Glutamic Decarboxylase Research Articles

Expression of GAD65/67 and VGLUT2 in Mediobasal Nuclei of Rat Hypothalamus during Aging.

The expression of glutamate decarboxylase GAD65/67, an enzyme of GABA synthesis, and vesicular glutamate transporter 2 (VGLUT2) in the arcuate, dorsomedial, and ventromedial nuclei of the hypothalamus of young (3 months), adult (12 months), and old male rats (24 months) was studied by Western blotting. In old rats, an increase in the expression of GAD65/67 in the arcuate and dorsomedial, VGLUT2 in the arcuate, dorsomedial, and ventromedial nuclei was observed. Thus, an increase in opposite processes of inhibition and excitation is observed in the hypothalamic nuclei during aging.

Biological characteristics of the enteroaggregative <i>escherichia coli </i>ont:h30 18-726 (no. B-8857) strain isolated from a patient with ulcerative colitis and a new method of pcr identification

The aim of the work is to assess biological properties of the enteroaggregativeEscherichia coli(EAgEC) strain isolated from the colon mucosa biopsy material from a patient with ulcerative colitis, and to develop a new method for PCR identification ofE. colistrains.
 Materials and methods.The study involved 46 patients with a verified ulcerative colitis. Isolation of 87E. colistrains was carried out by using colon biopsy material obtained during a standard endoscopic procedure. The description of the biochemical bacterial properties was combined with the molecular genetic detection of virulence determinants and presence of antibiotic resistance mechanisms. Using the “AmpliSense®Escherichiose — FL” reagent kit, a screening for the presence of diarrheagenicE. colistrains was performed, which revealed the presence of a single strain of EAgEC. The methodological approach to creating a new method for strain identification was based on the search for a unique genetic sequence within the glutamate decarboxylase(gad)gene.
 Results.E. coli18-726 can be described biochemically typical to its species. The studied strain was characterized by a multiple resistance phenotype (MDR) to antibiotics, as well as a lack of sensitivity to five bacteriophages. TheE. coli18-726 strain had a unique sequence of the gene encoding the O-antigen, which differed from 188 known O-antigens (ONT) and, by the identity of the nucleotide sequence of the gene encoding the synthesis of the H-antigen, the strain belonged to the H30 serovar. Thus, the antigenic formula of strain EAgEC 18-726 was expressed as ONT:H30. TheE. coli18-726 strain contained a significant spectrum of genes that enable properties of virulence(iss, capU, aggA, aggB, aggC, aggD, aap, aar)and resistance to antibiotics(blaCTX-M-15, blaTEM-1B, aadA1, aadA5, mph(A), catB3). A new method for identifying EAgEC in chronic bowel disease is based on the polymerase chain reaction method using primers for a specific region of bacterial glutamate decarboxylase(gad)gene.Conclusion.1) The biochemical, antigenic and molecular genetic properties ofE. coliONT:H30 18-726 strain (No. B-8857) isolated from a patient with histomorphologically diagnosed ulcerative colitis were analyzed. 2) A method for PCR identification of EAgEC strains based on the detection of a specific region within the bacterial genomic glutamate decarboxylase gene has been created and patented.

CmCML11 interacts with CmCAMTA5 to enhance γ-aminobutyric acid (GABA) accumulation by regulating GABA shunt in fresh-cut cantaloupe

The effect of calcium chloride (CaCl2) treatment on γ-aminobutyric acid (GABA) accumulation in fresh-cut cantaloupe and the involved mechanisms were investigated. The result showed that 1% (w/v) CaCl2 treatment increased GABA content and activities of glutamate decarboxylase (GAD) and succinate semialdehyde dehydrogenase (SSADH), while decreased glutamate (Glu) content and GABA transaminase (GABA-T) activities in fresh-cut cantaloupe. CmCML11 and CmCAMTA5 expressions of CaCl2-treated fruit increased by 187.4% and 165.6% than control fruit in the initial 6 h. Besides, expressions of GABA shunt genes, including CmGAD1, CmGAD2, CmGABA-T and CmSSADH were also up-regulated by CaCl2 treatment during early storage. Moreover, acting as a transcriptional activator, CmCAMTA5 could bind to the CG-box in promoters of CmGAD1, CmGABA-T and CmSSADH and activate their transcription. Furthermore, the interaction between CmCML11 and CmCAMTA5 could enhance the transcriptional activation on GABA shunt genes which were regulated by CmCAMTA5. Collectively, our findings revealed that CaCl2 treatment promoted GABA accumulation in fresh-cut cantaloupe via the combined effect of CmCML11 and CmCAMTA5 in the regulation of expressions of CmGAD1, CmGABA-T, and CmSSADH in GABA shunt.

