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Chiral resolution of n-methyl-d,l-aspartic acid using a predicted n-demethylase from Chloroflexi bacterium 54-19

Systems Microbiology and Biomanufacturing 2022年第3期2卷 507-522页

作者： Juanjuan du Liang Zhang national Engineering Laboratory for Cereal Fermentation Technology Jiangnan UniversityWuxi 214122China

n-methyl-d-aspartic acid(nMdA),an amino acid existing in human and animal central nervous system,exerts agonist action on one of the glutamate receptor subtypes and is clinically used for treatment of diabetes,Parkinson’s and Alzheimer’s syndrome.In this study,an enzymatic protocol for the chiral resolution of n-methyl-d,l-aspartic acid was built with a predicted n-demethylase(GenBank Id:OJV90073.1)from the genome of Chloroflexi bacterium 54-19.Through sequence alignment,the enzyme shares an identity of 32.19%to 2UZZ(PdB Id)with a conserved catalytic center.Recombinantly expressed in Bacillus subtilis WB600,the n-demethylase was characterized with optimal temperature and pH at 55℃and 7.5,and adaptive temperature and pH were 40-60℃ and 6-8.The effects of organic solvents and metal ions were investigated as well.Compared with other previously reported sarcosine oxidases,the enzyme showed a specific n-demethylation activity against n-methyl-l-aspartic acid according to the analysis by chiral liquid chromatography,LC-MS/MS and a detection by polarimeter.The results demonstrated 76%of 4.5 mM n-methyl-d,l-aspartic acid could be chirally separated by 7 mg·L^(−1) enzyme after reaction of 80 min.This work provided a foundation for mild synthesis of nMdA in industry.

关键词： n-methyl-d-aspartic acid Sarcosine oxidase n-demethylation Enantioselectivity Kinetic resolution

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Comparison of three administration modes for establishing a zebrafish seizure model induced by n-methyl-d-aspartic acid

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World Journal of Psychiatry 2020年第7期10卷 150-161页

作者： Xin-Yi Long Shuang Wang Zhi-Wen Luo Xu Zhang Hong Xu Queen Mary School of nanchang University Nanchang 330006Jiangxi ProvinceChina department of Sports Medicine Huashan HospitalFudan UniversityShanghai 200433China Affiliated Eye Hospital of nanchang University Nanchang 330006Jiangxi ProvinceChina Institute of Life Science Nanchang UniversityNanchang 330031Jiangxi ProvinceChina

BACKGROUnd Epilepsy is a complex neurological disorder characterized by recurrent,unprovoked seizures resulting from the sudden abnormal discharge of brain neurons.It leads to transient brain dysfunction,manifested by abnormal physical movements and consciousness.It can occur at any age,affecting approximately 65 million worldwide,one third of which are still estimated to suffer from refractory seizures.There is an urgent need for further establishment of seizure models in animals,which provides an approach to model epilepsy and could be used to identify novel anti-epileptic therapeutics in the future.AIM To compare three administration modes for establishing a seizure model caused by n-methyl-d-aspartic acid(nMdA)in zebrafish.METHOdS Three administration routes of nMdA,including immersion,intravitreal injection and intraperitoneal injection,were compared with regard to their effects on inducing seizure-like behaviors in adult zebrafish.We evaluated neurotoxicity by observing behavioral changes in zebrafish and graded those behaviors with a seizure score.In addition,the protective effects of MK-801(dizocilpine)and natural active constituent resveratrol against nMdA-induced alterations were studied.RESULTS The three nMdA-administration methods triggered different patterns of the epileptic process in adult zebrafish.Seizure scores were increased after increasing nMdA concentration regardless of the mode of administration.However,the curve of immersion continuously rose to a high plateau(after 50 min),while the curves of intravitreal injection and intraperitoneal injection showed a spike in the early stage(10-20 min)followed by a steady decrease in seizure scores.Furthermore,pretreatment with resveratrol and MK-801 significantly delayed seizure onset time and lowered seizure scores.COnCLUSIOn By comparing the three methods of administration,intravitreal injection of nMdA was the most suitable for establishing an acute epileptic model in zebrafish.Thus,intraperitoneal injection in zebrafish can be applied to simulate

关键词： Seizure Zebrafish n-methyl-d-aspartic acid Administration modes

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depression following a traumatic brain injury:uncovering cytokine dysregulation as a pathogenic mechanism

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neural Regeneration Research 2018年第10期13卷 1693-1704页

