D-丝氨酸在神经元-胶质细胞间通讯的新进展

D-丝氨酸(D-Ser)是一种重要的胶质细胞递质,也是N-甲基-D-天冬氨酸(NMDA)受体NR1亚基上"甘氨酸位点"的主要内源性配体,具有比甘氨酸更高的结合效能.D-Ser在体内主要由丝氨酸消旋酶将L-丝氨酸消旋而来,受多种因素调控,在中枢神经系统参与调节突触可塑性、感觉信息传递、神经发育及神经兴奋性毒性等生理及病理过程,并成为阿尔采末病(AD)等神经系统疾病新的治疗靶点.本文对D-Ser在中枢神经系统的产生、代谢、生理及病理作用的研究予以综述.     

D构象丝氨酸在中枢神经系统中的功能研究进展

哺乳动物中枢神经系统中D构象丝氨酸的区域性高浓度分布与N-甲基-D-天冬氨酸(NMDA)受体相一致.它主要由丝氨酸消旋酶将L丝氨酸直接消旋而来,也可能通过肠道菌群产生后吸收至体内,最终被D构象氨基酸氧化酶氧化.这种从胶质细胞而非神经元来源的“异常”构象氨基酸作为一种新型神经递质,不仅更新了传统“神经递质”的定义,而且为许多与NMDA受体过度兴奋或表达下调相关的神经系统疾病治疗提出了新的线索.     

小鼠丝氨酸消旋酶的克隆、表达与纯化

丝氨酸消旋酶(Serine Racemase,SR)是一种磷酸吡多醛依赖酶,在ATP和Mg2+的辅助下,催化L型丝氨酸转变为D型丝氨酸,D-丝氨酸通过结合在NMDA受体的Gly结合位点,调节其生理功能。本文将小鼠来源的丝氨酸消旋酶基因克隆至原核表达载体pMAL-C2上,构建重组质粒pMAL-C2-SR。将重组质粒转入E.coil BL21(DE3)宿主菌中,经IPTG诱导,获得重组表达。重组蛋白带有MBP标签,经Amylose亲合柱和Sephacryl S-200纯化,获得电泳纯的目的蛋白。     

特异性分布于心脏的丝氨酸蛋白酶--Corin

蛋白酶参与心血管活性肽类的活化和降解 ,对其生物学效应具有重要的调节意义。丝氨酸蛋白酶是一类II型跨膜嵌合蛋白酶超家族 ,参与体内多种重要的生理过程。Corin是新发现的第一个特异性分布于心脏并参与心血管活性肽前体原转化的丝氨酸蛋白酶 ,可将心钠素原和脑钠素原转化为心钠素和脑钠素 ,并通过调控心钠素和脑钠素的生成而间接调节血压。本文简要介绍Corin的分子特征及生理和病理生理意义     

鞘脂代谢关键酶丝氨酸棕榈酰转移酶的结构、功能及其相关疾病

鞘脂(sphingolipids)是生物细胞中最主要的膜脂之一，同时也作为信号分子介导细胞生长、增殖、迁移及死亡等重要的生理反应，异常鞘脂代谢经常与心血管疾病、糖尿病、癌症、神经变性病以及自身免疫性疾病等相关。丝氨酸棕榈酰转移酶(serine palmitoyltransferase, SPT)及其复合物是鞘脂从头合成途径的起始酶和关键酶，催化L-丝氨酸与棕榈酰辅酶A缩合形成3-酮二氢鞘氨醇，之后再经过一系列反应生成神经酰胺和其它重要的鞘脂，在鞘脂代谢和稳态调节方面发挥重要作用。本文基于国内外对SPT的研究，综述了SPT的构型、活性位点、底物结合位点等关键的结构信息，尤其近2年的研究发现，SPT是一种组成极其复杂的酶，各个亚基之间存在错综复杂的相互作用和高度调控。SPT具有重要的生物学功能，包括参与胚胎发育、调节内环境稳态、诱导细胞凋亡和参与机体免疫调节等。SPT还可以通过调节酶活性影响鞘脂代谢，进而影响血管疾病和肿瘤的发生发展，并有潜力成为肿瘤诊断和治疗的关键分子。此外，SPT突变体与神经变性病密切相关，本文着重介绍了遗传性感觉与自主神经病变1型(hereditary sensory...     