Two-component system LiaSR negatively regulated the acid resistance and pathogenicity of Listeria monocytogenes 10403S

The glutamate decarboxylase (GAD) system is one of the acid-resistant systems of Listeria monocytogenes (L. monocytogenes), while the regulatory mechanism of GadT2/GadD2, which plays the major role in the GAD system for acid resistance, is not clear. The two-component system (TCS) is a signal transduction system that is also involved in regulating acid resistance in bacteria. By screening the TCSs of L. monocytogenes 10403S, we found that knocking out the TCS LisSR (encoded by lmo1021/lmo1022) led to a significant increase in the transcription and expression of the gadT2/gadD2 cluster. Subsequently, we constructed a complemental strain CΔliaSR. and a complemental strain with LiaS His157 to Ala, which was designated as CΔliaSRH157A. Survival assay, transcriptional and expression analysis and pathogenicity assay revealed that liaSR deletion significantly enhanced the acid resistance and pathogenicity of 10403S and significantly increased the gadT2/gadD2 transcription and expression. Mutating LiaS His157 to Ala significantly enhanced the acid resistance and pathogenicity of CΔliaSR and significantly increased the gadT2/gadD2 transcription and expression. The results suggest that the two-component system LiaSR mediates the acid resistance and pathogenicity in 10403S by inhibiting the gadT2/gadD2 cluster, and the key activation site of LiaS is His157. This study provides novel knowledge on the regulation of GAD system and the control of this foodborne pathogen.

γ-Aminobutyric acid is involved in overlapping pathways against chilling injury by modulating glutamate decarboxylase and defense responses in papaya fruit.

The effect of γ-aminobutyric acid (GABA) treatment at two concentrations (1 mM or 5 mM) on papaya fruit stored at 4°C and 80%-90% relative humidity for 5 weeks was investigated. The application of GABA at 5 mM apparently inhibited chilling injury, internal browning, electrolyte leakage, malondialdehyde (MDA), hydrogen peroxide (H2O2), polyphenol oxidase (PPO), phospholipase D (PLD), and lipoxygenase (LOX) activities of papaya fruit. Fruit treated with 5 mM GABA enhanced the activities of ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), glutamate decarboxylase (GAD), and phenylalanine ammonia-lyase (PAL). In addition, GABA treatment significantly displayed higher levels of proline, endogenous GABA accumulation, phenolic contents, and total antioxidant activity than the nontreated papaya. The results suggested that GABA treatment may be a useful approach to improving the chilling tolerance of papaya fruit by reducing oxidative stress and enhancing the defense system.

Tumor-derived GABA promotes lung cancer progression by influencing TAMs polarization and neovascularization

BackgroundGamma-aminobutyric acid (GABA), a common neurotransmitter, has been found in various cancers but its origin and its role in the tumor immune microenvironment remains unclear. MethodsHere, we reported the expression of glutamate decarboxylase 1 (GAD1, converting glutamate into GABA) in lung cancer tissues based on the publicly available database, and explored the effects and underlying mechanism of GABA on lung cancer progression. ResultsCompared with normal tissues, GAD1 was aberrantly overexpressed in lung adenocarcinoma (LUAD) based on TCGA database. Furthermore, the LUAD patients’ overall survival was negatively correlated with the GAD1 expression levels. Our work found that a GABAa receptor inhibitor had a therapeutic effect on mouse tumors and significantly reduced tumor size and weight. Further experiments showed that GABA derived from tumor cells promoted tumor progression not by directly affecting cancer cells but by affecting macrophages polarization in the tumor microenvironment. We found that GABA inhibited the NF-κB pathway and STAT3 pathway to prevent macrophages from polarizing towards M1 type, while promoting macrophage M2 polarization by activating the STAT6 pathway. GABA was also found to promote tumor neovascularization by increasing the expression of FGF2 in macrophages. ConclusionsThese results suggest that GABA affects tumor progression by regulating macrophage polarization, and targeting GABA and its signaling pathway may represent a potential therapy for lung cancer.

