Engineered Biomimetic Nanovesicles Synergistically Remodel Folate-Nucleotide and γ-Aminobutyric Acid Metabolism to Overcome Sunitinib-Resistant Renal Cell Carcinoma.

Reprogramming of cellular metabolism in tumors promoted the epithelial-mesenchymal transition (EMT) process and established immune-suppressive tumor microenvironments (iTME), leading to drug resistance and tumor progression. Therefore, remodeling the cellular metabolism of tumor cells was a promising strategy to overcome drug-resistant tumors. Herein, CD276 and MTHFD2 were identified as a specific marker and a therapeutic target, respectively, for targeting sunitinib-resistant clear cell renal cell carcinoma (ccRCC) and its cancer stem cell (CSC) population. The blockade of MTHFD2 was confirmed to overcome drug resistance via remodeling of folate-nucleotide metabolism. Moreover, the manganese dioxide nanoparticle was proven here by a high-throughput metabolome to be capable of remodeling γ-aminobutyric acid (GABA) metabolism in tumor cells to reconstruct the iTME. Based on these findings, engineered CD276-CD133 dual-targeting biomimetic nanovesicle EMφ-siMTHFD2-MnO2@Suni was designed to overcome drug resistance and terminate tumor progression of ccRCC. Using ccRCC-bearing immune-humanized NPG model mice, EMφ-siMTHFD2-MnO2@Suni was observed to remodel folate-nucleotide and GABA metabolism to deactivate the EMT process and reconstruct the iTME thereby overcoming the drug resistance. In the incomplete-tumor-resection recurrence model and metastasis model, EMφ-siMTHFD2-MnO2@Suni reduced recurrence and metastasis in vivo. This work thus provided an innovative approach that held great potential in the treatment of drug-resistant ccRCC by remodeling cellular metabolism.

Triphenylamine Sensitized 8-Dimethylaminoquinoline: An Efficient Two-Photon Caging Group for Intracellular Delivery.

Triphenylamine-sensitized 8-dimethylaminoquinoline (TAQ) probes showed fair two-photon absorption and fragmentation cross sections in releasing kainate and GABA ligands. The water-soluble PEG and TEG-analogs allowed cell internalization and efficient light-gated liberation of the rhodamine reporter under UV and two-photon (NIR) irradiation conditions.

Intrahippocampal Supramolecular Assemblies Directed Bioorthogonal Liberation of Neurotransmitters to Suppress Seizures in Freely Moving Mice.

The precise delivery of anti-seizure medications (ASM) to epileptic loci remains the major challenge to treat epilepsy without causing adverse drug reactions. The unprovoked nature of epileptic seizures raises the additional need to release ASMs in a spatiotemporal controlled manner. Targeting the oxidative stress in epileptic lesions, here the reactive oxygen species (ROS) induced in situ supramolecular assemblies that synergized bioorthogonal reactions to deliver inhibitory neurotransmitter (GABA) on-demand, are developed. Tetrazine-bearing assembly precursors undergo oxidation and selectively self-assemble under pathological conditions inside primary neurons and mice brains. Assemblies induce local accumulation of tetrazine in the hippocampus CA3 region, which allows the subsequent bioorthogonal release of inhibitory neurotransmitters. For induced acute seizures, the sustained release of GABA extends the suppression than the direct supply of GABA. In the model of permanent damage of CA3, bioorthogonal ligation on assemblies provides a reservoir of GABA that behaves prompt release upon 365 nm irradiation. Incorporated with the state-of-the-art microelectrode arrays, it is elucidated that the bioorthogonal release of GABA shifts the neuron spike waveforms to suppress seizures at the single-neuron precision. The strategy of in situ supramolecular assemblies-directed bioorthogonal prodrug activation shall be promising for the effective delivery of ASMs to treat epilepsy.

Effect of terahertz waves on the aggregation behavior of neurotransmitters.