植物半胱氨酸合成及调控研究进展

硫是植物重要的营养元素。植物将氧化态硫吸收并还原后,首先合成半胱氨酸使其进入各种代谢途径。合成半胱氨酸的两种酶——丝氨酸乙酰转移酶和O-乙酰丝氨酸硫醇裂合酶均由多基因家族编码,并能可逆的结合形成二酶复合物进行有效的合成调节。本文对近年来半胱氨酸合成相关酶表达、定位、活性调控及转基因效果研究进展作了简要介绍,并对将来需要重点研究的方面作了展望。     

SOCS家族在中枢神经系统的研究进展

细胞因子信号抑制因子（SOCS）家族是一类对细胞因子信号通路具有负反馈调节作用的蛋白分子,参与多种细胞因子、生长因子和激素的信号调节。细胞因子对中枢神经系统中的各种生物效应具有广泛多样的调节作用,SOCS家族的许多成员在发育时期和成年的脑内均有表达,SOCS家族不仅与细胞因子信号调节及中枢神经系统多种功能的调节密切相关,而且可能是神经发育和分化的重要调控因子,并参与神经免疫内分泌调节。本文综述了SOCS家族的发现、结构特点、脑内分布以及在中枢神经系统中的功能等方面的研究进展。     

糜酶的研究进展

糜酶是一类丝氨酸蛋白酶,主要存在于肥大细胞的分泌颗粒和细胞间质。成熟的酶为一糖蛋白,由２２６个氨基酸组成,分子量为３０ｋＤ,其酶活力能被丝氨酸蛋白酶抑制剂所抑制,而不受血管紧张素Ⅰ转换酶抑制剂的影响。糜酶的ｃＤＮＡ和基因组ＤＮＡ均已被克隆和测序。该酶和神经炎症反应、血管活性肽代谢和细胞外基质代谢等有密切联系,在心脏血管紧张素Ⅱ形成中起重要作用。     

人丝氨酸蛋白酶抑制因子抗肿瘤作用的研究进展

丝氨酸蛋白酶抑制因子在不同的生命活动调节中均具有重要意义,可调节凝血(血栓形成和血栓溶解)、血管再生、神经生长、激素转运、血压、补体和炎症。不同的丝氨酸蛋白酶抑制因子与对应的不同恶性肿瘤的进展和缓解有一定关联,使之在肿瘤治疗和诊断中具有一定意义。开展对丝氨酸蛋白酶抑制因子介导抗肿瘤活性的疗效和机制的进一步研究,有望发展成为肿瘤治疗的新方法。     

L-丝氨酸检测技术的研究进展

L-丝氨酸作为一种非必需氨基酸,它在药物、化工产品以及食品等方面得到了广泛应用,是一种重要的工业产物,具有很重要的研究价值。快速、准确、高通量的检测L-丝氨酸含量的方法,能够为高通量菌种选育提供坚实的基础。本文介绍了目前检测L-丝氨酸含量的多种方法,包括变色酸-分光光度法、纸层析-分光光度法、荧光猝灭法、茚三酮显色法、高效液相色谱法、酶反应检测法及毛细管电泳-电致化学发光( CE-ECL)法,同时通过比较它们的优缺点,并针对L-丝氨酸检测中存在的各种问题进行讨论分析,对L-丝氨酸的检测技术进行展望。     