作者： Colleen n.Bodnar Josh M.Morganti Adam d.Bachstetter Spinal Cord & Brain Injury Research Center University of Kentucky department of neuroscience University of Kentucky Sanders-Brown Center on Aging University of Kentucky

A substantial number of individuals have long-lasting adverse effects from a traumatic brain injury（TBI）. depression is one of these long-term complications that influences many aspects of life. depression can limit the ability to return to work, and even worsen cognitive function and contribute to dementia. The mechanistic cause for the increased depression risk associated with a TBI remains to be defined. As TBI results in chronic neuroinflammation, and priming of glia to a secondary challenge, the inflammatory theory of depression provides a promising framework for investigating the cause of depression following a TBI. Increases in cytokines similar to those seen in depression in the general population are also increased following a TBI. Biomarker levels of cytokines peak within hours-to-days after the injury, yet pro-inflammatory cytokines may still be elevated above physiological levels months-to-years following TBI, which is the time frame in which post-TBI depression can persist. As tumor necrosis factor α and interleukin 1 can signal directly at the neuronal synapse, pathophysiological levels of these cytokines can detrimentally alter neuronal synaptic physiology. The purpose of this review is to outline the current evidence for the inflammatory hypothesis of depression specifically as it relates to depression following a TBI. Moreover, we will illustrate the potential synaptic mechanisms by which tumor necrosis factor α and interleukin 1 could contribute to depression. The association of inflammation with the development of depression is compelling; however, in the context of post-TBI depression, the role of inflammation is understudied. This review attempts to highlight the need to understand and treat the psychological complications of a TBI, potentially by neuroimmune modulation, as the neuropsychiatric disabilities can have a great impact on the rehabilitation from the injury, and overall quality of life.

关键词： concussion major-depressive disorder chronic traumatic encephalopathy inflammation tumor necrosis factor α interleukin 1 microglia astrocytes synaptic physiology n-methyl-d-aspartic acid

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Inhibition of n-methyl-d-aspartate-activated Current by Bis(7)-tacrine in HEK-293 Cells Expressing nR1/nR2A or nR1/nR2B Receptors

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2012年第6期32卷 793-797页

作者：刘宇炜李超英 Wuhan Institutes of Biomedical Sciences department of Anatomy School of Medicine Jianghan University

In normal rat forebrain, the nR1/nR2A and nR1/nR2B dimmers are the main constitutional forms of nMdA receptors. The present study was carried out to determine the functional properties of the heteromeric nMdA receptor subunits and their inhibition by bis(7)-tacrine (B7T). Rat nR1, nR2A and nR2B cdnAs were transfected into human embryonic kidney 293 cells (HEK-293).The inhibition of nMdA-activated currents by B7T was detected in HEK-293 cell expressing nR1/nR2A or nR1/nR2B receptors by using whole-cell patch-clamp techniques. The results showed that in HEK-293 cells expressing nR1/nR2A receptor, 1μmol/L B7T inhibited 30μmol/L nMdA- and 1000μmol/L nMdA-activated steady-state currents by 46% and 40%, respectively (P>0.05; n=5), suggesting that the inhibition of B7T on nR1/nR2A receptor doesn’t depend on nMdA concentration, which is consistent with a non-competitive mechanism of inhibition. But for the nR1/nR2B receptor, 1μmol/L B7T inhibited 30μmol/L nMdA- and 1000 μmol/L nMdA-activated steady-state currents by 61% and 13%, re-spectively (Pn=6), showing that B7T appears to be competitive with nMdA. In addition, simultaneous application of 1μmol/L B7T and 1000μmol/L nMdA produced a moderate inhibition of peak nMdA-activated current, followed by a gradual decline of the current to a steady state. However, the gradual onset of inhibition produced by B7T applied simultaneously with nMdA was eliminated when B7T was given 5s before nMdA. These results suggested that B7T inhibition of nMdA current mediated by nR1/nR2B receptor was slow onset, and it did not depend on the presence of the agonist. With holding potentials ranging from -50 to +50 mV, the B7T inhibition rate of nMdA currents didn’t change significantly, and neither did the reversal potential. We are led to conclude that the nR1/nR2B recombinant receptor can serve as a very useful model for studying the molecular mechanism of nMdA receptor inhibition by B7T.

关键词： bis(7)-tacrine n-methyl-d-aspartic acid nR1/nR2A nR1/nR2B

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