Understanding the dynamics of neurotransmitters is crucial for unraveling synaptic transmission mechanisms in neuroscience. In this study, we investigated the impact of terahertz (THz) waves on the aggregation of four common neurotransmitters through all-atom molecular dynamics (MD) simulations. The simulations revealed enhanced nicotine (NCT) aggregation under 11.05 and 21.44 THz, with a minimal effect at 42.55 THz. Structural analysis further indicated strengthened intermolecular interactions and weakened hydration effects under specific THz stimulation. In addition, enhanced aggregation was observed at stronger field strengths, particularly at 21.44 THz. Furthermore, similar investigations on epinephrine (EPI), 5-hydroxytryptamine (5-HT), and γ-aminobutyric acid (GABA) corroborated these findings. Notably, EPI showed increased aggregation at 19.05 THz, emphasizing the influence of vibrational modes on aggregation. However, 5-HT and GABA, with charged or hydrophilic functional groups, exhibited minimal aggregation under THz stimulation. The present study sheds some light on neurotransmitter responses to THz waves, offering implications for neuroscience and interdisciplinary applications.

Synthesis of R-GABA Derivatives via Pd(II) Catalyzed Enantioselective C(sp3)-H Arylation and Virtual Validation with GABAB1 Receptor for Potential leads.

GABA (γ-amino butyric acid) analogues like baclofen, tolibut, phenibut, etc., are well-known GABAB1 inhibitors and pharmaceutically important drugs. However, there is a huge demand for more chiral GABA aryl analogues with promising pharmacological actions. Here, we demonstrate the chiral ligand acetyl-protected amino quinoline (APAQ) mediated enantioselective synthesis of GABAB1 inhibitor drug scaffolds from easily accessible GABA via Pd-catalyzed C(sp3)-H activation. The synthetic methodology shows moderate to good yields, up to 74% of ee. We have successfully demonstrated the deprotection and removal of the directing group to synthesize R-tolibut in 86% yield. Further, we employed computation to probe the binding of R-GABA analogues to the extracellular domain of the human GABAB1 receptor. Our Rosetta-based molecular docking calculations show better binding for four R-enantiomers of GABA analogues than R-baclofen and R-phenibut. In addition, we employed GROMACS MD simulations and MMPB(GB)SA calculations to identify per-residue contribution to binding free energy. Our computational results suggest analogues (3R)-4-amino-3-(3,4-dimethylphenyl) butanoic acid, (3R)-4-amino-3-(3-fluorophenyl) butanoic acid, (3R)-3-(4-acetylphenyl)-4-aminobutanoic acid, (3R)-4-amino-3-(4-methoxyphenyl) butanoic acid, and (3R)-4-amino-3-phenylbutanoic acid are potential leads which could be synthesized from our methodology reported here.

Magnetic Resonance Spectroscopy Spectral Registration Using Deep Learning.

BACKGROUND: Deep learning-based methods have been successfully applied to MRI image registration. However, there is a lack of deep learning-based registration methods for magnetic resonance spectroscopy (MRS) spectral registration (SR). PURPOSE: To investigate a convolutional neural network-based SR (CNN-SR) approach for simultaneous frequency-and-phase correction (FPC) of single-voxel Meshcher-Garwood point-resolved spectroscopy (MEGA-PRESS) MRS data. STUDY TYPE: Retrospective. SUBJECTS: Forty thousand simulated MEGA-PRESS datasets generated from FID Appliance (FID-A) were used and split into the following: 32,000/4000/4000 for training/validation/testing. A 101 MEGA-PRESS medial parietal lobe data retrieved from the Big GABA were used as the in vivo datasets. FIELD STRENGTH/SEQUENCE: 3T, MEGA-PRESS. ASSESSMENT: Evaluation of frequency and phase offsets mean absolute errors were performed for the simulation dataset. Evaluation of the choline interval variance was performed for the in vivo dataset. The magnitudes of the offsets introduced were -20 to 20 Hz and -90° to 90° and were uniformly distributed for the simulation dataset at different signal-to-noise ratio (SNR) levels. For the in vivo dataset, different additional magnitudes of offsets were introduced: small offsets (0-5 Hz; 0-20°), medium offsets (5-10 Hz; 20-45°), and large offsets (10-20 Hz; 45-90°). STATISTICAL TESTS: Two-tailed paired t-tests for model performances in the simulation and in vivo datasets were used and a P-value <0.05 was considered statistically significant. RESULTS: CNN-SR model was capable of correcting frequency offsets (0.014 ± 0.010 Hz at SNR 20 and 0.058 ± 0.050 Hz at SNR 2.5 with line broadening) and phase offsets (0.104 ± 0.076° at SNR 20 and 0.416 ± 0.317° at SNR 2.5 with line broadening). Using in vivo datasets, CNN-SR achieved the best performance without (0.000055 ± 0.000054) and with different magnitudes of additional frequency and phase offsets (i.e., 0.000062 ± 0.000068 at small, -0.000033 ± 0.000023 at medium, 0.000067 ± 0.000102 at large) applied. DATA CONCLUSION: The proposed CNN-SR method is an efficient and accurate approach for simultaneous FPC of single-voxel MEGA-PRESS MRS data. EVIDENCE LEVEL: 4 TECHNICAL EFFICACY: Stage 2.