The GABA shunt contributes to ROS homeostasis in guard cells of Arabidopsis.

γ-Aminobutyric acid (GABA) accumulates rapidly under stress via the GABA shunt pathway, which has been implicated in reducing the accumulation of stress-induced reactive oxygen species (ROS) in plants. γ-Aminobutyric acid has been demonstrated to act as a guard-cell signal in Arabidopsis thaliana, modulating stomatal opening. Knockout of the major GABA synthesis enzyme Glutamate Decarboxylase 2 (GAD2) increases the aperture of gad2 mutants, which results in greater stomatal conductance and reduces water-use efficiency compared with wild-type plants. Here, we found that the additional loss of GAD1, GAD4, and GAD5 in gad2 leaves increased GABA deficiency but abolished the more open stomatal pore phenotype of gad2, which we link to increased cytosolic calcium (Ca2+ ) and ROS accumulation in gad1/2/4/5 guard cells. Compared with wild-type and gad2 plants, glutamate was ineffective in closing gad1/2/4/5 stomatal pores, whereas lowering apoplastic calcium, applying ROS inhibitors or complementation with GAD2 reduced gad1/2/4/5 guard-cell ROS, restored the gad2-like greater stomatal apertures of gad1/2/4/5 beyond that of wild-type. We conclude that GADs are important contributors to ROS homeostasis in guard cells likely via a Ca2+ -mediated pathway. As such, this study reveals greater complexity in GABA's role as a guard-cell signal and the interactions it has with other established signals.

Gamma-Aminobutyric Acid (GABA) Producing Lactobacillus plantarum TSUB-17 and Probiotic Properties for Using as Probiotics Additive in Swine Feed

Due to the use of probiotics in animal husbandry has recently increased, the selection of high-quality probiotic strains is crucial. Since the uses of probiotics for animal feed supplements have to be applied at high doses of bacteria to be effective, the cultures’ safety is essential. This study successfully isolated the novel Lactobacillus sp. from swine fecal samples and identified it as L. plantarum TSUB-17. The L. plantarum TSUB-17 exhibited excellent probiotic properties, including 100 % resistance to 3 % acids and 1 % bile salts, and effectively inhibits pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella Typhimurium (isolated ESBL strain). L. plantarum TSUB-17 was also found to adhere to the intestinal wall and form a biofilm. Furthermore, the bacteria have revealed some unique properties of GABA production and detected the glutamate decarboxylase B (gadB) gene similar to L. brevis both in nucleotide sequence and secondary structure of the glutamate decarboxylase enzyme that was deduced from the nucleotide. The isolated bacteria also have antioxidant activity, and most importantly, the isolated strain was safe; it is non-hemolysis and susceptible to the tested antimicrobials. Therefore, L. plantarum TSUB-17 is suitable for further development as a probiotic additive in animal feed. HIGHLIGHTS This study successfully isolated Lactobacillus plantarum TSUB-17 with a high potential to develop as a probiotic for animals. The isolated bacterium has excellent properties: It tolerates high levels of acids and bile salts. It is effective against many pathogenic bacteria, produces gamma-aminobutyric acid (GABA) and shows high levels of antioxidant activity. Moreover, L. plantarum TSUB-17 is very safe due to its non-hemolytic strain and susceptibility to numerous antimicrobial drugs. GRAPHICAL ABSTRACT

A novel RofA-family transcriptional regulator, GadR, controls the development of acid resistance in Listeria monocytogenes.

The ability to survive the acidic conditions found in the stomach is crucial for the food-borne pathogen Listeria monocytogenes to gain access to the mammalian gastrointestinal tract. Little is currently known about how acid resistance is regulated in this pathogen and why this trait is highly variable between strains. Here, we used comparative genomics to identify a novel RofA-family transcriptional regulator, GadR, that controls the development of acid resistance. The RofA family of regulators was previously found only in a small group of bacterial pathogens, including streptococci, where they regulate virulence properties. We show that gadR encodes the dominant regulator of acid resistance in L. monocytogenes and that its sequence variability accounts for previously observed differences between strains in this trait. Together, these findings significantly advance our understanding of how this important pathogen copes with acid stress and suggest a potential molecular target to aid its control in the food chain.

