Methods for the Determination of Stable Isotopes of Carbon and Nitrogen Directly in Valine, Proline, Glutamine, and Glutamic Acid

Amino acids are very important compounds for the body and are involved in important functions that keep us healthy. Amino acids are essential components such as valine, proline, glutamine and glutamic acid. They can be synthesized either naturally or artificially. To examine the metabolism and regulate the synthesis process, compounds labeled with nitrogen or carbon isotopes need to be used. These isotopic compounds allow for more extensive research and enable studies that would otherwise be impossible. However, their use is dependent on the availability of simple, efficient methods for isotopic analysis. Currently, the determination of the atomic fraction of carbon and nitrogen isotopes is only possible through their conversion into molecular nitrogen or carbon monoxide or carbon dioxide. This leads to the loss of information about isotopic enrichment in specific centers of the molecule. This article explores a new direct approach to determining the atomic fraction of carbon and nitrogen isotopes in the isotope-modified or identical centers of these compounds. This method eliminates the transfer process and dilution due to nitrogen and carbon impurities. It is now possible to simultaneously determine the atomic fraction of nitrogen and carbon isotopes in the research substance. This method can be applied to amino acids, making it an effective tool for proposing new research methods. Several articles [1] [2] [3] have proposed similar methods for organic compounds and amino acids.

Adsorption of glutamic acid from aqueous solution with calcined layered double Mg-Fe-CO_3 hydroxide

Layered double Mg-Fe-CO3 hydroxide （Mg-Fe-LDH） with a mole ratio of Mg to Fe of 3 was synthesized by coprecipitation method and calcined product Mg-Fe-CLDH was obtained by heating Mg-Fe-LDH at 500 ℃ for 6 h. The as prepared Mg-Fe-LDH and calcined Mg-Fe-CLDH were used for removal of glutamic acid （Glu） from aqueous solution, respectively. Batch studies were carried out to address various experimental parameters such as contact time, pH, initial glutamic acid （Glu） concentration, co-existing anions and temperature. Glu was removed effectively （99.9%） under the optimized experimental conditions with Mg-Fe-CLDH. The adsorption kinetics follows the Ho’s pseudo second-order model. Isotherms for adsorption with Mg-Fe-CLDH at different solution temperatures were well described using the Langmuir model with a good correlation coefficient. The intraparticle diffusion model fitted the data well, which suggests that the intraparticle diffusion is not only the rate-limiting step.

De novo transcriptome assembly of Aureobasidium melanogenum CGMCC18996 to analyze theβ-poly(L-malic acid)biosynthesis pathway under the CaCO_(3) addition

β-Poly(L-malic acid)(PMLA)is a water-soluble biopolymer used in food,medicine and other industries.To date,the biosynthesis pathway of PMLA has not been fully elucidated.In this study,we sequenced the transcriptom e of strain Aureobasidium melanogenum under 20 g/L CaCO_(3) addition.The resulting sequencing reads were assembled and annotated for the differentially expressed genes(DEGs)analysis and novel transcripts identification.The result indicated that with the CaCO_(3) addition,the tricarboxylic cycle(TCA)cycle and glyoxylate pathway were up-regulated,and it also found that a non-ribosomal peptide synthetase(NRPS)like protein was highly expressed.The DEGs analysis showed a high expression level of malate dehydrogenase(MDHC)and phosphoenolpyruvate carboxykinase(PCKA)in the CaCO_(3) group,which indicated a cytosolic malate activity.We speculated that the malate should be transported to or synthesized in the cytoplasm,which was then polymerized to PMLA by the NRPS-like protein,accompanied by the up-regulated TCA cycle providing ATP for the polymerization.Depending on the analysis,we assumed that an NRPS-like protein,the TCA cycle,and the cytosolic malate together are contributing to the PMLA biosynthesis.