Glutamate measurements using edited MRS.

PURPOSE: To demonstrate J-difference coediting of glutamate using Hadamard encoding and reconstruction of Mescher-Garwood-edited spectroscopy (HERMES). METHODS: Density-matrix simulations of HERMES (TE 80 ms) and 1D J-resolved (TE 31-229 ms) of glutamate (Glu), glutamine (Gln), γ-aminobutyric acid (GABA), and glutathione (GSH) were performed. HERMES comprised four sub-experiments with editing pulses applied as follows: (A) 1.9/4.56 ppm simultaneously (ONGABA /ONGSH ); (B) 1.9 ppm only (ONGABA /OFFGSH ); (C) 4.56 ppm only (OFFGABA /ONGSH ); and (D) 7.5 ppm (OFFGABA /OFFGSH ). Phantom HERMES and 1D J-resolved experiments of Glu were performed. Finally, in vivo HERMES (20-ms editing pulses) and 1D J-resolved (TE 31-229 ms) experiments were performed on 137 participants using 3 T MRI scanners. LCModel was used for quantification. RESULTS: HERMES simulation and phantom experiments show a Glu-edited signal at 2.34 ppm in the Hadamard sum combination A+B+C+D with no overlapping Gln signal. The J-resolved simulations and phantom experiments show substantial TE modulation of the Glu and Gln signals across the TEs, whose average yields a well-resolved Glu signal closely matching the Glu-edited signal from the HERMES sum spectrum. In vivo quantification of Glu show that the two methods are highly correlated (p < 0.001) with a bias of ∼10%, along with similar between-subject coefficients of variation (HERMES/TE-averaged: ∼7.3%/∼6.9%). Other Hadamard combinations produce the expected GABA-edited (A+B-C-D) or GSH-edited (A-B+C-D) signal. CONCLUSION: HERMES simulation and phantom experiments show the separation of Glu from Gln. In vivo HERMES experiments yield Glu (without Gln), GABA, and GSH in a single MRS scan.

Experimental Substantiation of the Use of Phenibut Combinations with Salicylic, Nicotinic, and Glutamic Acids in Cerebral Ischemia.

BACKGROUND: One of the possible ways to increase the effectiveness of drugs based on gamma-aminobutyric acid derivatives is to introduce biologically active acids into their structure. In this regard, the compositions of phenibut with organic acids, which have a more pronounced psychotropic activity, low toxicity, and good tolerability, are of interest. The purpose of this study is to experimentally substantiate the use of phenibut combinations with organic acids in various forms of cerebral ischemia. METHODS: The study was performed on 1210 male Wistar rats weighing 180-220 g each. The cerebroprotective activities of phenibut combinations with salicylic acid (2:1, doses of 15, 30, and 45 mg/kg), nicotinic acid (2:1, doses of 25, 50, and 75 mg/kg), and glutamic acid (2:1, doses of 25, 50, and 75 mg/kg) have been studied. The study involved a single prophylactic administration of phenibut combinations with organic acids and a 7 days course of the combination treatment administered at doses that proved the most effective according to the results of a single prophylactic administration. The rate of local cerebral blood flow and the vasodilating function of cerebral endothelium were measured, and the researchers evaluated the effects of the studied phenibut combinations on biochemical parameters in rats with focal ischemia. RESULTS: Compositions of phenibut with salicylic, nicotinic, and glutamic acids in subtotal and transient cerebral ischemia were found to have the most pronounced cerebroprotective effect in doses of 30, 50 and 50 mg/kg, respectively. Under reversible 10 min occlusion of the common carotid arteries, prophylactic administration of the studied phenibut compositions prevented a decrease in cerebral blood flow during ischemia and reduced the severity of postischemic hypoperfusion and hyperperfusion. At a course of 7 days of therapeutic administration of compounds, their pronounced cerebroprotective effect was observed. CONCLUSIONS: The data obtained can be considered as promising the pharmacological search in this series of substances for the treatment of patients with cerebrovascular disease.

