Gamma-aminobutyric acid A receptor and N-methyl-D-aspartate receptor subunit expression in rat spiral ganglion neurons

BACKGROUND： Gamma-aminobutyric acid A （GABAA） and N-methyl-D-aspartate （NMDA） receptors are significant receptors in the central nervous system. An understanding of GABAA and NMDA receptor expression in spiral ganglion neurons （SGN） provides information for the functional role of these receptors in the auditory system. OBJECTIVE： To investigate mRNA expression of GABAA receptor （GABAAR） and NMDA receptor （NMDAR） subunits in the rat SGN. DESIGN, TIME AND SETTING： This in vitro, molecular biological study was performed at the Laboratory of Otolaryngology-Head and Neck Surgery, Guangxi Medical University, China from July 2007 to May 2008. MATERIALS： Reverse Transcriptase Kit and Taq DNA polymerase were purchased from Fermentas Burlington, ON, Canada; GABAAR and NMDAR primers were purchased from Shanghai Sangon, Shanghai, China. METHODS： SGN from 3-5 day postnatal Wistar rats was collected for primary cultures, mRNA expression of GABAAR and NMDAR subunits in the SGN was determined by reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURES： Expression levels of GABAAR and NMDAR subunits were determined by quantitative analysis. RESULTS： GABAAR subunits （αl 6, β1 3, and y1 3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were detected in the SGN. In α subunit genes of GABAAR, α1 and α3 expression was similar （P 〉 0.05） and greater than the other subunits. Of the β subunit genes, β1 subunit mRNA levels were greater than β2 and β3. Of the y subunit genes, y2 subunit mRNA levels were greater than y1 and y3. NR1 mRNA expression was the greatest of NMDAR subunits. CONCLUSION： GABAAR subunits （α1 6, β1-3, and y1-3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were expressed in the rat SGN. Through comparison of GABAAR and NMDAR subunit expression, possible GABAAR combinations, as well as highly expressed subunit combinations, were estimated, which provided information for pharmacological and electrophysiological characteristics of GABAAR in the auditory system.

N-methyl-D-aspartate (NMDA) mediates vascular relaxation via nitric oxide (NO) in rats but not in mice

Amperometric studies have indicated that substance P as well as NMDA stimulates release of NO in rat aortic rings. These data have been confirmed by functional observations of vaso-relaxant action of NMDA within noradrenaline pre-contracted aortic rings, supporting the presence of NMDA receptor in rat aortic rings. It is known that the enzyme endothelial NO synthase (eNOS) mediates vasodilatation not only in rats, but also in C57BL6 mice aortic ring, indicating that in this blood vessel NO is the endogenous endothelium-derived vasodilator. In this work, amperometry together with specifically nitrites insensitive micro-biosensors have been applied to examine the effect of NMDA and substance P upon NO release in rat and in two strains of mice aortic rings. The electrochemical data monitored demonstrate that NMDA mediates vascular relaxation via NO in rats but not in mice. These results are supported by functional data, therefore they suggest that NMDA receptors are “not responding” within these experimental conditions in mice aortic rings.

D-Aspartate, a Key Element for the Improvement of Sperm Quality

Introduction: D-Aspartate is an endogenous amino acid involved in LH and testosterone release in humans. In this study we investigate the impact of nutritional supplementation of sodium D-aspartate on the improvement of sperm quality in sub-fertile patients and the rate of pregnancies that occurred with their partners. Materials and Methods: A group of 30 patients affected by oligo-asthenozoospermia and a group of 30 patients affected by asthenozoospermia were treated with a daily dose of sodium D-aspartate for 90 days. After which, the change in spermatozoa concentration and their motility and the pregnancies that occurred with their partners were recorded. Results: We found that the supplementation of D-aspartate significantly increased the concentration and the motility of spermatozoa. In oligo- asthenozoospermic patients the increase of sperm concentration was found to be 2.0-fold, P Conclusions:Treatment of sub-fertile patients with sodium D-aspartate improved the number and the motility of the spermatozoa and consequently improved the rate of pregnancies of their partners.

