In silico insight into Amurensinine - an N-Methyl-D-Aspartate receptor antagonist

BACKGROUND Some isopavines can exhibit important biological activity in the treatment of neurological disorders since it is considered an antagonist of the specific Nmethyl-D-Aspartate(NMDA)receptor.Amurensinine is an isopavine which still has few studies.In view of the potential of isopavines as NMDA receptor antagonists,theoretical studies using bioinformatics were carried out in order to investigate whether Amurensinine binds to the NMDA receptor and to analyze the receptor/Ligand complex.This data can contribute to understanding of the onset of neurological diseases and contribute to the planning of drugs for the treatment of neurological diseases involving the NMDA receptor.AIM To investigate the interaction of the antagonist Amurensinine on the GluN1A/GluN2B isoform of the NMDA receptor using bioinformatics.METHODS The three-dimen-sional structure of the GluN1A/GluN2B NMDA receptor was selected from the Protein Data Bank(PDB)-PDB:4PE5,and the three-dimensional structure of Amurensinine(ligand)was designed and optimized using ACD/SchemsketchTM software.Prediction of the protonation state of Amurensinine at physiological pH was performed using MarvinSketch software(ChemAxon).Protonated and non-protonated Amurensin were prepared using AutoDock Tools 4 software and simulations were performed using Autodock Vina v.1.2.0.The receptor/Ligand complexes were analyzed using PyMol(Schrödinger,Inc)and BIOVIA Discovery Studio(Dassault Systemes)software.To evaluate the NMDA receptor/Amurensinine complex and validate the molecular docking,simulations using NMDA receptor and Ifenprodil antagonist were performed under the same conditions.Ifenprodil was also designed,optimized and protonated,under the same conditions as Amurensinine.RESULTS Molecular docking simulations showed that both non-protonated and protonated Amurensinine bind to the amino terminal domain(ATD)domain of the GluN1A/GluN2B NMDA receptor with significant affinity energy,-7.9 Kcal/mol and-8.1 Kcal/mol,respectively.The NMDA receptor/non-protonated Amurensinine complex was stabilized by 15 bonds,while the NMDA receptor/protonated Amurensinine complex was stabilized by less than half,6 bonds.Despite the difference in the number of bonds,the variation in bond length and the average bond length values are similar in both complexes.The complex formed by the NMDA receptor and Ifenprodil showed an affinity energy of-8.2 Kcal/mol,a value very close to that obtained for the NMDA receptor/Amurensinine complex.Molecular docking between Ifenprodil and the GluN1A/GluN2B NMDA receptor demonstrated that this antagonist interacts with the ATD of the receptor,which validates the simulations performed with Amurensinine.CONCLUSION Amurensinine binds to the NMDA receptor on ATD,similar to Ifenprodil,and the affinity energy is closer.These data suggest that Amurensinine could behave as a receptor inhibitor,indicating that this compound may have a potential biological application,which should be evaluated by in vitro and preclinical assays.

Importance of human leukocyte antigen antibodies and leukocyte antigen/killer-cell immunoglobulin-like receptor genes in liver transplantation

Many mechanisms have been proposed to explain the hypothetical state of hepatic tolerance,which is described by eventual imbalances or deregulation in the balance of cytokines,mediators,effectors,and regulatory cells in the complex milieu of the liver.In this section,we will comment on the importance of donorspecific anti-human leukocyte antigen(HLA)antibodies(DSA)as well as the compatibility and pairings of HLA and killer-cell immunoglobulin-like receptor(KIR)genotypes in the evolution of liver transplantation.Thus,HLA compatibility,viral infections,and HLA-C/KIR combinations have all been linked to liver transplant rejection and survival.There have been reports of increased risk of acute and chronic rejection with ductopenia,faster graft fibrosis,biliary problems,poorer survival,and even de novo autoimmune hepatitis when DSAs are present in the recipient.Higher mean fluorescence intensity(MFI)values of the DSAs and smaller graft size were associated with poorer patient outcomes,implying that high-risk patients with preformed DSAs should be considered for selecting the graft placed and desensitization methods,according to the investigators.Similarly,in a combined kidney-liver transplant,a pretransplant with a visible expression of several DSAs revealed that these antibodies were resistant to treatment.The renal graft was lost owing to antibody-mediated rejection(AMR).The HLA antigens expressed by the transplanted liver graft influenced antibody elimination.Pathologists are increasingly diagnosing AMR in liver transplants,and desensitization therapy has even been employed in situations of AMR,particularly in patients with DSAs in kidney-hepatic transplants and high-class II MFI due to Luminex.In conclusion,after revealing the negative impacts of DSAs with high MFI,pretransplant virtual crossmatch techniques may be appropriate to improve evolution;however,they may extend cold ischemia periods by requiring the donor to be typed.