Asymmetric Michael Addition in Synthesis of ß-Substituted GABA Derivatives.

γ-Aminobutyric acid (GABA) represents one of the most prolific structural units widely used in the design of modern pharmaceuticals. For example, ß-substituted GABA derivatives are found in numerous neurological drugs, such as baclofen, phenibut, tolibut, pregabalin, phenylpiracetam, brivaracetam, and rolipram, to mention just a few. In this review, we critically discuss the literature data reported on the preparation of substituted GABA derivatives using the Michael addition reaction as a key synthetic transformation. Special attention is paid to asymmetric methods featuring synthetically useful stereochemical outcomes and operational simplicity.

The ability of carbon nanoparticles to increase transmembrane current of cations coincides with impaired synaptic neurotransmission.

Here, carbon nanodots synthesized from ß-alanine (Ala-CDs) and detonation nanodiamonds (NDs) were assessed using (1) radiolabeled excitatory neurotransmitters L-[14C]glutamate, D-[2,33H]aspartate, and inhibitory ones [3H]GABA, [3H]glycine for registration of their extracellular concentrations in rat cortex nerve terminals; (2) the fluorescent ratiometric probe NR12S and pH-sensitive probe acridine orange for registration of the membrane lipid order and synaptic vesicle acidification, respectively; (3) suspended bilayer lipid membrane (BLM) to monitor changes in transmembrane current. In nerve terminals, Ala-CDs and NDs increased the extracellular concentrations of neurotransmitters and decreased acidification of synaptic vesicles, whereas have not changed sufficiently the lipid order of membrane. Both nanoparticles, Ala-CDs and NDs, were capable of increasing the conductance of the BLM by inducing stable potential-dependent cation-selective pores. Introduction of divalent cations, Zn2+ or Cd2+ on the particles` application side (cis-side) increased the rate of Ala-CDs pore-formation in the BLM. The application of positive potential (+100 mV) to the cis-chamber with Ala-CDs or NDs also activated the insertion as compared with the negative potential (-100 mV). The Ala-CD pores exhibited a wide-range distribution of conductances between 10 and 60 pS and consecutive increase in conductance of each major peak by ~10 pS, which suggest the clustering of the same basic ion-conductive structure. NDs also formed ion-conductive pores ranging from 6 pS to 60 pS with the major peak of conductance at ~12 pS in cholesterol-containing membrane. Observed Ala-CDs and NDs-induced increase in transmembrane current coincides with disturbance of excitatory and inhibitory neurotransmitter transport in nerve terminals.

Enantiomers of 4-aminopentanoic acid act as false GABAergic neurotransmitters and impact mouse behavior.