Production, purification and characterization of D-aspartate oxidase from the fungus <i>Trichoderma harzianum</i>SKW-36

Trichoderma harzianum Rifai SKW-36 produced two kinds of D-amino acid oxidizing enzymes. One enzyme was D-aspartate oxidase acting on acidic D-amino acids such as D-aspartate and D-glutamate and another one was D-amino acid oxidase acting on neutral D-amino acid such as D-phenylalanine and D-methionine. These enzymes in the cell-free extract were separated by DEAE-Toyopearl ion-exchange column chromatography. Casamino acids, peptone, and yeast extract as carbon and nitrogen sources were effective for the production of the enzymes. No D-amino acid tested induced the production of the enzymes. Casamino acid (0.33%) as carbon and nitrogen source gave a highest specific activity of D-aspartate oxidase among media tested. D-Aspartate oxidase, which was purified by four-step column chromatography in addition to ammonium sulfate precipitation, exhibited a subunit molecular mass of 40 kDa by SDS-PAGE analysis. D-Aspartate, D-glutamate and N-methyl-D- aspartate were oxidized as substrates with the specific activities of 7.80 U/mg, 4.90 U/mg, and 4.22 U/mg, respectively. D-Asparagine, D-glutamine, D-alanine, and D-valine were slightly oxidized. No other D- amino acids tested were inert. The enzyme exhibited relatively wide substrate specificity compared to D-aspartate oxidases reported so far. The pH and temperature optima were 7.5 - 8.0 and 35°C, respectively. The enzyme was stable at pH 6.0 - 9.0. About 75% of the enzyme activity was retained even after treating the enzyme at 50°C for 10 min. The enzyme activity was inhibited not by benzoate and tartrate, but 60% and 24% by fumarate and malonate, respectively.

Dissolution Properties of 2-(Dinitromethylene)-5-methyl-1,3-diazacyclopentane in Dimethyl Sulfoxide and N-Methyl Pyrrolidone

The molar enthalpies of dissolution for 2-（dinitromethylene）-5-methyl-1,3-diazacyclopentane（DNMDZ） in dimethyl sulfoxide（DMSO） and N-methyl pyrrolidone（NMP） were measured using an RD496-2000 Calvet microcalorimeter at 298.15 K under atmospheric pressure.Empirical formulae for the calculation of the molar enthalpies of dissolution（Δ diss H） were obtained from the experimental data of the dissolution processes of DNMDZ in DMSO or NMP.The relationships between the rate constant（k） and the molality（b） and between the reaction order（n） and the molality（b） were determined.The corresponding kinetic equations describing the two dissolution processes were dα/dt=10^-2.16（1-α） ^1.01 for the dissolution of DNMDZ in DMSO,and dα/dt=10^-2.02（1-α）^ 0.85 for the dissolution of DNMDZ in NMP,respectively.

Synthesis and Structure Analysis of a Tripeptide Containing N-methyl Group Amino Acid

A convenient method of synthesizing a tripeptide-containing N-methyl group amino acid was developed using O-benzotriazole-N,N,N＇,N＇-tetramethyluronium-hexafluorophosphate as the condensing agent. The crystals of tripeptide had white needles belonging to the orthorhombic space group P2_12_12_1. The conformational preference for homochiral tripeptides with one N-methylated amide bond was also investigated. Crystal-structure analysis showed that homochiral tripeptides with an internal N-methylated amide bond preferred a trans-amide form, thereby giving the peptide β-fold characteristics. Intermolecular C-H···O and N-H···O hydrogen bonds linked the molecules into a one-dimensional chain and stabilized the structure.

Studies on the cyclization reaction of D-aspartic acid

The cyclization reaction of D-aspartic acid was studied, the carboxyl groups of D-aspartic acid were protected by benzyl alcohol to give compound D-dibenzyl aspartate. Then （4R）-benzyl azetidine-2-one-4-carboxylate and meso-3,6-disubstituted piperazine-2,5-diones were synthesized via intramolecular cyclization and intermolecular cycfization of D-dibenzyl aspartate, respectively, and their structures were confirmed by ^1 H NMR and MS. Both cyclization reaction conditions were also investigated in detail.

RE-DISTRIBUTION OF THE N-METHYL GROUP IN GELSEMINE

The N_(b)-bromomethyl derivative (2) of gelsemine,a quaternary ammonium salt,was found to be recalcitrant toward Hofmann type alkaline degradation which has been successful in other cases.Instead,a re-distribution of the N-methyl group took place,giving rise to 3a,1,3b and 3c,in descending order of R_f values.