C-X-C chemokine receptor type 7 antibody enhances neural plasticity after ischemic stroke

Stromal cell-derived factor-1 and its receptor C-X-C chemokine receptor 4(CXCR4) have been shown to regulate neural regeneration after stroke.Howeve r,whether stromal cell-derived factor-1 receptor CXCR7,which is widely distributed in the develo ping and adult central nervous system,participates in neural regeneration remains poorly unde rstood.In this study,we established rat models of focal cerebral ischemia by injecting endothelin-1 into the cerebral co rtex and striatum.Starting on day 7 after injury,CXCR7-neutralizing antibody was injected into the lateral ventricle using a micro drug delivery system for 6 consecutive days.Our results showed that CXCR7-neutralizing antibody increased the total length and number of sprouting co rticospinal tra ct fibers in rats with cerebral ischemia,increased the expression of vesicular glutamate transporter 1 and growth-related protein 43,marke rs of the denervated spinal cord synapses,and promoted the differentiation and maturation of oligodendrocyte progenitor cells in the striatum.In addition,CXCR7 antibody increased the expression of CXCR4 in the striatum,increased the protein expression of RAS and ERK1/2 associated with the RAS/ERK signaling pathway,and im proved rat motor function.These findings suggest that CXCR7 improved neural functional recovery after ischemic stroke by promoting axonal regeneration,synaptogenesis,and myelin regeneration,which may be achieved by activation of CXCR4 and the RAS/ERK1/2 signaling pathway.

N-methyl-D-aspartate receptor subunit 1 regulates neurogenesis in the hippocampal dentate gyrus of schizophrenia-like mice

N-methyl-D-aspartate receptor hypofunction is the basis of pathophysiology in schizophrenia. Blocking the N-methyl-D-aspartate receptor impairs learning and memory abilities and induces pathological changes in the brain. Previous studies have paid little attention to the role of the N-methyl-D-aspartate receptor subunit 1 (NR1) in neurogenesis in the hippocampus of schizophrenia. A mouse model of schizophrenia was established by intraperitoneal injection of 0.6 mg/kg MK-801, once a day, for 14 days. In N-methyl-D-aspartate-treated mice, N-methyl-D-aspartate was administered by intracerebroventricular injection in schizophrenia mice on day 15. The number of NR1-, Ki67- or BrdU-immunoreactive cells in the dentate gyrus was measured by immunofluorescence staining. Our data showed the number of NR1-immunoreactive cells increased along with the decreasing numbers of BrdU- and Ki67-immunoreactive cells in the schizophrenia groups compared with the control group. N-methyl-D-aspartate could reverse the above changes. These results indicated that NR1 can regulate neurogenesis in the hippocampal dentate gyrus of schizophrenia mice, supporting NR1 as a promising therapeutic target in the treatment of schizophrenia. This study was approved by the Experimental Animal Ethics Committee of the Ningxia Medical University, China (approval No. 2014-014) on March 6, 2014.

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis

Morphine is a frequently used analgesic that activates the mu-opioid receptor(MOR),which has prominent side effects of tolerance.Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance,currently,there is no effective therapy to treat morphine tolerance.In the current study,we aimed to develop a monoclonal antibody(mAb)precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms.We successfully prepared a mAb targeting MOR,named 3A5C7,by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization,and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation.Treatment of two cell lines,HEK293T and SH-SY5Y,with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2(GRK2)/b-arrestin2-dependent mechanism,as demonstrated by immunofluorescence staining,flow cytometry,Western blotting,coimmunoprecipitation,and small interfering ribonucleic acid(siRNA)-based knockdown.This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR.We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid.Western blot,enzyme-linked immunosorbent assays,and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase,the in vitro biomarker of morphine tolerance,via the GRK2/b-arrestin2 pathway.Furthermore,in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice,and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence.Finally,intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/b-arrestin2 pathway.Collectively,our study provided a therapeutic mAb,3A5C7,targeting MOR to treat morphine tolerance,mediated by enhancing morphine-induced MOR endocytosis.The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