Imbalance in the metabolic pathway linking excitatory and inhibitory neurotransmission has been implicated in multiple psychiatric and neurologic disorders. Recently, we described enantiomer-specific effects of 2-methylglutamate, which is not decarboxylated to the corresponding methyl analogue of gamma-aminobutyric acid (GABA): 4-aminopentanoic acid (4APA). Here, we tested the hypothesis that 4APA also has enantiomer-specific actions in brain. Mouse cerebral synaptosome uptake (nmol/mg protein over 30 min) of (R)-4APA or (S)-4APA was time and temperature dependent; however, the R enantiomer had greater uptake, reduction of endogenous GABA concentration, and release following membrane depolarization than did the S enantiomer. (S)-4APA exhibited some weak agonist (GABAA α4ß3δ, GABAA α5ß2γ2, and GABAB B1/B2) and antagonist (GABAA α6ß2γ2) activity while (R)-4APA showed weak agonist activity only with GABAA α5ß2γ2. Both 4APA enantiomers (100 mg/kg IP) were detected in mouse brain 10 min after injection, and by 1 hr had reached concentrations that were stable over 6 hr; both enantiomers were cleared rapidly from mouse serum over 6 hr. Two-month-old mice had no mortality following 100-900 mg/kg IP of each 4APA enantiomer but did have similar dose-dependent reduction in distance moved in a novel cage. Neither enantiomer at 30 or 100 mg/kg impacted outcomes in 23 measures of well-being, activity chamber, or withdrawal from hot plate. Our results suggest that enantiomers of 4APA are active in mouse brain, and that (R)-4APA may act as a novel false neurotransmitter of GABA. Future work will focus on disease models and on possible applications as neuroimaging agents.

Fermented date residue extract mix containing gamma-aminobutyric acid augments the immune function of mouse splenocytes.

An extract of date (fruit of a palm tree) residue plus food-grade glutamate, acetic acid, and yeast extract (date residue extract mix, DREM) has been successfully fermented with using Lactobacillus brevis JCM 1059T to produce gamma-aminobutyric acid (GABA). Here, mouse splenocytes were found to be viable when supplemented with DREM and fermented DREM containing GABA (fDREM). The addition of DREM and fDREM resulted in the secretion of tumor necrosis factor (TNF)-α from the splenocytes, fDREM being more effective than DREM. The TNF-α secretion with DREM was elevated by exogenous addition of GABA and that with fDREM was in part mediated via A-type GABA receptors. Contrary to general understanding of the suppressive effects of GABA on various biological functions, our findings suggest that GABA-containing fDREM arguments the immune function as a food and pharmaceutical material.

COR758, a negative allosteric modulator of GABAB receptors.

Allosteric modulators of G protein coupled receptors (GPCRs), including GABABRs (GABABRs), are promising therapeutic candidates. While several positive allosteric modulators (PAM) of GABABRs have been characterized, only recently the first negative allosteric modulator (NAM) has been described. In the present study, we report the characterization of COR758, which acts as GABABR NAM in rat cortical membranes and CHO cells stably expressing GABABRs (CHO-GABAB). COR758 failed to displace the antagonist [3H]CGP54626 from the orthosteric binding site of GABABRs showing that it acts through an allosteric binding site. Docking studies revealed a possible new allosteric binding site for COR758 in the intrahelical pocket of the GABAB1 monomer. COR758 inhibited basal and GABABR-stimulated O-(3-[35Sthio)-triphosphate ([35S]GTPγS) binding in brain membranes and blocked the enhancement of GABABR-stimulated [35S]GTPγS binding by the PAM GS39783. Bioluminescent resonance energy transfer (BRET) measurements in CHO-GABAB cells showed that COR758 inhibited G protein activation by GABA and altered GABABR subunit rearrangements. Additionally, the compound altered GABABR-mediated signaling such as baclofen-induced inhibition of cAMP production in transfected HEK293 cells, agonist-induced Ca2+ mobilization as well as baclofen and the ago-PAM CGP7930 induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in CHO-GABAB cells. COR758 also prevented baclofen-induced outward currents recorded from rat dopamine neurons, substantiating its property as a NAM for GABABRs. Altogether, these data indicate that COR758 inhibits G protein signaling by GABABRs, likely by interacting with an allosteric binding-site. Therefore, COR758 might serve as a scaffold to develop additional NAMs for therapeutic intervention.

NMR-Based Metabolomic Analyses to Identify the Effect of Harvesting Frequencies on the Leaf Metabolite Profile of a Moringa oleifera Cultivar Grown in an Open Hydroponic System.