Lactococcus lactis CNCM I-5388 versus NCDO2118 by its GABA hyperproduction ability, counteracts faster stress-induced intestinal hypersensitivity in rats.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by its main symptom, visceral hypersensitivity (VH), which is aggravated by stress. Gut-brain interactions and gut bacteria may alleviate IBS symptoms, including VH. γ-amino butyric acid (GABA), produced notably by lactic acid bacteria (LAB), shows promising result in IBS symptoms treatment. In bacteria, GABA is generated through glutamate decarboxylase (GAD) metabolism of L-glutamic acid, maintaining intracellular pH. In mammals, GABA acts as an inhibitory neurotransmitter, modulating pain, stress, and anxiety. Therefore, utilizing GABA-producing LAB as a therapeutic approach might be beneficial. Our previous work showed that a GABA-producing Lactococcus lactis strain, NCDO2118, reduced VH induced by acute stress in rats after a 10-day oral treatment. Here, we identified the strain CNCM I-5388, with a four-fold higher GABA production rate under the same conditions as NCDO2118. Both strains shared 99.1% identical GAD amino acid sequences and invitro analyses revealed the same optimal pH for GAD activity; however, CNCM I-5388 exhibited 17 times higher intracellular GAD activity and increased resistance to acidic pH. Additionally, invivo experiments have demonstrated that CNCM I-5388 has faster anti-VH properties in rats compared with NCDO2118, starting from the fifth day of treatment. Finally, CNCM I-5388 anti-VH effects partially persisted after 5-day treatment interruption and after a single oral treatment. These findings highlight CNCM I-5388 as a potential therapeutic agent for managing VH in IBS patients.

Glutamate decarboxylase-1 is essential for efficient acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation.

Glutamate decarboxylase (GAD) is a Ca2+ -calmodulin-activated, cytosolic enzyme that produces γ-aminobutyrate (GABA) as the committed step of the GABA shunt. This pathway bypasses the 2-oxoglutarate to succinate reactions of the tricarboxylic acid (TCA) cycle. GABA also accumulates during many plant stresses. We tested the hypothesis that AtGAD1 (At5G17330) facilitates Arabidopsis acclimation to Pi deprivation. Quantitative RT-PCR and immunoblotting revealed that AtGAD1 transcript and protein expression is primarily root-specific, but inducible at lower levels in shoots of Pi-deprived (-Pi) plants. Pi deprivation reduced levels of the 2-oxoglutarate dehydrogenase (2-OGDH) cofactor thiamine diphosphate (ThDP) in shoots and roots by > 50%. Growth of -Pi atgad1 T-DNA mutants was significantly attenuated relative to wild-type plants. This was accompanied by: (i) an > 60% increase in shoot and root GABA levels of -Pi wild-type, but not atgad1 plants, and (ii) markedly elevated anthocyanin and reduced free and total Pi levels in leaves of -Pi atgad1 plants. Treatment with 10 mM GABA reversed the deleterious development of -Pi atgad1 plants. Our results indicate that AtGAD1 mediates GABA shunt upregulation during Pi deprivation. This bypass is hypothesized to circumvent ThDP-limited 2-OGDH activity to facilitate TCA cycle flux and respiration by -Pi Arabidopsis.

The regulatory role of central neuropeptide-S in locomotion

Central exogenous Neuropeptide-S (NPS) was demonstrated to increase locomotor activity (LMA) in rodent studies. NPS receptor (NPSR) is produced in locomotion-related brain regions including basal ganglia while NPS mediates dopaminergic neurotransmission suggesting that endogenous brain NPS is involved in the regulation of locomotion. Aim of the study was to elucidate whether antagonism of NPSR impairs locomotion and to determine the neurochemical profile of NPSR-expressing cells in basal ganglia network. In the rats received intracerebroventricular injection of selective non-peptide NPSR antagonist ML154 (20 nmol/5 µL) or vehicle, in addition to measurement of catalepsy, motor performance, and motor coordination were evaluated by assessment of LMA and RR test, respectively. The immunoreactivities for NPSR, tyrosine hydroxylase (TH), glutamate decarboxylase 67 (GAD67), and choline acetyltransferase (ChAT) were detected by immunofluorescence in frozen sections. Compared to the control rats, total LMA was significantly declined following ML154 administration. The ML154-injected rats were more prone to fall in rotarod (RR) test, while they exhibited remarkably high catalepsy time. The most robust immunoreactivity for NPSR was detected in globus pallidus externa (GPe), while moderate levels of NPSR expression were observed in substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA), but not in striatum. The NPSR-ir cell bodies were found to express GAD67 in GPe and TH in SNpc and VTA, respectively. NPSR expression was detected in SNpc-projecting pallidal cells. The present findings indicate the regulatory role of central endogenous NPS in the control of locomotion. NPSR may be a potential therapeutic target for the treatment of movement disorders.

