生物降解性聚[N-(2-羟乙基)-L-谷酰胺]的合成研究

本文以L-谷氨酸为原料，经γ-羧基的苄基保护．与光气-甲苯液反应制备谷氨酸苄酯的N-羧酸酐(NCA)．均聚得聚谷氨酸节酯(PBLG)，然后由2-氨基乙醇胺解得水溶性的聚[N-(2-羟乙基)-L-谷酰胺](PHEG)，并对其进行了UV、IR、分子量、羟基含量的表征。     

儿童1型糖尿病的抗体检测及临床意义

目的　了解胰岛细胞抗体 (ICA)和谷氨酸脱羧酶抗体 (GADA)阳性率与患儿胰岛功能的关系 ,明确ICA和GADA检测对 1型糖尿病的临床意义。方法　儿童 1型糖尿病 31例均采用ELISA法测定GADA ,间接免疫荧光法测定ICA ,化学发光法测定血清C肽、胰岛素 (DPC -IMMULITE机器 )。结果　ICA和GADA的阳性率分别为 4 5 .2 %和 38.7% ,儿童 1型糖尿病患者ICA阳性率高于GADA。抗体阳性患儿空腹和餐后C肽(0 .99± 0 .36ng/ml和 2 .10± 0 .99ng/ml)及餐后胰岛素 (11.5 3± 7.11μIU/ml)明显低于抗体阴性患儿空腹C肽 (2 .19± 1.33)ng/ml,餐后C肽 (6 .18± 3.91)ng/ml和餐后胰岛素 (18.6 0± 10 .5 9) μIU/ml,P <0 .0 5。结论　青少年糖尿病患者中ICA阳性率高于GADA。自身抗体阳性患儿胰岛功能明显低于阴性患儿 ,提示自身抗体阳性患儿胰岛功能损伤更明显。由于ICA、GADA两种抗体的阳性结果常不一致 ,联合检测可提高诊断率     

复方灯盏花滴丸对海马神经元一氧化氮和一氧化氮合酶的影响

目的研究复方灯盏花滴丸(FDD)对谷氨酸致大鼠原代海马神经元的NO和一氧化氮合酶的影响。方法取SD大鼠32只,每8只分别用生理盐水、尼莫地平、FDD高、低剂量灌胃,85h后,取每只大鼠股动脉血,制成5%的含药血清。另取新生24h SD乳鼠大脑原代培养海马神经元并鉴定后,随机分为对照组、尼莫地平组、FDD高、低剂量组和模型组,前4组分别加入上述配制对应的药物血清,模型组不加含药血清。以谷氨酸制成损伤模型。采用MTT法测定细胞存活率,酶法检测细胞释放NO量,细胞内总一氧化氮合酶(tNOS)和诱导型一氧化氮合酶(iNOS)活性。结果与对照组比较,模型组细胞存活率下降,NO、tNOS和iNOS活性增加(P0.01),与模型组比较,FDD高、低剂量组均能明显增加细胞存活率、减少NO释放、降低tNOS活性和iNOS活性(P0.05,P0.01)。结论 FDD对谷氨酸致原代培养大鼠海马神经元损伤有保护作用,其作用机制可能与降低一氧化氮合酶的活性、尤其iNOS活性及减少NO释放有关。     

Comparison of effects induced by toxic applications of kainate and glutamate and by glucose deprivation on area CA1 of rat hippocampal slices