Serine Racemase Regulated by Binding to Stargazin and PSD-95: POTENTIAL N-METHYL-d-ASPARTATE-α-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID (NMDA-AMPA) GLUTAMATE NEUROTRANSMISSION CROSS-TALK*

d-Serine, an endogenous co-agonist for the glycine site of the synaptic NMDA glutamate receptor, regulates synaptic plasticity and is implicated in schizophrenia. Serine racemase (SR) is the enzyme that converts l-serine to d-serine. In this study, we demonstrate that SR interacts with the synaptic proteins, postsynaptic density protein 95 (PSD-95) and stargazin, forming a ternary complex. SR binds to the PDZ3 domain of PSD-95 through the PDZ domain ligand at its C terminus. SR also binds to the C terminus of stargazin, which facilitates the cell membrane localization of SR and inhibits its activity. AMPA receptor activation internalizes SR and disrupts its interaction with stargazin, therefore derepressing SR activity, leading to more d-serine production and potentially facilitating NMDA receptor activation. These interactions regulate the enzymatic activity as well as the intracellular localization of SR, potentially coupling the activities of NMDA and AMPA receptors. This shuttling of a neurotransmitter synthesizing enzyme between two receptors appears to be a novel mode of synaptic regulation.     

Hippocampal Glutamate NMDA Receptor Loss Tracks Progression in Alzheimer’s Disease: Quantitative Autoradiography in Postmortem Human Brain

Early Alzheimer''s disease (AD) is characterized by memory loss and hippocampal atrophy with relative sparing of basal ganglia. Activation of glutamate NMDA receptors in the hippocampus is an important step in memory formation. We measured the density of NMDA receptors in samples of hippocampus, entorhinal cortex and basal ganglia obtained from subjects who died with pathologically confirmed AD and age- and sex- matched non-demented controls. We found significant decreases in NMDA receptor density in the hippocampus and entorhinal cortex but not in the basal ganglia. Loss of NMDA receptors was significantly correlated with neuropathological progression as assessed by Braak staging postmortem. The same samples were probed for neuroinflammation by measuring the density and gene expression of translocator protein 18kDA (TSPO), an established marker of microglial activation. Unlike NMDA receptor loss, increased densities of TSPO were found in all of the brain regions sampled. However hippocampal, but not striatal TSPO density and gene expression were inversely correlated with NMDA receptor density and positively correlated with Braak stage, suggesting NMDA receptors exacerbate neuroniflammatory damage. The high correlation between hippocampal NMDA receptor loss and disease progression supports the use of non invasive imaging with NMDA receptor tracers and positron emission tomography as a superior method for diagnosis, staging and treatment monitoring of AD in vivo.     

Cyclopropane derivatives as potential human serine racemase inhibitors: unveiling novel insights into a difficult target

d-Serine is the co-agonist of NMDA receptors and binds to the so-called glycine site. d-Serine is synthesized by human serine racemase (SR). Over activation of NMDA receptors is involved in many neurodegenerative diseases and, therefore, the inhibition of SR might represent a novel strategy for the treatment of these pathologies. SR is a very difficult target, with only few compounds so far identified exhibiting weak inhibitory activity. This study was aimed at the identification of novel SR inhibitor by mimicking malonic acid, the best-known SR inhibitor, with a cyclopropane scaffold. We developed, synthesized, and tested a series of cyclopropane dicarboxylic acid derivatives, complementing the synthetic effort with molecular docking. We identified few compounds that bind SR in high micromolar range with a lack of significant correlation between experimental and predicted binding affinities. The thorough analysis of the results can be exploited for the development of more potent SR inhibitors.     

In silico and pharmacological screenings identify novel serine racemase inhibitors

d-Serine is a coagonist of the N-methyl-d-aspartate (NMDA)-type glutamate receptor and its biosynthesis is catalyzed by serine racemase (SR). The overactivation of the NMDA receptor has been implicated in the development of neurodegenerative diseases, strokes, and epileptic seizures, thus, the inhibitors of SR have potential against these pathological states. Here, we have developed novel inhibitors of SR by in silico screening and in vitro enzyme assay. The newly developed inhibitors have lower IC₅₀ value comparing with that of malonate, one of the standard SR inhibitor. The structural features of novel inhibitors suggest the importance of central amide structure having a phenoxy substituent in their structure for the SR inhibitory activity. The present findings suggest the importance and rational development of new drugs for diseases of NMDAR overactivation.     