Identification of N-Methyl Bis(2-(Alkyloxy-Alkylphosphoryloxy)Ethyl) Amines by LC-HRMS/MS

N-Methyl bis(2-(alkyloxy-alkylphosphoryloxy)ethyl)amines, which are abbreviated as PNPs, are a series of new skeleton chemicals belonging to schedule 2.B.04 chemicals of Chemical Weapons Convention (CWC). PNPs are important markers of chemical warfare agents because they are structurally relative to both nerve agents and N-mustards. In this study, fragmentation pathways of the most characteristic fragment ions in Q-TOF mass spectrometry were proposed based on the information from accurate mass and secondary fragmentations of product ions scan experiments. Results indicated that the base ion in LC/HRMS was the quasi-molecular ion [M+H]+. In LC-HRMS/MS, it was [M+H-CnH2n+1P(O)(OH)CmH2m+1O]+ fragment ion which was formed by losing an alkyloxy alkylphosphoryloxy group from the quasi-molecular ion. The diagnostic ion m/z84.0814 was identified as [C5H10N]+, which was the group of (CH2=CH)2N+(H)CH3. PNPs have two protonated centers. One is on the N atom, the other is on the O atom (P=O). O-n-propyl PNPs generally exhibited two fragmentation pathways. Firstly, the quasi-molecular ion [M+H]+ lost a propoxy alkylphosphoryloxy group to produce [R1P(OH+)(O-n-C3H7)OCH2CH2N(CH3)CH=CH2]+, which could be fragmented further to produce [C5H10N]+ ion. Secondly, [R1P(OH+)(O-n-C3H7) OCH=CH2]+ ions were produced from [M+H]+ and fragmented further to produce the abundant ions [R1P(OH+)(OH)OCH =CH2]. However, O-isopropyl PNPs characteristically produced weak fragment ions [M+H-C3H6]+, which were presumably formed via loss of CH3CH=CH2 from [M+H]+. Other PNPs showed similar fragmentation pathways as O-n-propyl PNPs. On the summarization of the MS fragmentation pathways of PNPs, LC-HRMS/MS quantitative and qualitative methods were developed and applied to analyze N-Methyl bis(2-(butoxy-methylphosphoryloxy)ethyl]amine in high background organic samples. The analytical results had successfully supported the sample preparation for the 33rd official proficiency test of Organization for Prohibition of Chemical Weapons (OPCW).

Bifunctional flame retardant solid-state electrolyte toward safe Li metal batteries

Solid polymer electrolytes(SPEs)are one of the most promising alternatives to flammable liquid electrolytes for building safe Li metal batteries.Nevertheless,the poor ionic conductivity at room temperature(RT)and low resistance to Li dendrites seriously hinder the commercialization of SPEs.Herein,we design a bifunctional flame retardant SPE by combining hydroxyapatite(HAP)nanomaterials with Nmethyl pyrrolidone(NMP)in the PVDF-HFP matrix.The addition of HAP generates a hydrogen bond network with the PVDF-HFP matrix and cooperates with NMP to facilitate the dissociation of Li TFSI in the PVDF-HFP matrix.Consequently,the prepared SPE demonstrates superior ionic conductivity at RT,excellent fireproof properties,and strong resistance to Li dendrites.The assembled Li symmetric cell with prepared SPE exhibits a stable cycling performance of over 1200 h at 0.2 m A cm^(-2),and the solid-state LiFePO_4||Li cell shows excellent capacity retention of 85.3%over 600 cycles at 0.5 C.

Selective N-Methylation of N-Methylaniline with CO_(2) and H_(2) over Cu/In_(2)O_(3) Catalyst

N-Methylation of amines with CO_(2) and H2 is a potential approach for CO_(2) utilization because N-methylated amines can be used as solvents and organic intermediates. In_(2)O_(3)-supported Cu (Cu/In_(2)O_(3)) acts as an effective heterogeneous catalyst for N-methylation reaction of N-methylaniline (MA) with CO_(2) and H_(2),showing higher N,N-dimethylaniline (DMA) selectivity than other supported Cu catalysts. On one hand,the dispersion of Cu can be improved by the defective In_(2)O_(3) support. On the other hand,In_(2)O_(3) support is active in the dissociative adsorption of CO_(2) through C—O bond breaking. In addition,the H_(2) dissociation ability of In_(2)O_(3) can also be enhanced by Cu. The combination of Cu and In_(2)O_(3) is effective in the activation of CO_(2),the adsorption of intermediate N-methylformanilide (MFA),the hydrogenation of MFA to DMA and the prohibition of C—N bond cleavage side reactions,thereby enhancing the reaction rate of MA conversion and the selectivity to DMA.

