhibition of tau and GSK3β phosphorylations correlated with increase in Pin1 activation in NSE/hTau- cells. Thus, these observations suggest that Pin1 might have an inhibitive role in phosphorylating tau and GSK3β for protecting against Alzheimer’s disease.     

Anthelmintic efficacy of flemingia vestita (fabaceae): Genistein-induced alterations in the esterase activity in the cestode,raillietina echinobothrida

To ascertain the anthelmintic efficacy ofFlemingia vestita (an indigenous leguminous plant of Meghalaya, having putative anthelmintic usage), its crude root-tuber peel extract and active chemical principle, genistein, were testedin vitro with reference to esterase activity in the fowl tapeworm,Raillietina echinobothrida. With the localization of non-specific esterases (NSE) and cholinesterase (ChE), the organization of the cholinergic components of the nervous system in toto could be visualized in the cestodeo The specific ChE in the parasite is acetylcholinesterase (AChE). Both NSE and ChE were found in close association with the central and peripheral nervous components, besides being present in the tegument and muscular parts of the terminal male genitalia. The whole tissue homogenate of the parasite also showed a high AChE activity. After exposure to the crude peel extract (50 mg/ml of the incubation medium) and to genistein (0.5 mg/ml), a pronounced decline in the visible stain intensity in the cholinergic components of the nervous system and in the tegument was noticeable, indicating extremely reduced activity of NSE and ChE in these sites. The total AChE activity was also reduced to 4907% and 56–77%, following treatment with the peel extract and genistein, respectively. The reference drug, praziquantel (0.01 mg/ml) also caused reduction in the enzyme activity, somewhat at par with the genistein treatment. Genistein appears to have a transtegumental mode of action. Alteration in the AChE activity points towards acetylcholine, an inhibitory neurotransmitter in cestodes, as the potential target of action.     

Frequent expression of neuroendocrine markers in mucinous tubular and spindle cell carcinoma of the kidney

Mucinous tubular and spindle cell carcinoma (MTSCC) is a new tumorous entity which has been recently established. In this article, we examined the expression of neuroendocrine markers including neuron specific enolase (NSE), chromogranin A and synaptophysin in 16 cases of MTSCC using immunohistochemistry. The sex ratio (male: female) of the patients was 4:12. In normal kidney, distal tubules or collecting ducts were positive for NSE, but no structures were positive for chromogranin A or synaptophysin. All MTSCCs showed a positive reaction for NSE. Additionally, fifteen of sixteen neoplasms (93.8%) with MTSCC showed the expression of either chromogranin A or synaptophysin or both. Finally, it is possible that MTSCC may be one of renal neoplasms which frequently exhibit the neuroendocrine differentiation.     

血清NSE、LP-PLA2联合CAR对老年股骨粗隆间骨折患者PFNA内固定术后谵妄的预测价值

摘要 目的：探讨血清神经元特异性烯醇化酶（NSE）、脂蛋白相关磷脂酶A2（LP-PLA2）联合C-反应蛋白与白蛋白比值（CAR）对老年股骨粗隆间骨折患者闭合复位防旋髓内钉（PFNA）内固定术后谵妄的预测价值。方法：选择2020年4月至2023年2月北京中医药大学第三附属医院收治的209例老年股骨粗隆间骨折患者。所有患者均行PFNA内固定术治疗，术前检测血清NSE、LP-PLA2、CAR，术后根据是否发生谵妄分为谵妄组和非谵妄组。多因素Logistic回归分析术后谵妄的因素。受试者工作特征（ROC）曲线分析NSE、LP-PLA2、CAR预测术后谵妄的价值。结果：术后43例发生谵妄，发生率为20.57%。谵妄组血清NSE、LP-PLA2、CAR高于非谵妄组（P＜0.05）。多因素Logistic回归分析显示年龄偏大、手术时间过长、高NSE、高LP-PLA2、高CAR是老年股骨粗隆间骨折患者术后谵妄的危险因素（P＜0.05）。NSE、LP-PLA2、CAR预测老年股骨粗隆间骨折患者术后谵妄的曲线下面积（AUC）为0.784、0.808、0.820，联合预测的AUC 为0.907，高于单独预测。结论：老年股骨粗隆间骨折PENA术后谵妄患者血清NSE、LP-PLA2、CAR增高，高水平NSE、LP-PLA2、CAR是术后谵妄的主要危险因素。联合NSE、LP-PLA2、CAR对术后谵妄风险有较高预测价值。     

Neuron-specific enolase and serotonin in the Merkel cells of conger-eel (Conger conger) epidermis

Summary Immunocytochemical techniques were used to investigate the distribution and co-localization of neuronspecific enolase (NSE) and serotonin (5-HT) in the skin of the conger eel, Conger conger. NSE and 5-HT immunoreactivity were found in Merkel cells; these cells were also identified at the electron-microscope level by the presence of characteristic granules and their association with an intraepithelial nerve ending. For the first time, it was demonstrated that Merkel-cell granules of vertebrate skin exhibit in immunoreaction with 5-HT. The production of amines may indicate that the Merkel cells of C. conger have both secretory capabilities and transduction functions.However, immunocytochemical investigation of the synaptic zones at the electron microscope level will be necessary to confirm this hypothesis.The present histochemical results suggest that NSE and 4-HT may be marker substances for Merkel cells, and that immunocytochemistry is a useful tool for the light-microscopic localization of these cells.