Propofol effectively inhibits lithium-pilocarpine-induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine.The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior,electroencephalography and 24-hour survival rate.Propofol（12.5-100 mg/kg） improved status epilepticus in a dose-dependent manner,and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection.Western blot results showed that,24 hours after induction of status epilepticus,the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus.Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels,but not the increase in N-methyl-D-aspartate receptor 2A subunit levels.The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine.This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures.

Discrimination between leucine-rich glioma-inactivated 1 antibody encephalitis and gamma-aminobutyric acid B receptor antibody encephalitis based on ResNet18

This study aims to discriminate between leucine-rich glioma-inactivated 1(LGI1)antibody encephalitis and gammaaminobutyric acid B(GABAB)receptor antibody encephalitis using a convolutional neural network(CNN)model.A total of 81 patients were recruited for this study.ResNet18,VGG16,and ResNet50 were trained and tested separately using 3828 positron emission tomography image slices that contained the medial temporal lobe(MTL)or basal ganglia(BG).Leave-one-out cross-validation at the patient level was used to evaluate the CNN models.The receiver operating characteristic(ROC)curve and the area under the ROC curve(AUC)were generated to evaluate the CNN models.Based on the prediction results at slice level,a decision strategy was employed to evaluate the CNN models’performance at patient level.The ResNet18 model achieved the best performance at the slice(AUC=0.86,accuracy=80.28%)and patient levels(AUC=0.98,accuracy=96.30%).Specifically,at the slice level,73.28%(1445/1972)of image slices with GABAB receptor antibody encephalitis and 87.72%(1628/1856)of image slices with LGI1 antibody encephalitis were accurately detected.At the patient level,94.12%(16/17)of patients with GABAB receptor antibody encephalitis and 96.88%(62/64)of patients with LGI1 antibody encephalitis were accurately detected.Heatmaps of the image slices extracted using gradient-weighted class activation mapping indicated that the model focused on the MTL and BG for classification.In general,the ResNet18 model is a potential approach for discriminating between LGI1 and GABAB receptor antibody encephalitis.Metabolism in the MTL and BG is important for discriminating between these two encephalitis subtypes.

Influences of levodopa on expression of N-methyl-D-aspartate receptor-1-subunit in the visual cortex of monocular deprivation rats

AIM: Many studies have demonstrated N-methyl-D-aspartate receptor-1-subunit (NMDAR1) is associated with amblyopia. The effectiveness of levodopa in improving the visual function of the children with amblyopia has also been proved. But the mechanism is undefined. Our study was to explore the possible mechanism. METHODS: Sixty 14-day-old healthy SD rats were randomly divided into 4 groups, including normal group, monocular deprivation group, levodopa group and normal saline group, 15 rats each. We sutured all the rats' unilateral eyelids except normal group to establish the monocular deprivation animal model and raise them in normal sunlight till 45-day-old. NMDAR1 was detected in the visual cortex with immunohistochemistry methods, Western Blot and Real time PCR. LD and NS groups were gavaged with levodopa (40mg/kg) and normal saline for 28 days respectively. NMDAR1 was also detected with the methods above. RESULTS: NMDAR1 in the visual cortex of MD group was less than that of normal group. NMDAR1 in the visual cortex of LD group was more than that of NS group. CONCLUSION: NMDAR1 is associated with the plasticity of visual development. Levodopa may influence the expression of NMDAR1 and improve visual function, and its target may lie in the visual cortex.

Autoantibodies related to ataxia and other central nervous system manifestations of gluten enteropathy

Gluten ataxia and other central nervous system disorders could be linked to gluten enteropathy and related autoantibodies.In this narrative review,we focus on the various neuro-logical manifestations in patients with gluten sensitivity/celiac disease,immunological and autoimmune mechanisms of ataxia in connection to gluten sensitivity and the autoantibodies that could be used as a biomarker for diagnosing and following.We focused on the anti-gliadin antibodies,antibodies to different isoforms of tissue transglutaminase(TG)(anti-TG2,3,and 6 antibodies),anti-glycine receptor antibodies,anti-glutamine acid decarboxylase antibodies,anti-deamidated gliadin peptides antibodies,etc.Most studies found a higher prevalence of these antibodies in patients with gluten sensitivity and neurological dysfunction,presented as different neurological disorders.We also discuss the role of a gluten-free diet on the clinical improvement of patients and also on imaging of these disorders.