Tanshinone IIa antagonizes spinal ischemia/reperfusion injury by interfering with upstream glutamic acid factors

Based on the hypothesis that upstream factor inhibition results in better treatment effects than downstream factor inhibition,the present study interfered with glutamic acid（Glu）-released upstream factors,such as Glu transporter function and Na＋-K＋-adenosine triphosphatases（ATPase）activity relativly.Rats with spinal cord ischemia/reperfusion injury received intraperitoneal injections of tanshinone Ila and Glu uptake and Na＋-K＋-ATPase activity were increased.Results showed that tanshinone Ila influenced Glu-released upstream factors following spinal ischemia/reperfusion injury and protected against spinal ischemia/reperfusion injury.

Metal ion-binding properties of L-glutamic acid and L-aspartic acid, a comparative investigation

A comparative research has been developed for acidity and stability constants of M(Glu)1, M(Asp)2 and M(Ttr)3 complexes, which have been determined by potentiometric pH titration. Depending on metal ion-binding properties, vital differences in building complex were observed. The present study indicates that in M(Ttr) com-plexes, metal ions are arranged to the carboxyl groups, but in M(Glu) and M(Asp), some metal ions are able to build chelate over amine groups. The results mentioned-above demonstrate that for some M(Glu) and M(Asp) complexes, the stability constants are also largely determined by the affinity of metal ions for amine group. This leads to a kind of selectivity of metal ions, and transfers them through building complexes accompanied with glutamate and aspartate. For heavy metal ions, this building complex helps the absorption and filtration of the blood plasma, and consequently, the excursion of heavy metal ions takes place. This is an important method in micro-dialysis. In this study the different as-pects of stabilization of metal ion complexes regarding to Irving-Williams sequence have been investigated.

Sequential elevation of autoantibodies to thyroglobulin and glutamic acid decarboxylase in type 1 diabetes

We have previously reported the high levels of glutamic acid decarboxylase 65 autoantibodies(GAD65A)in patients with type 1 diabetes and autoimmune thyroid disease.Here we describe a 32-year-old Japanese female with a thirteen-year history of type 1 diabetes whose levels of GAD65A were elevated just after the emergence of anti-thyroid autoimmunity.At 19 years of age,she developed diabetic ketoacidosis and was diagnosed with type 1 diabetes.She had GAD65A,insulinoma-associated antigen-2 autoantibodies(IA-2A),and zinc transporter-8 autoantibodies(ZnT8A),but was negative for antibodies to thyroid peroxidase(TPOAb)and thyroglobulin(TGAb)at disease onset.ZnT8A and IA-2A turned negative 2-3 years after the onset,whereas GAD65A were persistently positive at lower level(approximately 40 U/mL).However,just after the emergence of TGAb at disease duration of 12.5 years,GAD65A levels were reelevated up to5717 U/mL in the absence of ZnT8A and IA-2A.Her thyroid function was normal and TPOAb were consistently negative.She has a HLA-DRB1*03:01/*04:01-DQB1*02:01/*03:02 genotype.Persistent positivity for GAD65A might be associated with increased risk to develop anti-thyroid autoimmunity.

Granulocyte colony-stimulating factor increases extracellular glutamic acid uptake and suppresses free radicals in an experimental model of amyotrophic lateral sclerosis

Excitatory amino acid toxicity and free radical damage play important roles in amyotrophic lateral sclerosis. Granulocyte colony-stimulating factor （G-CSF） protects nerve cells exposed to high-concentrations of glutamic acid, suggesting positive effects in the treatment of amyotrophic lateral sclerosis. The present study induced in vitro motor neuron injury using glutamic acid excitotoxicity, and the biochemical effects of G-CSF on glutamic acid concentration were determined. In addition, the effects of G-CSF on superoxide dismutase, glutathione peroxidase activity in motor neurons, and malondialdehyde and nitric oxide contents were analyzed. Immunohistochemistry was performed to measure neuronal survival. Results revealed that G-CSF significantly suppressed free radical activity, inhibited excitotoxicity, and reduced apoptosis and loss of motor neurons in the anterior horn of the spinal cord.