NMDA Receptor-Mediated Regulation of AMPA Receptor Properties in Organotypic Hippocampal Slice Cultures

Abstract: Activation of the calcium-dependent protease calpain has been proposed to be a necessary step in the formation of long-term potentiation (LTP) in the hippocampus, and stimulation of N-methyl-d -aspartate (NMDA) receptors leads to an increase in intracellular calcium concentration, calpain activation, proteolysis of cytoskeletal elements, and modification of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor properties. In the present study, we evaluated the effects of NMDA treatment of cultured hippocampal slices on the properties of AMPA receptors. Cultured hippocampal slices were treated with NMDA (100 µM) for 15 min and [³H]AMPA binding to membrane fractions was measured. NMDA-treated slices exhibited an increase in both “high-affinity” and “low-affinity” [³H]-AMPA binding, with smaller changes in 6-cyano-7-nitro[³H]quinoxaline-2,3-dione binding. The increase in [³H]AMPA binding was significantly reduced by preincubation of cultures with calpain inhibitor I or calpeptin (100 µM). Furthermore, NMDA exposure decreased the number of GluR1 subunits of AMPA receptors detected by an antibody against the C-terminal domain of the subunit in western blots and resulted in the formation of a lower molecular weight species detected by an antibody against the N-terminal domain. Both effects were completely prevented by calpain inhibitors. These results indicate that NMDA receptor activation produces calpain activation and complex modifications of AMPA receptor properties, which could be involved in NMDA receptor-mediated changes in synaptic efficacy.     

迷走神经切断和NK3受体拮抗剂干预在辣椒素诱导小鼠咳嗽中的作用

目的:观察右侧迷走神经切断及NK3受体拮抗剂对辣椒素诱导小鼠咳嗽的作用及其机制。方法:将48只小鼠随机分为4组,分别为:右侧迷走神经切断术组、右侧假手术组、SR 142801腹腔注射组和生理盐水对照组。辣椒素雾化制作小鼠咳嗽模型后,分别进行迷走神经切断术、假手术、SR142801腹腔注射及生理盐水腹腔注射,SR142801组及生理盐水对照组腹腔注射每日1次,连续7天。第8天计数各组所有小鼠咳嗽次数,检测所有小鼠肺组织中SP(substance P,P物质)、NKA(neurokinin A,神经肽A)、NKB(neurokinin B,神经肽B)表达水平。结果:右侧迷走神经切断组术后咳嗽次数[(6.92±1.78)次]较术前[(7.83±2.48)次]显著降低(P0.01),较假手术组[(7.58±2.43)次]降低(P0.05)。右侧迷走神经切断组术后SP、NKA、NKB水平较对照组显著降低(P0.05),SR 142810组干预后咳嗽次数[(8.67±1.37)次]较干预前[(8.33±2.15)次]无明显降低(P0.05)。SR 142801组腹腔注射后NKB[(8.32±0.86)pg/m L]较生理盐水对照组[(8.83±0.80)pg/m L]无明显降低(P0.05)。结论:迷走神经切断可以抑制辣椒素诱导的咳嗽,其机制主要与减少迷走神经相关神经肽SP、NKA、NKB的表达有关,而NK3受体拮抗剂SR142801对辣椒素诱导的咳嗽无明显抑制作用。     

Activation of synaptic receptors and its allosteric regulation

This review considers activation of synaptic receptors (SR) as a process of transduction of the energy of interaction between an agonist (A) and an agonist-recognizing module (R) toward an ion-conducting module (channel,Ch) orG protein within respective complexes (ARCh orARG). The agonist provides functional and, in the case of metabotropic SR, spatial association of protein components in these complexes. Conformational transformations of the latter, which are sufficient for dissociation ofG protein into subunits or opening of an ion-conducting channel, sometimes need additional energy expenditure. The source for this energy can be interaction with one more ligand: guanosine-5′-triphosphate (in the case of metabotropic SR) or glycine (in case of NMDA receptors), which act as co-agonists. The activation of SR is regulated by their allosteric modulators, which affect either the kinetics of interaction between the agonist and receptor by changing the rate of dissociation with the agonist or the kinetics of conformational transitions of the components into the activated or desensitized state.     