Genome-wide association studies reveal the genetic basis of amino acid content variation in tea plants

Tea is one of the most popular non-alcoholic beverages in the world, and free amino acids, especially theanine, make a major contribution to the umami taste of tea. However, the genetic basis of the variation in amino acid content in tea plants remains largely unknown. Here, we measured the free amino acid content in fresh leaves of 174 tea accessions over two years using a targeted metabolomics approach and obtained genotype data via RNA sequencing. Genome-wide association studies were conducted to investigate loci affecting the content of free amino acids. A total of 69 quantitative trait loci (–log10(P-value)>5) were identified. Functional annotation revealed that branched-chain amino acid aminotransferase, glutamine synthetase, nitrate transporter, and glutamate decarboxylase might be important for amino acid metabolism. Two significant loci, glutamine synthetase (Glu1, P=3.71×10−4; Arg1, P=4.61×10−5) and branched-chain amino acid aminotransferase (Val1, P=4.67×10−5; I_Leu1, P=3.56×10−6), were identified, respectively. Based on the genotyping result, two alleles of CsGS (CsGS-L and CsGS-H) and CsBCAT (CsBCAT-L and CsBCAT-H) were selected to perform function verification. Overexpression of CsGS-L and CsGS-H enhanced the contents of glutamate and arginine in transgenic plants, and overexpression of CsBCAT-L and CsBCAT-H promoted the accumulation of valine, isoleucine and leucine. Enzyme activity assay uncovered that SNP1054 is important for CsGS catalyzing glutamate into glutamine. Furthermore, CsGS-L and CsGS-H differentially regulated the accumulation of glutamine, and CsBCAT-L and CsBCAT-H differentially regulated the accumulation of branched-chain amino acids. In summary, the findings in our study would provide new insights into the genetic basis of amino acids contents variation in tea plants and facilitate the identification of elite genes to enhance amino acids content.

The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations.

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala.

GHRH Neurons from the Ventromedial Hypothalamic Nucleus Provide Dynamic and Sex-Specific Input to the Brain Glucose-Regulatory Network.

The ventromedial hypothalamic nucleus (VMN) controls glucose counter-regulation, including pituitary growth hormone (GH) secretion. VMN neurons that express the transcription factor steroidogenic factor-1/NR5A1 (SF-1) participate in glucose homeostasis. Research utilized in vivo gene knockdown tools to determine if VMN growth hormone-releasing hormone (Ghrh) regulates hypoglycemic patterns of glucagon, corticosterone, and GH outflow according to sex. Intra-VMN Ghrh siRNA administration blunted hypoglycemic hypercorticosteronemia in each sex, but abolished elevated GH release in males only. Single-cell multiplex qPCR showed that dorsomedial VMN (VMNdm) Ghrh neurons express mRNAs encoding Ghrh, SF-1, and protein markers for glucose-inhibitory (γ-aminobutyric acid) or -stimulatory (nitric oxide; glutamate) neurotransmitters. Hypoglycemia decreased glutamate decarboxylase67 (GAD67) transcripts in male, not female VMNdm Ghrh/SF-1 neurons, a response that was refractory to Ghrh siRNA. Ghrh gene knockdown prevented, in each sex, hypoglycemic down-regulation of Ghrh/SF-1 nerve cell GAD65 transcription. Ghrh siRNA amplified hypoglycemia-associated up-regulation of Ghrh/SF-1 neuron nitric oxide synthase mRNA in male and female, without affecting glutaminase gene expression. Ghrh gene knockdown altered Ghrh/SF-1 neuron estrogen receptor-alpha (ERα) and ER-beta transcripts in hypoglycemic male, not female rats, but up-regulated GPR81 lactate receptor mRNA in both sexes. Outcomes infer that VMNdm Ghrh/SF-1 neurons may be an effector of SF-1 control of counter-regulation, and document Ghrh modulation of hypoglycemic patterns of glucose-regulatory neurotransmitter along with estradiol and lactate receptor gene transcription in these cells. Co-transmission of glucose-inhibitory and -stimulatory neurochemicals of diverse chemical structure, spatial, and temporal profiles may enable VMNdm Ghrh neurons to provide complex dynamic, sex-specific input to the brain glucose-regulatory network.