Analysis of Glutamic Acid in Cerebrospinal Fluid by Capillary Electrophoresis with High Frequency Conductivity Detection

A rapid method to determine glutamic acid (Glu) in cerebrospinal fluid (CSF) by capillaryelectrophoresis with high frequency conductivity detection (contactless conductivity detection) wasdescribed. The CSF sample was pretreated with silver cation resin to remove high concentration ofCl- ions in CSF. The separation was achieved in the buffer solution of 10 mmol/L Tris and 8mmol/L boric acid at the separation voltage of 20.0 kV. Glu showed linear response in the range of5.0×10-6 to 6.0×10-3 mol/L, the limit of detection was 1.0×10-6 mol/L. The method was used foranalysis Glu in CSF satisfactorily with a recovery of 97.8-98.8%.

Pathological Changes in Rabbit Retina and Its Relationship with Glutamic Acid after Injuries from High-Speed Bullets

Purpose:To observe the pathological changes in rabbit retinas and the measure of glutamic acid levels in the vitreous body after suffering from high-speed bullet injuries.Methods:Rabbits eyeball contusion models were established with high-speed bullets,i.e,the rabbits eyes were shot with a fixed air rifle at a speed of 90 m/s.(using plastic bullets,weighing 0.201 g,on average).Retinal tissues treated with HE staining and were prepared for light microscopy examination and glutamate levels were tested at different time points after the injury.Results:Edema,exudation,hemorrhage,and rupture were evident in rabbit retinas following bullet injuries.Meanwhile,glutamate levels gradually increased as time proceeded.Conclusion:Visual impairment is related with retinal damages after high-speed bullet injuries.Increased glutamate concentration serves as a potential factor for aggravating retinal injuries.

Increased excitatory amino acid transporter 2 levels in basolateral amygdala astrocytes mediate chronic stress–induced anxiety-like behavior

The conventional perception of astrocytes as mere supportive cells within the brain has recently been called into question by empirical evidence, which has revealed their active involvement in regulating brain function and encoding behaviors associated with emotions.Specifically, astrocytes in the basolateral amygdala have been found to play a role in the modulation of anxiety-like behaviors triggered by chronic stress. Nevertheless, the precise molecular mechanisms by which basolateral amygdala astrocytes regulate chronic stress–induced anxiety-like behaviors remain to be fully elucidated. In this study, we found that in a mouse model of anxiety triggered by unpredictable chronic mild stress, the expression of excitatory amino acid transporter 2 was upregulated in the basolateral amygdala. Interestingly, our findings indicate that the targeted knockdown of excitatory amino acid transporter 2 specifically within the basolateral amygdala astrocytes was able to rescue the anxiety-like behavior in mice subjected to stress. Furthermore, we found that the overexpression of excitatory amino acid transporter 2 in the basolateral amygdala, whether achieved through intracranial administration of excitatory amino acid transporter 2agonists or through injection of excitatory amino acid transporter 2-overexpressing viruses with GfaABC1D promoters, evoked anxiety-like behavior in mice. Our single-nucleus RNA sequencing analysis further confirmed that chronic stress induced an upregulation of excitatory amino acid transporter 2 specifically in astrocytes in the basolateral amygdala. Moreover, through in vivo calcium signal recordings, we found that the frequency of calcium activity in the basolateral amygdala of mice subjected to chronic stress was higher compared with normal mice.After knocking down the expression of excitatory amino acid transporter 2 in the basolateral amygdala, the frequency of calcium activity was not significantly increased, and anxiety-like behavior was obviously mitigated. Additionally, administration of an excitatory amino acid transporter 2 inhibitor in the basolateral amygdala yielded a notable reduction in anxiety level among mice subjected to stress. These results suggest that basolateral amygdala astrocytic excitatory amino acid transporter 2 plays a role in in the regulation of unpredictable chronic mild stress-induced anxiety-like behavior by impacting the activity of local glutamatergic neurons, and targeting excitatory amino acid transporter 2 in the basolateral amygdala holds therapeutic promise for addressing anxiety disorders.