Baseline and stimulus-induced changes in [Ca²⁺]_o and [K⁺]_o as well as field potentials (fp's) were studied during application of the excitatory amino acids kainate or glutamate, or during glucose deprivation in area CA1 and CA3 of rat hippocampal slices. Bath application of kainate in concentrations of 1, 2, 5, 8 and 10 mM induced a sudden rapid fall of [Ca²⁺]_o in area CA1, associated with a negative shift of the slow fp. Kainate induced disappearance of stratum radiatum (SR) as well as alveus stimulation-evoked postsynaptic fp's, with partial recovery after application of up to 2 mM kainate, but no recovery after 5 mM kainate. Only afferent volleys and repetitive SR stimulation-induced decreases of [Ca²⁺]_o recovered after 5 mM kainate. Similar observations were made with glutamate. Only when glutamate was applied with 20 mM, irreversible disappearance of postsynaptic fp's was noted. Glucose deprivation for 60–90 min led to an initial slow decline of [Ca²⁺]_o in area CA1 and CA3, associated with increases in [K⁺]_o, but no significant changes in the fp baseline. Before reaching the lowest level in [Ca²⁺]_o, stimulation of afferent and efferent fibres in area CA1 and CA3 evoked epileptiform discharges. After reaching the lowest level in [Ca²⁺]_o, all postsynaptic potential components were irreversibly abolished, sparing afferent volleys and SR stimulation-induced decreases in [Ca²⁺]_o. The application of the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 μM) and

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-2-amino-5-phosphonovalerate (2APV, 30 μM) during glucose deprivation did not prevent irreversible loss of alveus and SR stimulation-induced postsynaptic signals. These findings suggest that glutamate release during glucose deprivation is not the main factor of acute cell damage.     

绞股蓝皂甙XL Ⅲ对小鼠脑内谷氨酸和γ-氨基丁酸的影响

目的研究绞股蓝皂甙ＸＬⅢ（ｇｙｎｏｓａｐｏｎｉｎ，ＧｙｎＸＬⅢ）对小鼠学习记忆获得的影响和其作用机制—对小鼠脑内谷氨酸（Ｇｌｕ）／γ－氨基丁酸（ＧＡＢＡ）的影响。方法跳台法研究ＧｙｎＸＬⅢ对小鼠的学习记忆获得过程的影响；薄层层析法测试小鼠脑内谷氨酸和γ－氨基丁酸的含量。结果Ｇｙｎ－ＸＬⅢ１５ｍｇ·ｋｇ－１，ｉｐ对小鼠的学习记忆获得过程无影响（Ｐ＞０．０５），３０ｍｇ·ｋｇ－１可增强小鼠的学习记忆获得过程１９９７－０８－０６收稿，１９９７－１０－０５修回＊广东药学院科研基金资助课题，Ｎｏ９５１０３３作者简介：冯冰虹，男，３４岁，讲师；杜淇璋，男，６３岁，教授，中国药理学会理事，广东省药理学会专家委员会副主任（Ｐ＜０．０５），并显著提高脑内Ｇｌｕ水平（Ｐ＜０．０５），明显降低ＧＡＢＡ水平（Ｐ＜０．０５）；东莨菪碱（Ｓｃｏｐ）０．３ｍｇ·ｋｇ－１，ｉｐ的结果相反，显著降低Ｇｌｕ水平（Ｐ＜０．０１），提高ＧＡＢＡ水平（Ｐ＜０．０５）；预先ＧｙｎＸＬⅢ３０ｍｇ·ｋｇ－１，ｉｐ，５ｍｉｎ后ｉｐ东莨菪碱０．３ｍｇ·ｋｇ－１，结果显示，ＧｙｎＸＬⅢ扭转Ｓｃｏｐ的升高ＧＡＢＡ作用，使之显著下降（Ｐ＜０．０５），对Ｇｌｕ的影响?     