A Novel Method for γ-Aminobutyric Acid Biosynthesis Using Glutamate Decarboxylase Entrapped in Polyvinyl Alcohol-Sodium Alginate Capsules.

γ-aminobutyric acid (GABA) has essential physiological functions in the human body. A novel method using glutamate decarboxylase (GAD) entrapped in polyvinyl alcohol (PVA)-sodium alginate (SA) capsules provides a green biological strategy for GABA synthesis. In this investigation, the stability range of immobilized GAD was effectively broadened, and immobilized GAD could be repeatedly used as a batch and fixed-bed column catalyst. The immobilized enzymes were stable and retained 89% of their activity in a pH range of 4.0-5.6, while there was an approximately 50% decrease in free GAD activity in the pH range of 4.8 ± 0.4. The immobilized GAD affinity to the substrate improved, and this was evidenced by the apparent decrease in Km to 13.3 mmol/L from the 30.9 mmol/L for free GAD. The immobilized GAD retained >90.6% activity after eight cycles and a near-100% enzyme activity retention after 120 h of a continuous fixed-bed column catalyst operation. This study has thus presented an effective PVA-SA-GAD immobilization method that could be used to continuously scale-up GABA biosynthesis.

Gad1 knock-out rats exhibit abundant spike-wave discharges in EEG, exacerbated with valproate treatment.

To elucidate the functional role of gamma-aminobutyric acid (GABA)-ergic inhibition in suppressing epileptic brain activities such as spike-wave discharge (SWD), we recorded electroencephalogram (EEG) in knockout rats for Glutamate decarboxylase 1 (Gad1), which encodes one of the two GABA-synthesizing enzymes in mammals. We also examined how anti-epileptic drug valproate (VPA) acts on the SWDs present in Gad1 rats and affects GABA synthesis in the reticular thalamic nucleus (RTN), which is known to play an essential role in suppressing SWD. Chronic EEG recordings were performed in freely moving control rats and homozygous knockout Gad1 (-/-) rats. Buzzer tones (82 dB) were delivered to the rats during EEG monitoring to test whether acoustic stimulation could interrupt ongoing SWDs. VPA was administered orally to the rats, and the change in the number of SWDs was examined. The distribution of GABA in the RTN was examined immunohistochemically. SWDs were abundant in EEG from Gad1 (-/-) rats as young as 2 months old. Although SWDs were universally detected in older rats irrespective of their Gad1 genotype, SWD symptom was most severe in Gad1 (-/-) rats. Acoustic stimulation readily interrupted ongoing SWDs irrespective of the Gad1 genotype, whereas SWDs were more resistant to interruption in Gad1 (-/-) rats. VPA treatment alleviated SWD symptoms in control rats, however, counterintuitively exacerbated the symptoms in Gad1 (-/-) rats. The immunohistochemistry results indicated that GABA immunoreactivity was significantly reduced in the somata of RTN neurons in Gad1 (-/-) rats but not in their axons targeting the thalamus. VPA treatment greatly increased GABA immunoreactivity in the RTN neurons of Gad1 (-/-) rats, which is likely due to the intact GAD2, another GAD isozyme, in these neurons. Our results revealed two opposing roles of GABA in SWD generation: suppression and enhancement of SWD. To account for these contradictory roles, we propose a model in which GABA produced by GAD1 in the RTN neuronal somata is released extrasynaptically and mediates intra-RTN inhibition.

GAD1-mediated GABA elicits aggressive characteristics of human oral cancer cells

Studies suggest that the expression of glutamate decarboxylase 1 (GAD1), γ-aminobutyric acid (GABA), and GABA receptors are involved in tumor progression. However, the underlying mechanisms of high expression and potential functions of GAD1 and GABA in oral squamous cell carcinoma (OSCC) are not known. In this study, we found that the expressions of GAD1 and GABA were considerably increased in OSCC samples, which were closely associated with clinical stage and lymph node metastasis. The knockdown of GAD1 expression significantly inhibited the proliferation, migration and invasion abilities of OSCC cells by reducing the expression of GABA-mediated GABAB receptors, which could be reversed by exogenous GABA, but did not cause excessive OSCC cell proliferation. And GABA secreted by OSCC cells promoted M2 macrophage polarization for inhibiting anti-tumor immunity by activating GABBR1/ERK/Ca2+. In addition, GABA/GABABR promoted the proliferation and progression of OSCC xenograft tumor. Altogether, our results showed that GAD1 synthetized GABA to promote the malignant progression of OSCC and limits the anti-tumor immunity of macrophages, thereby targeting GABA can be a novel strategy for treating OSCC.