Preparation and Evaluation of Poly-γ-Glutamic Acid Hydrogel Mixtures with Basic Drugs or Acidic Drugs: Effect on Ease of Swallowing and Taste Masking

The purpose of this study was to prepare a poly-γ-glutamic acid hydrogel (PGA gel), to examine its ease of swallowing using texture profile analysis (TPA) and to evaluate its taste-masking effects on basic or acidic drugs using the artificial taste sensor. Using TPA, 0.5% and 1.0% PGA gels, 0.5% and 1.0% agar and 1.0% ι-carrageenan in the absence of drug was examined the hardness, adhesiveness and cohesiveness, ranked according to permission criteria published by the Japanese Consumers Affairs Agency. 0.5% PGA gel and 1.0% agar were classified into grade II. In the taste sensor measurement, the bitterness suppressions by 0.5% PGA gel were larger than that by 1.0% agar in all drugs and the bitterness suppressions of basic drugs in 0.5% PGA gel were more potent than those of acidic drugs in 0.5% PGA gel. 1H-nuclear magnetic resonance spectroscopic analysis was carried out to examine the difference in mechanism of bitterness suppression between basic drugs and acidic drugs mixed with PGA gel. The signals of the proton nearest to the nitrogen atom of basic drugs shifted clearly upfield, suggesting an interaction between the amino group of basic drugs and the carboxyl group of PGA gel. In conclusion, PGA gel is expected to be a useful excipient in formulations contained various drugs, especially basic drugs;it also has advantage for not only increasing ease of swallowing but also masking the bitterness of drugs even though a small amount of a single drug dose might be preferred.

In Vitro Biomineralization of Glutaraldehyde Crosslinked Chitosan/Glutamic Acid Films

In vitro biomineralization of glutaraldehyde crosslinked chitosan/glutamic acid films were studied. IR and ESCA （electron spectroscopy for chemical analysis） determinations confirm that chitosan and glutamic acid are successfully crosslinked by glutaraldehyde to form chitosan-glutamic acid surfaces. Composite films were soaked in saturated Ca（OH）2 solution for 8 d and then immersed in simulated body fluid （SBF） for more than 20 d. Morphological characterizations and structure of cal-cium phosphate coatings deposited on the films were studied by SEM, XRD, and EDAX （energy dispersive X-ray analysis）. Initially, the treatment in SBF results in the formation of single-layer cal-cium phosphate particles over the film surface. As immersion time increases, further nucleation and growth produce the simulated calcium-carbonate hydroxyapatite coating. ICP results show Ca/P ratio of calcium phosphate coating is a function of SBF immersion time. The inducing of glutamic acid improves the biomineralization property of chitosan films.

Study on the Dissolution of Dusts in Glutamic Acid

This article describes the characteristics of natural dusts, artificial dusts and industrial dusts, such as mineral phases, chemical components, morphological observation and size. Quartz and calcite are the main phases of natural dusts and industrial dusts with high SiO2, CaO and low K2O, Na2O on the chemical composition. Natural dusts are mainly irregular shaped and some particle aggregation made of small dusts on the surface of large dust. Industrial dusts are globular and blob-like, but artificial dusts are columnar and fibrous. The fine particles are mainly in the range of 0.3-5 μm,of which the dusts of less than 5 μm are over 99%.The dissolution and electrochemical action of dusts in glutamic acid liquor at the simulated human body temperature (37 ℃) in 32 hours were investigated. The changes of pH values and electric conductivity of those dusts were similar, increased slowly in first 8 hours, and then the pH values increased rapidly. The total amount of dissolved ions of K, Ca, Na, Mg was 35.4-429 mg/L, particularly Ca was maximal of 20-334 mg/L. The total amount of dissolved ions of Fe, Zn, Mn, Pb, Ba was 0.18-5.59 ppm and the Al, Si was 3.0-21.7 mg/L. Each element dissolved rapidly relatively in first 16 hours. The relative solubility order of dusts in glutamic acid are: wollastonite > serpentine > sepiolite, the cement plant industrial dusts > power plant industrial dusts, and natural dusts have similar solubility. The wollastonite and power plant industrial dusts have highest solubility, which have high content of CaO; this shows there are a poorer corrosion-resisting ability and lower bio-resistibility. Sepiolite and cement plant industrial dusts have lowest solubility, which have high content of SiO2; this shows there are a higher corrosion-resisting ability and stronger bio-resistibility.