Changes in synaptic and extrasynaptic N-methyl-D-aspartate receptor-mediated currents at early-stage epileptogenesis in adult mice

Previous reports have shown that N-methyl-D-aspartate （NMDA） receptors are extensively involved in epilepsy genesis and recurrence. Recent studies have shown that synaptic and extrasynaptic NMDA receptors play different, or even opposing, roles in various signaling pathways, including synaptic plasticity and neuronal death. The present study analyzed changes in synaptic and extrasynaptic NMDA receptor-mediated currents during epilepsy onset. Mouse models of lithium chloride pilocarpLne-induced epilepsy were established, and hippocampal slices were prepared at 24 hours after the onset of status epilepticus. Synaptic and extrasynaptic NMDA receptor-mediated excitatory post-synaptic currents （NMDA-EPSCs） were recorded in CA1 pyramidal neurons by whole-cell patch clamp technique. Results demonstrated no significant difference in rise and delay time of synaptic NMDA-EPSCs compared with normal neurons. Peak amplitude, area-to-peak ratio, and rising time of extrasynaptic NMDA-EPSCs remained unchanged, but decay of extrasynaptic NMDA-EPSCs was faster than that of normal neurons, These results suggest that extrasynaptic NMDA receptors play a role in epileptogenesis.

Kappa opioid receptor antagonist and N-methyl-D-aspartate receptor antagonist affect dynorphin-induced spinal cord electrophysiologic impairment

The latencies of motor- and somatosensory-evoked potentials were prolonged to different degrees, and wave amplitude was obviously decreased, after injection of dynorphin into the rat subarachnoid cavity. The wave amplitude and latencies of motor- and somatosensory-evoked potentials were significantly recovered at 7 and 14 days after combined injection of dynorphin and either the kappa opioid receptor antagonist nor-binaltorphimine or the N-methyl-D-aspartate receptor antagonist MK-801. The wave amplitude and latency were similar in rats after combined injection of dynorphin and nor-binaltorphimine or MK-801. These results suggest that intrathecal injection of dynorphin causes damage to spinal cord function. Prevention of N-methyl-D-aspartate receptor or kappa receptor activation lessened the injury to spinal cord function induced by dynorphin.

Dose-dependent and combined effects of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters

This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine （L-NA） on the survival of retinal ganglion cells （RGCs） at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 （1.0, 3.0 and 4.5 mg/kg） and L-NA （1.5, 3.0, 4.5 and 6.0 mg/kg） increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.

N-methyl-D-aspartate receptor subtype 3A promotes apoptosis in developing mouse brain exposed to hyperoxia

In the present study, 7 day postnatal C57/BL6 wild-type mice （hyperoxia group） and 7 day postnatal N-methyI-D-aspartate receptor subtype 3A knockout mice （NR3A KO group） were exposed to 75% oxygen and 15% nitrogen in a closed container for 5 days. Wild-type mice raised in normoxia served as controls. TdT-mediated dUTP nick end labeling （TUNEL）/neuron-specific nuclear protein （NeuN） and 5-bromo-2＇-deoxyuridine （BrdU）/NeuN immunofluorescence staining showed that the number of apoptotic cells and the number of proliferative cells in the dentate subgranular zone significantly increased in the hyperoxia group compared with the control group. However, in the same hyperoxia environment, the number of apoptotic cells and the number of proliferative cells significantly decreased in the NR3A KO group compared with hyperoxia group. TUNEL＋/NeuN＋ and BrdU＋/NeuN~ cells were observed in the NR3A KO and the hyperoxia groups. These results demonstrated that the NR3A gene can promote cell apoptosis and mediate the potential damage in the developing brain induced by exposure to non-physiologically high concentrations of oxygen.