Moringa oleifera Lam. is one of the world's most useful medicinal plants. Different parts of the M. oleifera tree contain a rich profile of important minerals, proteins, vitamins, and various important bioactive compounds. However, there are differences in the phytochemical composition of the medicinal plant's raw materials due to seasonal variation, cultivation practices, and post-harvest processing. The main objective of this study was therefore to determine the effect of harvesting frequencies on selected bioactive compounds of a M. oleifera cultivar (PKM1) grown in a hydroponic system under a shade net structure. Three harvesting frequency treatments were applied in the study, with the plants harvested at every 30 days (high frequency), 60 days (intermediate frequency), and 90 days (low frequency) respectively. 1H-NMR was used for data acquisition, and multivariate data analysis by means of principal component analysis (PCA), partial least square discriminatory analysis (PLS-DA), and orthogonal partial least square discriminatory analysis (OPLS-DA) were applied to determine the changes in the leaf metabolite profile, and also to identify the spectral features contributing to the separation of samples. Targeted metabolite analysis was used to match the NMR peaks of the compounds with the NMR chemical shifts of the contribution plot. The contribution plot showed that the increase in concentration of some compounds in aliphatic, sugar and aromatic regions contributed to the separation of the samples. The results revealed that intermediate and low harvesting frequencies resulted in a change in the leaf metabolite profile. Compounds such as chlorogenic acid, ferulic acid, vanillic acid, wogonin, esculetin, niazirin, and gamma-aminobutyric acid (GABA) showed an increase under intermediate and low harvesting frequencies. These results provide insight into the effect of harvesting frequencies on the metabolite profile and associated medicinal activity of M. oleifera.

Effect of RGPU-260, a Novel GABA Derivative, on Functional Reserves of Rat Heart after Chronic Alcohol Intoxication.

The effect of a new derivative of GABA, the compound RGPU-260, on the heart contractility of rats exposed to chronic alcohol intoxication (10% ethanol solution for 24 weeks) was studied. In comparison with intact rats, the alcoholized ones were characterized with smaller increments in the rates of myocardial contraction (+dP/dtmax) and relaxation (-dP/dtmax), left ventricular pressure, and maximum intensity of functioning of structures during the load tests (volume load/preload, stimulation of the cardiac adrenergic receptors, and occlusion of the ascending aorta/afterload) attesting to degraded cardiac contractility function. In rats treated with RGPU-260 (daily, 25 mg/kg intragastrically during 14 days), these parameters were higher in comparison with control values indicating a positive action of the examined agent on inotropic function of the heart. Effectiveness of cardiotropic action of RGPU-260 was comparable to that of the reference drug mildronate.

Synergistic integration of dihydro-artemisinin with γ-aminobutyric acid results in a more potential anti-depressant.

Three hybrids of dihydro-artemisinin (DHA) with ß-aminopropionic acid, γ-aminobutyric acid, and histamine have been designed and synthesized. The conjugate of DHA with GABA labelled as 5b was confirmed the most active candidate against both Cort- and SNP-induced PC12 cell impairments with EC50 value of 8.04 ± 0.35, and 9.38 ± 0.56 µM, respectively. 5b was clearly highlighted as a good modulator on protein expression of Akt, Bcl-2, and Bax, indicating its functions against programmed cell apoptosis. 5b significantly reversed the Cort-induced excessive calcium influx and release from internal organelles. It was demonstrated the ability to express increased levels of ß-tubulin III and to up-regulate phosphorylation level of cAMP response element-binding protein (CREB), leading to cell differentiation. It can penetrate blood - brain barrier (BBB) with propriate stability. Altogether, these data strongly support that 5b is a potential anti-depressant.

α-Amino Acids and Peptides as Bifunctional Reagents: Carbocarboxylation of Activated Alkenes via Recycling CO2.