Integrated transcriptomics and metabolomics reveals that regulating biosynthesis and metabolism of HCN and GABA plays a key role in drought resistance of wild soybean

Wild soybeans [Glycine soja (Siebold & Zucc.)] grown in arid areas develop adaptive traits and they are important wild plant resources. In order to reveal the drought-tolerance mechanism in wild soybean, we studied the changes of growth parameters, photosynthetic physiology, and ion physiology of two wild soybean ecotypes under drought stress, and integratedly analyzed the transcriptome and metabolome changes in their leaves. Drought-tolerant wild soybean had greater root/shoot ratio, water use efficiency, and K+ and Ca2+ contents than common wild soybean. In drought-tolerant wild soybean, the gene encoding (R)-mandelonitrile lyase in the pathway of hydrogen cyanide (HCN) biosynthesis and metabolism and the genes encoding serine O-acetyltransferase and aminocyclopropanecarboxylate oxidase in the pathway of 1-aminocyclopropane-1-carboxylate oxidation to produce HCN were down-regulated. In HCN biosynthesis and metabolic pathways of drought-tolerant wild soybean, the content of cysteine as a raw material was significantly reduced, while the content of 3-cyanoalanine as a product was significantly increased. In addition, drought-tolerant wild soybeans up-regulated genes for glutamate synthase and glutamate decarboxylase during synthesis of 4-aminobutyric acid (GABA), accumulating aspartic acid, asparagine, glutamic acid, and GABA; and down-regulated the gene for 4-aminobutyrate-pyruvate transaminase in GABA metabolism. Integrated analysis showed that drought-resistant wild soybean weakened the production of HCN and enhanced the utilization of HCN to avoid HCN toxicity under drought stress. At the same time, it also accumulated GABA to maintain carbon/nitrogen balance and amino acid metabolism. This study provides insights into the genetic mechanism of adaptation to drought by wild soybean, which will be helpful for screening of excellent wild plant resources.

Integrative transcriptomic analysis unveils lncRNA-miRNA-mRNA interplay in tomato plants responding to Ralstonia solanacearum

Ralstonia solanacearum, a bacterial plant pathogen, poses a significant threat to tomato (Solanum lycopersicum) production through destructive wilt disease. While noncoding RNA has emerged as a crucial regulator in plant disease, its specific involvement in tomato bacterial wilt remains limited. Here, we conducted a comprehensive analysis of the transcriptional landscape, encompassing both mRNAs and noncoding RNAs, in a tomato resistant line (‘ZRS_7’) and a susceptible line (‘HTY_9’) upon R. solanacearum inoculation using high-throughput RNA sequencing. Differential expression (DE) analysis revealed significant alterations in 7506 mRNAs, 997 lncRNAs, and 69 miRNAs between ‘ZRS_7’ and ‘HTY_9’ after pathogen exposure. Notably, 4548 mRNAs, 367 lncRNAs, and 26 miRNAs exhibited genotype-specific responses to R. solanacearum inoculation. GO and KEGG pathway analyses unveiled the potential involvement of noncoding RNAs in the response to bacterial wilt disease, targeting receptor-like kinases, cell wall-related genes, glutamate decarboxylases, and other key pathways. Furthermore, we constructed a comprehensive competing endogenous RNA (ceRNA) network incorporating 13 DE-miRNAs, 30 DE-lncRNAs, and 127 DEGs, providing insights into their potential contributions to the response against bacterial inoculation. Importantly, the characterization of possible endogenous target mimics (eTMs) of Sly-miR482e-3p via VIGS technology demonstrated the significant impact of eTM482e-3p-1 silencing on tomato's sensitivity to R. solanacearum. These findings support the existence of an eTM482e-3p-1-Sly-miR482e-3p-NBS-LRRs network in regulating tomato's response to the pathogen. Collectively, our findings shed light on the intricate interactions among lncRNAs, miRNAs, and mRNAs as underlying factors in conferring resistance to R. solanacearum in tomato.