人参皂苷Rb1对胎鼠背根神经节损伤的保护作用

目的观察不同浓度人参皂苷Rb1对损伤胎鼠背根神经节（DRG）神经元形态学改变的影响,探讨人参皂苷Rb1对谷氨酸引起的兴奋性神经毒损伤的保护作用,为Rb1的进一步研究和临床应用提供理论依据。方法选择胎龄为15 d的SD大鼠,获取DRG神经元并进行体外分散培养48 h后,随机分为对照组、谷氨酸损伤组、谷氨酸损伤＋低浓度Rb1（10μg/ml）保护组和谷氨酸损伤＋高浓度Rb1（100μg/ml）保护组,继续培养12 h。终止培养后,倒置相差显微镜观察各组神经元的生长状态,MTT检测不同浓度人参皂苷Rb1孵育的背根神经节神经元的凋亡增殖情况。结果对照组DRG神经元细胞贴壁呈单层散在分布,少部分出现细胞聚集现象,突起较长且互相交织形成网状;谷氨酸损伤组DRG神经元细胞聚集现象明显,神经元突起变短、断裂甚至消失;谷氨酸损伤＋Rb1保护组DRG神经元细胞部分呈簇状聚集,部分呈单个散在分布,突起仍然相互交织。MTT结果显示谷氨酸损伤＋Rb1保护组细胞存活率均高于谷氨酸损伤组,但高浓度Rb1保护组与低浓度Rb1保护组之间差异无统计学意义。结论人参皂苷Rb1可以影响损伤胎鼠DRG神经元的形态学改变和凋亡增殖情况,对胎鼠背根神经节神经元具有一定的保护作用。     

Glycine and GABA(A) receptor subunits on Renshaw cells: relationship with presynaptic neurotransmitters and postsynaptic gephyrin clusters

Inhibitory synapses with large and gephyrin-rich postsynaptic receptor areas are likely indicative of higher synaptic strength. We investigated the presynaptic inhibitory neurotransmitter content (GABA, glycine, or both) and the presence and subunit composition of GABA(A) and glycine postsynaptic receptors in one example of gephyrin-rich synapses to determine neurochemical characteristics that could also contribute to enhance synaptic strength. Hence, we analyzed subunit receptor expression in gephyrin patches located on Renshaw cells, a type of spinal interneuron that receives powerful excitatory and inhibitory inputs and displays many large gephyrin patches on its surface. GABA(A) and glycine receptors were almost always colocalized inside Renshaw cell gephyrin clusters. According to the subunit-immunoreactivities detected, the composition of GABA(A) receptors was inferred to be either alpha(3)beta((2or3))gamma(2), alpha(5)beta((2or3))gamma(2), alpha(3)alpha(5)beta((2or3))gamma(2) or a combination of these. The types of neurotransmitters contained inside boutons presynaptic to Renshaw cell gephyrin patches were also investigated. The majority (60-75%) of terminals presynaptic to Renshaw cell gephyrin patches contained immunocytochemical markers for GABA as well as glycine, but a proportion contained markers only for glycine. Significantly, 40% of GABA(A) receptor clusters were opposed to presynaptic boutons that contained only glycinergic markers. We postulate that GABA and glycine corelease, and the presence of alpha3-containing GABA(A) receptors can enhance the postsynaptic current and contribute to strengthen inhibitory input on Renshaw cells. In addition, a certain degree of imprecision in the localization of postsynaptic GABA(A) receptors in regard to GABA release sites onto adult Renshaw cells was also found.     

Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons

The inferior colliculus (IC) is partitioned into three subdivisions: the dorsal and lateral cortices (DC and LC) and the central nucleus (ICC), and serves as an integration center of auditory information. Recent studies indicate that a certain population of IC neurons may represent the non‐GABAergic phenotype, while they express well‐established cortical/hippocampal GABAergic neuron markers. In this study we used the optical disector to investigate the phenotype of IC neurons expressing parvalbumin (PV) and/or nitric oxide synthase (NOS) in C57BL/6J mice during the late postnatal period. Four major types of IC neurons were defined by the presence (+) or absence (–) of PV, NOS, and glutamic acid decarboxylase 67 (GAD67): PV⁺/NOS^?/GAD67⁺, PV⁺/NOS⁺/GAD67⁺, PV⁺/NOS^?/GAD67^?, and PV^?/NOS⁺/GAD67^?. Fluorescent in situ hybridization for vesicular glutamate transporter 2 mRNA indicated that almost all GAD67^? IC neurons represented the glutamatergic phenotype. The numerical densities (NDs) of total GAD67⁺ IC neurons remained unchanged in all subdivisions. The NDs of PV⁺/NOS^?/GAD67⁺ neurons and PV^?/NOS⁺/GAD67^? neurons were reduced with age in the ICC, while they remained unchanged in the DC and LC. By contrast, the NDs of PV⁺/NOS⁺/GAD67⁺ neurons and PV⁺/NOS^?/GAD67^? neurons were increased with age in the ICC, although there were no changes in the DC and LC. The cell body size of GAD67⁺ IC neurons did not vary according to the expression of PV with or without NOS. The present findings indicate that the expression of PV and NOS may shift with age within the GABAergic and glutamatergic phenotypes of IC neurons during the late postnatal period. J. Comp. Neurol. 525:868–884, 2017. © 2016 Wiley Periodicals, Inc.     

Effect of intravenous transplantation of bone marrow mesenchymal stem cells on neurotransmitters and synapsins in rats with spinal cord injury

Bone marrow mesenchymal stem cells were isolated, purified and cultured in vitro by Percoll density gradient centrifugation combined with the cell adherence method. Passages 3-5 bone marrow mesenchymal stem cells were transplanted into rats with traumatic spinal cord injury via the caudal vein. Basso-Beattie-Bresnahan scores indicate that neurological function of experimental rats was significantly improved over transplantation time (1-5 weeks). Expressions of choline acetyltransferase, glutamic acid decarboxylase and synapsins in the damaged spinal cord of rats was significantly increased after transplantation, determined by immunofluorescence staining and laser confocal scanning microscopy. Bone marrow mesenchymal stem cells that had migrated into the damaged area of rats in the experimental group began to express choline acetyltransferase, glutamic acid decarboxylase and synapsins, 3 weeks after transplantation. The Basso-Beattie- Bresnahan scores positively correlated with expression of choline acetyltransferase and synapsins. Experimental findings indicate that intravenously transplanted bone marrow mesenchymal stem cells traverse into the damaged spinal cord of rats, promote expression of choline acetyltransferase, glutamic acid decarboxylase and synapsins, and improve nerve function in rats with spinal cord injury.     

Nattokinase (Bac s 1), a subtilisin family serine protease,is a novel allergen contained in the traditional Japanese fermented food natto

BackgroundImmediate allergy caused by natto, a popular Japanese food prepared by fermenting soybeans with Bacillus subtilis var. natto, has been reported. Polygamma glutamic acid (PGA) in the sticky substance around natto beans has been reported to be a causative allergen of natto allergy. However, some of our patients with natto allergy were negative for PGA in the skin prick test (SPT). The sticky substance of natto beans contains a subtilisin family serine protease, nattokinase, along with PGA. In this study, we aimed to examine the antigenicity of nattokinase in natto allergy.MethodsEight patients, who developed symptoms after ingesting natto and positively reacted to natto (seven to the sticky substance in natto and one to the whole natto product) in their SPT, were enrolled in this study. To analyze IgE reactivity, we performed immunoblotting, ELISA, and SPT for natto (bean and sticky substance), and/or PGA, and/or nattokinase and/or cultured B. subtilis var. natto extract.ResultsIn the SPT, four cases each were PGA-positive and PGA-negative. Immunoblotting of the sera from PGA-negative patients showed a protein band at 30 kDa, which was identified as nattokinase. Three PGA-negative cases, but not three PGA-positive cases, showed a positive reaction to nattokinase in the SPT and had a history of atopic dermatitis. The ELISA for nattokinase revealed a positive reaction of PGA-negative cases and negative reaction of PGA-positive cases in the SPT.ConclusionsWe identified a subtilisin family serine protease, nattokinase, as a novel allergen in natto allergy patients unsensitized to PGA.