海马长时程增强与26S蛋白酶复合体活性变化的关系

实验观察了大鼠海马脑片上突触传递长时程增强(long term potentiation,LTP)的产生和维持中26S蛋白酶复合体活性的动态变化过程,初步分析了介导其变化的受体途径。结果显示:强直刺激前,26S蛋白酶复合体活性为190±14.3 cpm/(100 μg·2 h),强直刺激诱导fEPSP斜率增加10 min时,其活性升为273±18.3 epm/(100μg·2 h),强直刺激诱导fEPSP斜率增加60 min时,26S蛋白酶复合体活性又降为210±12.8 cpm/(100μg·2 h)。NMDA受体特异阻断剂AP-5在损害L1P产生的同时,抑制26S蛋白酶复合体活性升高。实验结果提示:大鼠海马LTP产生过程中,26S蛋白酶复合体活性存在一个短时间的,依赖于N-methyl-D-aspartate(NMDA)受体的升高过程。

Synthesis of 4-methyl Guanidine Butyric Acid

N-methyl pyrrolidone,hydrochloric acid and thiourea dioxide were adopted as the raw material,and 4-methyl guanidine butyric acid was synthesized through two-step reaction.The optimum synthesis condition for the first step was as follows：n（N-methyl pyrrolidone）∶n（10% HCl）= 1∶2.0,reaction temperature 135 ℃,reaction time 5 h;at that moment,the yield of intermediate 4-methyl-amino butyric acid hydrochloride was 72.89%.The optimum synthesis condition for the second step was as follows：n（4-methyl-amino butyric acid hydrochloride）∶n（thiourea dioxide）= 1∶2.0,reaction temperature 25 ℃,reaction time 12 h,at that moment,the yield of target product was 82.68%.Structure characterization on the intermediates and the target products were carried out through Fourier transform infrared spectroscopy and elemental analysis.

Role of D-aspartate on biosynthesis,racemization,and potential functions:A mini-review

D-aspartate, a natural and endogenous amino acid, widely exists in animal tissues and can be synthesized through aspartate racemase and transformed by D-aspartate oxidase(DDO). D-aspartate mainly serves as a neurotransmitter and has been demonstrated to exhibit various physiological functions,including nutritional potential, regulation on reproduction and hormone biology, and neuron protection.This article mainly reviews the synthesis, racemization, and physiological functions of D-aspartate with emphasis on the potential in diseases.

Physicochemical properties of β-cyclodextrin solutions and precipitates prepared from injectable vehicles

β-Cyclodextrin( β-CyD) is cyclic oligosaccharide of a glucopyranose, containing a relatively hydrophobic central cavity and hydrophilic outer surface. However, the usefulness of β-CyD is limited owing to its low aqueous solubility whereas we found that its apparent high solubility was evident in some injectable solvents including 2-pyrrolidone(PYR), Nmethyl pyrrolidone(NMP) and dimethyl sulfoxide(DMSO). Therefore, in the present study, the physicochemical properties of the 30–60% w/w β-CyD in PYR, NMP and DMSO were investigated such as viscosity, water resistant, matrix formation rate and syringeability. The higher the concentration of β-CyD resulted in the increased viscosity and the higher force and energy of syringeability. β-CyD in PYR gave the highest viscosity which contributed to the lowest syringeability while β-CyD in DMSO exhibited the highest syringeability. The β-CyD in DMSO and NMP exhibited the higher rate of matrix formation. β-CyD in PYR showed the highest water resistant for phase separation while β-CyD in NMP gave the faster de-mixing rate compared to that from PYR. The difference in physicochemical properties of β-CyD dried ppts studied by scanning electron microscope(SEM), differential scanning calorimetry(DSC), X-ray diffraction(XRD), Fourier-transform infrared spectroscopy(FT-IR) and thermogravimetric analysis(TGA) revealed that there was partial complexation of β-CyD with respective solvents. Both solution and precipitate characteristic properties will be useful for using β-CyD in further investigation as matrix material dissolved in the injectable vehicles as the in situ forming gel for periodontitis treatment.