Double Labeling Immulloelectron Microscopic Study on the Synaptic Connections between Glutamic Acid Neurons and GABA Neurons in the Hippocampus of Rats

Summary: In order to explore the roles of different neurotransmitters in epileptic pathogenesis, the synaptic connections between glutamic acid (Gin) neurons and GABA neurons in normal rat hippocampus were studied by pre-embedding double labeling immunoelectron microscopy. The GABA immunoreaction was first demonstrated by chromogen DAB, then the Gin immunoreaction was demonstrated by molybdic acid-TAB method. After being stabilized by DAB-cobalt chloride. the sections were processed for electron microscopic embedding. Under electron microscope, there were many Gin immunoreaction-positive neurons in the pyramidal layer of hippocampal CA1 area and some GABA immunoreaction-positive neurons with pyramidal or polygonal perikarya in the pyramidal, polymorphic and radiant layer of CA1 area. There were also symmetric dendro-axonic synapses formed by GABA-positive dendrites and Glu-positive a-cons in the polymorphic layer and symmetric axo-dendritic synapses formed by GABA-positive axons and Glu-positive dendrites in the radiant layer. In addition, there were symmetric autoregulatory axo-dendritic synapses be- tween Gin-positive axons and dendrites and autoregulatory axo-axonic synapses (both symmetric and asymmetric) between GABA-positive axons. Above mentioned results, for the first time, showed that there were complex synaptic regulatory relationships between excitatory Glu neurons and inhibitory GABA neurons in the hippocampal CA1 area, thereby, providing ultrastructural evidence for different neurotransmitters participating in epileptic pathogenesis.

Determination of Enantiomeric Excess of Glutamic Acids by Lab-made Capillary Array Electrophoresis

Simulated enantiomeric excess of glutamic acid was determined by a lab-made sixteen-channel capillary array electrophoresis with confocal fluorescent rotary scanner. The experimental results indicated that the capillary array electrophoresis method can accurately determine the enanfiomefic excess of glutamic acid and can be used for high-throughput screening system for combinatorial asymmetric catalysis.

Development of a Freeze-Dried Skin Care Product Composed of Hyaluronic Acid and Poly(γ-Glutamic Acid) Containing Bioactive Components for Application after Chemical Peels

Eight types of spongy sheet were prepared by freeze-drying aqueous solutions of hyaluronic acid (HA) and poly(γ-glutamic acid) (PGA) with or without bioactive components including vitamin C derivative (VC), glucosylceramide (GC), and epidermal growth factor (EGF). Spongy sheets were categorized into the following groups: Group I (HA/PGA), Group II (HA/PGA + VC), Group III (HA/PGA + GC), Group IV (HA/PGA + VC, GC), Group V (HA/PGA + EGF), Group VI (HA/PGA + VC, EGF), Group VII (HA/PGA + GC, EGF), and Group VIII (HA/PGA + VC, GC, EGF). In the first experiment, we examined fibroblast proliferation in conditioned medium that had been prepared by immersing each spongy sheet in a conventional culture medium. EGF-incorporating spongy sheets (Groups V-VIII) enhanced fibroblast proliferation more than EGF-free spongy sheets (Groups I-IV). In the second experiment, cytokine production by fibroblasts was evaluated using a wound surface model. This involved elevation of fibroblasts-incorporating collagen gel sheets to the air-liquid interface, on which a spongy sheet (Groups I, IV, V and VIII) was placed and cultured for 1 week. EGF-incorporating spongy sheets (Groups V and VIII) enhanced the production of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by fibroblasts more than EGF-free spongy sheets (Groups I and IV). The effect of these four types of spongy sheet on wounds was investigated in animal experiments. Chemical peel was performed by contacting 50% trichloroacetic acid (TCA) on the dorsal region of mice, after which a spongy sheet was placed, and the wound condition was then observed in a two-week period. Angiogenesis was facilitated to a greater degree in Group VIII compared with Groups I, IV and V. This finding indicates that Group VIII spongy sheet is a promising aid for skin recovery after chemical peel.