Cell Selective Conditional Null Mutations of Serine Racemase Demonstrate a Predominate Localization in Cortical Glutamatergic Neurons

d-Serine, which is synthesized by the enzyme serine racemase (SR), is a co-agonist at the N-methyl-d-aspartate receptor (NMDAR). Crucial to an understanding of the signaling functions of d-serine is defining the sites responsible for its synthesis and release. In order to quantify the contributions of astrocytes and neurons to SR and d-serine localization, we used recombinant DNA techniques to effect cell type selective suppression of SR expression in astrocytes (aSRCKO) and in forebrain glutamatergic neurons (nSRCKO). The majority of SR is expressed in neurons: SR expression was reduced by ~65% in nSRCKO cerebral cortex and hippocampus, but only ~15% in aSRCKO as quantified by western blots. In contrast, nSRCKO is associated with only modest decreases in d-serine levels as quantified by HPLC, whereas d-serine levels were unaffected in aSRCKO mice. Liver expression of SR was increased by 35% in the nSRCKO, suggesting a role for peripheral SR in the maintenance of brain d-serine. Electrophysiologic studies of long-term potentiation (LTP) at the Schaffer collateral–CA1 pyramidal neuron synapse revealed no alterations in the aSRCKO mice versus wild-type. LTP induced by a single tetanic stimulus was reduced by nearly 70% in the nSRCKO mice. Furthermore, the mini-excitatory post-synaptic currents mediated by NMDA receptors but not by AMPA receptors were significantly reduced in nSRCKO mice. Our findings indicate that in forebrain, where d-serine appears to be the endogenous co-agonist at NMDA receptors, SR is predominantly expressed in glutamatergic neurons, and co-release of glutamate and d-serine is required for optimal activation of post-synaptic NMDA receptors.     

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells

Background aimsCholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA.MethodsTo overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28.ResultsInitially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8⁺ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed.ConclusionTaken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.     

Striatal glutamate release evoked in vivo by NMDA is dependent upon ongoing neuronal activity in the substantia nigra, endogenous striatal substance P and dopamine

The aim of the present microdialysis study was to investigate whether the increase in striatal glutamate levels induced by intrastriatal perfusion with NMDA was dependent on the activation of extrastriatal loops and/or endogenous striatal substance P and dopamine. The NMDA-evoked striatal glutamate release was mediated by selective activation of the NMDA receptor-channel complex and action potential propagation, as it was prevented by local perfusion with dizocilpine and tetrodotoxin, respectively. Tetrodotoxin and bicuculline, perfused distally in the substantia nigra reticulata, prevented the NMDA-evoked striatal glutamate release, suggesting its dependence on ongoing neuronal activity and GABA(A) receptor activation, respectively, in the substantia nigra. The NMDA-evoked glutamate release was also dependent on striatal substance P and dopamine, as it was antagonized by intrastriatal perfusion with selective NK(1) (SR140333), D(1)-like (SCH23390) and D(2)-like (raclopride) receptor antagonists, as well as by striatal dopamine depletion. Furthermore, impairment of dopaminergic transmission unmasked a glutamatergic stimulation by submicromolar NMDA concentrations. We conclude that in vivo the NMDA-evoked striatal glutamate release is mediated by activation of striatofugal GABAergic neurons and requires activation of striatal NK(1) and dopamine receptors. Endogenous striatal dopamine inhibits or potentiates the NMDA action depending on the strength of the excitatory stimulus (i.e. the NMDA concentration).