RESEARCH ON CONFORMATION OF CAPROLACTAM AND ITS ALKYL DERIVATIVES

Caprolactam is treated with dimethyl sulfate in benzene medium under conditions of various molar ratios to yield two different methylatcd products. The homogeneous reaction of caprolactam with dimethyl sulfate without any medium can only givc one methylated derivative. In a similar condition to that mentioned above, the reaction of caprolactam with diethylsulfate forms also one product, O-ethyl caprolactim. In this articlc NMR spectrum is applied to further identify the molecular structures of two methylated derivatives held by predecessors, and, applied the chemical shift reagent to induce the NMR and combined the field scanning and decoupling method to confirm the classification of signals, as a consequence the preferred conformation of caprolactam and its alkyl derivatives are proposed.

Zwitterionic cellular polymer enabled reductive fixation of CO_(2) for N-methylation of amines

Using heterogeneous catalysts to promote the construction of the C–N bond between amines and CO_(2) under mild conditions is a challenge yet.Herein,we synthesized a novel zwitterionic polymer(PDDC)with a cellular structure via self-complexation of copolymer bearing both quaternary ammonium cation and carboxylate anion.PDDC was employed as a recyclable catalyst for N-methylation of atmospheric CO_(2) and various amines with hydrosilane as a reductant,affording more than 17 N-methylamines in good to excellent yields(up to 99%)under mild conditions.The behavior of PDDC in the reaction medium was disclosed by using a dynamic light scattering study,revealing that the decreased hydraulic radius of particle size contributes to exposing more active sites and increasing reaction activity.Utilizing a series of designed experiments and density functional theory calculations uncovered the crucial role of the prepared zwitterionic polymer during the reaction procedure of CO_(2) conversion.

Air-tolerant direct reductive N-methylation of amines using formic acid via simple inorganic base catalysis

The construction of N-methyl amine moieties is an important reaction that has found numerous applications.Development of new methylation agents that are more environmentally benign than classical agents,such as iodomethane and methyl sulfate,is still highly desirable.Herein,we report a convenient protocol for direct reductive N-methylation of amines using formic acid as the methylation agent via simple inorganic base catalysis.The present protocol operates under transition-metal-free and air-tolerant conditions.Both the catalyst,K2 HPO4,and the reductant,polymethylhydrosiloxane(PMHS),are cheap and easily separable from the crude reaction product mixture.Mechanistic investigations suggest that the reaction occur through the formation of an acetal inte rmediate followed by the C-N bond formation.

Scalable direct N-methylation of drug-like amines using 12CO2/13CO2 by simple inorganic base catalysis

With the growing urgency of potential catastrophic climate changes due to anthropogenic CO2 emissions,numerous efforts have been devoted to development of synthetic protocols using CO2 as a building block in organic reactions, but the general applicability to complex drug-like substrates remains a challenge.We develop a general protocol for scalable direct N-methylation of a wide-scope drug-like amines using CO2 and polymethylhydrosiloxane-a nontoxic, aerobically-stable hydrosilane considered as an industrial waste-via simple inorganic base catalysis. A rare application of the Sabatier principle in organic chemistry led to the discovery of cheap, nontoxic K3PO4 as an efficient catalyst. Preparations of a wide-scope drug-like amines with carbon-isotope label were also successfully achieved, enabling direct use of CO2 in studies of drug absorption, distribution, metabolism and excretion.

Theoretical Studies of the Aminolysis for N-Methyl β-Sultam in Solution

The aminolysis and the effect of water on the aminolysis processes of n-methyl β-sultam have been studied using density functional theory （DFF） method at the B3LYP/6-31G＊ level. The stationary structures and energies have been investigated for both reactions to find two different reaction channels. Specific and general solvent effects have been evaluated and the most favored pathway was found. The presence of solvent disfavors the reaction, whereas the participation of water in the aminolysis reaction plays a positive role and reduces the activation energy greatly. All transition states in the assisted aminolysis are 35-70 kJ/mol lower than those for the non-assisted reaction.