Controlling N-methyl-D-aspartate receptor subunit 1 with calcitonin gene related peptide after cerebral ischemic injury

BACKGROUND: Activation of N-methyl-D-aspartate receptor (NMDAR) is a key link of exitotoxicity at the phase of cerebral ischemic injury. Because NMDAR is a main way to mediate internal flow of Ca2+ among glutamic acid receptors, over-excitation can cause neuronal apoptosis. Calcitonin gene related peptide has a strongly biological activity. On one hand, it can protect ischemic neurons through inhibiting the expression of NMDAR1 mRNA; on the other hand, it can play the protective effect through down-regulating the expression of NMDAR1 mRNA by exogenous calcitonin gene related peptide. OBJECTIVE: To observe the expression of NMDAR1 and the regulatory effect of calcitonin gene related peptide on the expression of NMDAR1 mRNA and protein in the cerebral cortex of rats with focal cerebral ischemia/reperfusion (I/R). DESIGN: Randomized controlled animal study. SETTING: China Medical University. MATERIALS: A total of 216 healthy male Wistar rats, general grade, weighing 250-280 g, were selected in this study. Twelve rats were randomly selected to regard as control group; meanwhile, other 204 rats were used to establish middle cerebral artery occlusion/reperfusion (MACO) models. The main reagents were detailed as follows: calcitonin gene related peptide (Sigma Company); calcitonin gene related peptide kit (Boster Company); antibody Ⅰ, Ⅱ and antibody β-actin Ⅰ, Ⅱ of NMDAR1 mRNA and chemiluminescence reagent (Santa Cruz Company, USA). METHODS: The experiment was carried out in the Laboratory of Neurobiology of China Medical University from August 2005 to June 2006. ① Right MCAO models of rats were established to cause focal ischemia and scored based on Zea Longa five-grade scale. If the scores were 1, 2 and 3 after wakefulness, the MACO models were established successfully and involved in the experiment. A total of 120 rats with successful modeling were randomly divided into I/R group and administration group with 60 in each group. All rats in the both groups were observed at five time points, including 6, 12, 24, 48 and 72 hours after reperfusion and after 2-hour ischemia, with 12 experimental animals at each time point. Six rats were prepared for detection of hybridization in situ, and the other 6 were used for Western blotting histochemical detection. Rats in the control group were opened their skin to separate common carotid artery and not treated with line and drugs. In addition, rats in the I/R group were treated with 1 mL saline at 2 hours after focal cerebral ischemia, and then, rats in the administration group were treated with 1 mL (1 g/L) calcitonin gene related peptide at 2 hours after focal cerebral ischemia. ② The expression of NMDAR1 mRNA was detected with hybridization in situ at various time points; moreover, the expression of NMDAR1 protein was measured with Western blotting method at various time points. The results were analyzed with Metamoph imaging analytical system. MAIN OUTCOME MEASURES: The expression of NMDAR1 mRNA and its protein in cortical neurons of rats at various time points. RESULTS: A total of 84 rats were excluded because of non-symptoms, exanimation or death; and then, 132 rats were involved in the final analysis. The expression of NMDAR1 mRNA and its protein in cortical neurons of rats in the control group was 0.205±0.001 and 0.184±0.001, respectively; after I/R, expression of NMDAR1 mRNA and its protein was up-regulated, especially, expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.245±0.003, 0.287±0.004, 0.354±0.008, 0.284±0.002 and 0.217±0.006, respectively; moreover, expression of protein at 6, 12, 24, 48 and 72 hours was 0.222±0.003, 0.261±0.028, 0.311±0.004, 0.259±0.013 and 0.210±0.008, respectively. There was significant difference between the two groups (0.205±0.001, P < 0.01). The expression was up-related in the former 24 hours, reached peak at 24 hours, down-regulated, and decreased to the level of control group at 72 hours. Except 72 hours, the expression of NMDAR1 mRNA and its protein was lower in administration group than that in I/R group at other four time points. In addition, the expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.223±0.005, 0.243±0.001, 0.292±0.002, 0.250±0.003 and 0.213±0.003, respectively; moreover, the expression of protein at 6, 12, 24, 48 and 72 hours was 0.216±0.006, 0.245±0.025, 0.276±0.003, 0.241±0.045 and 0.202±0.013, respectively. There was significant difference at various time points (P < 0.05). CONCLUSION: The expressions of NMDAR1 mRNA and its protein of peripheral cortical neurons are up-related in ischemic area after focal cerebral I/R. Meanwhile, exogenous calcitonin gene related peptide can protect cortical neurons through inhibiting expression of NMDAR1 mRNA and its protein after focal cerebral I/R.