Carboxylic acids, including amino acids (AAs), have been widely used as reagents for decarboxylative couplings. In contrast to previous decarboxylative couplings that release CO2 as a waste byproduct, herein we report a novel strategy with simultaneous utilization of both the alkyl and carboxyl components from carboxylic acids. Under this unique strategy, carboxylic acids act as bifunctional reagents in the redox-neutral carbocarboxylation of alkenes. Diverse, inexpensive, and readily available α-AAs take part in such difunctionalizations of activated alkenes via visible-light photoredox catalysis, affording a variety of valuable but otherwise difficult to access γ-aminobutyric acid derivatives (GABAs). Additionally, a series of dipeptides and tripeptides also participate in this photocatalytic carbocarboxylation. Although several challenges exist in this system due to the low concentration and quantitative amount of CO2, as well as unproductive side reactions such as hydrodecarboxylation of the carboxylic acids and hydroalkylation of the alkenes, excellent regioselectivity and moderate to high chemoselectivity are achieved. This process features low catalyst loading, mild reaction conditions, high step and atom economy, and good functional group tolerance, and it is readily scalable. The resulting products are subject to efficient derivations, and the overall process is amenable to applications in the late-stage modification of complex compounds. Mechanistic studies indicate that a carbanion is generated catalytically and it acts as the key intermediate to react with CO2, which is also generated catalytically in situ and thus remains in low concentration. The overall transformation represents an efficient and sustainable system for organic synthesis, pharmaceutics, and biochemistry.

Metabolism of glutamic acid to alanine, proline, and γ-aminobutyric acid during takuan-zuke processing of radish root.

Takuan-zuke is a traditional Japanese fermented pickle, prepared by dehydration of radish root (daikon) by salt-pressing or sun-drying followed by aging with salt. We previously reported that alanine, proline, and γ-aminobutyric acid (GABA) accumulate during daikon dehydration, whereas the level of glutamic acid, their precursor, decreases. We have also reported that dehydration and salt-aging markedly influence the dynamics of free amino acids. In this study, we quantitatively analyzed free amino acid levels, enzyme activity, and gene expression to characterize takuan-zuke amino acid metabolism. Enzyme activities related to alanine, proline, GABA, and glutamic acid metabolism were sustained during dehydration. Moreover, genes encoding alanine, proline, and GABA synthases (ALT1, P5CS1, and GAD4) were significantly upregulated during dehydration. These effects may represent cellular stress responses to the dehydration process. The biological response of daikon contributes to the healthy functional aspects that characterize takuan-zuke. These findings could guide the selection of suitable vegetable varieties to produce pickled vegetables with health-promoting properties. PRACTICAL APPLICATION: The fermented pickle takuan-zuke, prepared by dehydration of radish root (daikon), accumulates amino acids, such as alanine, proline, and GABA, during preparation that provide taste and health benefits. In this study, the aforementioned amino acids were found to accumulate because of the stress response of daikon during the dehydration process and not because of the action of microorganisms during fermentation. Takuan-zuke processing is a method for improving the nutrition of daikon.

Simultaneous determination of five amino acid neurotransmitters in rat and porcine blood and brain by two-dimensional liquid chromatography.

A method for the simultaneous determination of aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), taurine (Tau) and gamma-aminobutyric acid (GABA) in animal blood and brain by two-dimensional liquid chromatography (2D-LC) combined with ultraviolet detection was established for the first time. First, the amino acid neurotransmitters (AANTs) were labeled on the corresponding fluorescent derivatives with 4-fluoro-7-nitrobenzofurazan (NBD-F), enriched on the extraction column and automatically transferred to the analytical column to achieve on-line extraction and complete separation of the target components. This method exhibited good selectivity, and the correlation coefficients for the analyte calibration curves of were > 0.99. The intra- and inter-day precisions were ≤ 16.03, and the accuracies were in the range of 70.59-116.20%. The system realizes the rapid detection and stability quantification of the five AANTs, which proves that the alternative dilution method is feasible. The results show that the system has high loading capacity, excellent resolution, and good peak shape and is not affected by other endogenous substances. Moreover, the developed method has been successfully applied to the analysis of biological samples in the blood and whole brain of rats and pigs. The content of AANTs in the hippocampus and cortex of rats was higher than that in those of pigs. This method is expected to provide applicability for the determination of AANTs in pharmacological, pharmaceutical and clinical research in nervous science.

Functional Characterization of the γ-Aminobutyric Acid Transporter from Mycobacterium smegmatis MC2 155 Reveals Sodium-Driven GABA Transport.

Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential γ-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported γ-aminobutyric acid both in vitro and when overexpressed in E. coli Additionally, transport was greatly reduced in the presence of ß-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active γ-aminobutyric acid transport protein from mycobacteria.IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport ß-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.