Tb(lll) and Ca(ll) Ion Equilibria in the Presence of Glutamic Acid and Glutamine

Tb(111) and Ca(11) ion equilibria in the presence of glutamic acid and glutamine were studied by potentiometric titration at 37'C and an ionic strength of 0. 15mol/L(NaCl) .The stability constants for Tb(111) and Ca(11)complexes in the systems were obtained. The species and their distribution in the systems were discussed.

Preparation and Characterization of Orally Fast-Disintegrating Mini-Tablets Containing Diphenhydramine Hydrochloride and Aspartic or Glutamic Acid as an Umami Amino Acid

The aim of this study was to prepare diphenhydramine hydrochloride (DPH)-loaded orally fast-disintegrating mini-tablets (OFDMTs) containing either L-aspartic acid (Asp) or L-glutamic acid (Glu) as bitterness-suppressant, to characterize the prepared tablets and to evaluate their bitterness under conditions mimicking those of the oral cavity. The preparation of five formulation batches of the OFDMTs involved mixing DPH, with or without two different concentrations of Asp or Glu, and a premix containing a disintegrating agent. When all ingredients were well mixed, the mixture was directly compacted to form small (4 mm diameter) DPH-loaded OFDMTs. There were only small differences between the tablets with respect to mass, diameter, width and hardness. The disintegration times of the five formulation batches of DPH-loaded OFDMTs were measured using the OD-mate, a disintegration test apparatus in which conditions resemble those of the oral cavity. The disintegration times were all within 10 s of exposure to a medium representing the inside of the oral cavity. Rapid release profiles were observed for DPH, Asp and Glu in these dissolution tests. The taste sensor outputs of samples taken at different times (5 - 30 s) from the dissolution test solutions of the four DPH-loaded OFDMTs containing Asp or Glu were significantly inhibited compared with those of control DPH-loaded OFDMT. These results suggest that the inclusion of Asp or Glu in DPH-loaded OFDMTs is sufficient to mask bitterness in the oral cavity for the first 30 s after the tablet is placed in the mouth. It is anticipated that swallowing will have taken place within 30 s.

Effects of Glutamic Acid on C-type Inactivation of Kv1.4ΔN Channel

Objectives Acidosis has an inhibitory effect on the inactivation of Kv1.4 ΔN channel through the position H508. So in order to show the effects of glutamic acid on the mutant Kv 1.4 channel that lacks N-type inactivation （Kv1.4 Δ2-146）, we studied in the expression system of the Xenopus oocytes. Methods The two-electrode voltage-clamp technique （TEV） was used to record the currents. Results Acidosis increased fKv1.4 Δ2-146 C-type inactivation. After application of glutamic acid （1 mmol/L） to Kv1.4 Δ2-146 increased C-type inactivation further, changed inactivation time constants from （2.02 ± 0.39 s ） to （1.71 ± 0.23 s） （P〈 0.05） at ＋50mv, and shifted the steady-state inactivation curves of Kv1.4 ΔN to positive potential, which was from （-44.30 ± 0.59 mV） to （-39.88 ± 0.29 mV）（P〈0.05）. and slowed the rate of recovery from inactivation, which was from （1.64 ± 0.19 s） to （1.91 ± 0.23 s）（P〈 0.05）. Conclusions Together, these results suggest that 1 mmol/L glutamic acid accelerates the C-type inactivation of Kv1.4 ΔN in pH 6.8.

STUDY ON THE SEPARATION OF GLUTAMIC ACID BY ION-EXCHANGE

The feasibility of recavering glutamic acid by ion exchange method with macroporous resins was investigated. Their adsorption properties in static state and the effective factors,such as pH, concentration of feed and the ratio of ammonium ion toglutamic acid,were systematically explored. The best conditions of separating glutamic acid from mother liquid were obtained.