N-methyl-D-aspartate glutamate receptors (NMDARs) in stroke pathogenesis and treatments

N-methyl-D-aspartate glutamate receptors(NMDARs)play crucial roles in the pathogenesis of neuronal injuries following a stroke insult;therefore,a plethora of preclinical studies focus on better understanding functions of NMDARs and their associated signaling pathways.Over the past decades,NMDARs have been found to exert dual effects in neuronal deaths signaling and neuronal survival signaling during cerebral ischemia.Moreover,many complex intracellular signaling pathways downstream of NMDAR activation have been elucidated,which provide novel targets for developing much-needed neuro-protectants for patients with stroke.In this review,we will discuss the recent progress in understanding the underlying mechanisms of stroke related to NMDAR activation and the potential therapeutic strategies based on these discoveries.

Changes in soluble interleukin-2 receptor level in serum and Na^+ -K^+ -exchanging ATPase activity in semen of infertile men caused by antisperm antibody

Aim: To explore the possible mechanisms of male infertility caused by antisperm antibody (AsAb). Methods: Thesoluble interleukin-2 receptor (sIL-2R) level in serum was analyzed by ELISA and Na^+ -K^+ -exchanging ATPase activi-ty in semen by phosphorus (Pi) assay. Results: The slL-2R level in serum was significantly higher and the Na^+ -K^+ -exchanging ATPase activity in semen significantly lower in AsAb positive infertile men when compared with thecontrols. Conclusion: The AsAb titer varies with the slL-2R level in serum. A decrease in Na^+ -K^+ -exchangingATPase activity in semen may play a role in male infertility caused by AsAb.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.

Effect of Xingnaojing Injection(醒脑静注射液)on Hippocampal N-methyl-D-aspartic Acid Receptors of Focal Cerebral Ischemia in Rats

Objective: To observe and elucidate the neuroprotective effect of Xingnaojing (XNJ) injection on hippocampal N-methyl-D-aspartic acid (NMDA) receptors of focal cerebral ischemia in rats. Methods: Cerebral ischemia was established by occluding the middle cerebral artery with an intraluminal suture technique in rats. Neurological deficit score, infarct volume and quantity of NMDA receptors were estimated in all groups and compared. Results: After being treated with XNJ, the score decreased in the initial 6 hours and infarct volume decreased in 24 hours. And within 24 hours, the quantity of NMDA receptors obviously decreased compared with the model group (P<0. 01) It indicated that XNJ could ameliorate neurological behavior of middle cerebral artery occlusion rats and down-regulate the expression of hippocampal NMDA receptors. Conclusion: The neuroprotective effect of XNJ on focal cerebral ischemia is possibly related to down-regulating the expression of NMDA receptors in rats.

Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan

The objective of this study is to express the carbohydrate recognition domain （CRD） of the asialoglycoprotein receptor （ASGPR） H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21（DE3）plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay （ELISA）, Western blotting and immunohistochemical staining （IHC）. The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marrnota himalayan—— for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

Adult celiac disease with acetylcholine receptor antibody positive myasthenia gravis

Celiac disease has been associated with some autoimmune disorders. A 40-year-old competitive strongman with celiac disease responded to a gluten-free diet, but developed profound and generalized motor weakness with acetylcholine receptor antibody positive myasthenia gravis, a disorder reported to occur in about 1 in 5000. This possible relationship between myasthenia gravis and celiac disease was further explored in serological studies. Frozen stored serum samples from 23 acetylcholine receptor antibody positive myasthenia gravis patients with no intestinal symptoms were used to screen for celiac disease. Both endomysial and tissue transglutaminase antibodies were examined. One of 23 (or, about 4.3%) was positive for both IgA-endomysial and IgA tissue transglutaminase antibodies. Endoscopic studies subsequently showed duodenal mucosal scalloping and biopsies confirmed the histopathological changes of celiac disease. Celiac disease and myasthenia gravis may occur together more often than is currently appreciated. The presence of motor weakness in celiac disease may be a clue to occult myasthenia gravis, even in the absence of intestinal symptoms.