Glutamic Decarboxylase Research Articles

A GAD1 inhibitor suppresses osteosarcoma growth through the Wnt/β-catenin signaling pathway

BackgroundAs a marker of the GABAergic system, the expression of glutamate decarboxylase 1 (GAD1) is mainly restricted to the central nervous system. Emerging studies have shown that aberrant expression of GAD1 in tumor tissues may promote tumor cell growth. The role of GAD1 in the development of osteosarcoma (OS) remains unclear, so this study sought to investigate the expression status of GAD1 and the effect of its specific inhibitor 3-mercaptopropionic acid (3-MPA) on OS. MethodsThe R2 database was used to analyze the relationship between the expression of GAD1 and clinical prognosis in OS patients. Immunohistochemistry was used to compare the expression profile of GAD1 between OS and matched neighboring tissues. The potential antitumor effects of 3-MPA on cell viability, colony formation and the cell cycle were examined. Moreover, the in vivo effect of 3-MPA on tumor growth was investigated using tumor-bearing nude mice. ResultsThe expression level of GAD1 was aberrantly upregulated in OS tissues, but almost no expression of GAD1 was found in matched neighboring tissues. Western blotting analyses showed upregulation of GAD1 in OS cells compared to human osteoblast cells. In vitro and in vivo, 3-MPA significantly suppressed the growth of OS. Regarding the mechanism, 3-MPA inhibited β-catenin and cyclin D1 in OS cells, thereby inactivating the Wnt/β-catenin pathway. ConclusionsOS displays increased expression of the GABAergic neuronal marker GAD1, and 3-MPA significantly reduces OS growth by inhibiting the Wnt/β-catenin pathway.

Angiotensin-(1-7) Mas Receptors are Highly Colocalized to GABAergic Proopiomelanocortin Neurons in the Arcuate Nucleus of the Hypothalamus

Previous studies from our laboratory have shown that circulating levels of angiotensin (Ang)-(1-7), a protective hormone of the renin-angiotensin system, are greatly reduced in obese hypertensive mice. Additionally, restoration of this hormone reduces cardiovascular sympathetic tone and lowers blood pressure in this model suggesting a central mechanism of action. In support of this, our recent data show that the sympatho-inhibitory and blood pressure lowering effects of Ang-(1-7) involve activation of mas receptors (masR) within the arcuate nucleus of the hypothalamus (ARC). More specifically, we have found that masR are highly colocalized to proopiomelanocortin (POMC) neurons in the ARC. Due to the heterogeneous nature of POMC neuron subtypes, we wanted to explore the neurotransmitter phenotype of these POMCmasR neurons. We hypothesized that due to the blood pressure lowering effects of Ang-(1-7), POMCmasR are likely colocalized to inhibitory GABAergic neurons. To test this, we performed RNAscope in-situ hybridization in male C57Bl/6J mice after twelve weeks of either a 60% high fat diet (HFD) or matched control diet (n=12-18/group). We used RNAscope to colocalize expression of POMC, masR, and the vesicular GABA transporter, Vgat, mRNA transcripts within the ARC. Colocalization of POMCmasR and Vgat was quantified and percent-colocalization compared between control and HFD groups. We found that POMCmasR are highly colocalized with the GABAergic marker, Vgat, to a similar extent in control and HFD groups (84±4 vs. 90±2% of neurons, respectively; p=0.294 unpaired t-test). The population of POMC neurons that did not express masR also did not colocalize with Vgat. As an initial step to determine if masR modulates GABAergic signaling in the ARC, we assessed gene expression of glutamate decarboxylase 2 (Gad2; a GABA synthetic enzyme) in the ARC following chronic six-week treatment with systemic Ang-(1-7) versus saline infusion in male mice fed a control diet versus HFD (n=9-13/group). We found that Ang-(1-7) increases ARC Gad2 mRNA in HFD mice (1.03±0.09 control diet vs. 0.93±0.07 HFD vs. 1.23±0.07 HFD plus Ang-(1-7); p=0.023 one-way ANOVA). Supporting specificity of these actions, Ang-(1-7) did not alter POMC mRNA in the ARC. These overall data suggest that masR primarily localize to GABA-expressing POMC neurons in the ARC, and Ang-(1-7) may directly modulate GABA synthesis pathways in this brain region. Further studies are needed to elucidate the functional importance of these ARC POMC GABAergic neurocircuits to the depressor effects of Ang-(1-7) in obesity. Funding: NIH R01 HL156986. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Integrative metabolomics-genomics analysis identifies key networks in a stem cell-based model of schizophrenia

Schizophrenia (SCZ) is a neuropsychiatric disorder, caused by a combination of genetic and environmental factors. The etiology behind the disorder remains elusive although it is hypothesized to be associated with the aberrant response to neurotransmitters, such as dopamine and glutamate. Therefore, investigating the link between dysregulated metabolites and distorted neurodevelopment holds promise to offer valuable insights into the underlying mechanism of this complex disorder. In this study, we aimed to explore a presumed correlation between the transcriptome and the metabolome in a SCZ model based on patient-derived induced pluripotent stem cells (iPSCs). For this, iPSCs were differentiated towards cortical neurons and samples were collected longitudinally at various developmental stages, reflecting neuroepithelial-like cells, radial glia, young and mature neurons. The samples were analyzed by both RNA-sequencing and targeted metabolomics and the two modalities were used to construct integrative networks in silico. This multi-omics analysis revealed significant perturbations in the polyamine and gamma-aminobutyric acid (GABA) biosynthetic pathways during rosette maturation in SCZ lines. We particularly observed the downregulation of the glutamate decarboxylase encoding genes GAD1 and GAD2, as well as their protein product GAD65/67 and their biochemical product GABA in SCZ samples. Inhibition of ornithine decarboxylase resulted in further decrease of GABA levels suggesting a compensatory activation of the ornithine/putrescine pathway as an alternative route for GABA production. These findings indicate an imbalance of cortical excitatory/inhibitory dynamics occurring during early neurodevelopmental stages in SCZ. Our study supports the hypothesis of disruption of inhibitory circuits to be causative for SCZ and establishes a novel in silico approach that enables for integrative correlation of metabolic and transcriptomic data of psychiatric disease models.

Risk predictors of glycaemic control in children and adolescents with type 1 diabetes: A systematic review and meta-analysis.

To conduct systematic evaluation of the risk predictors of glycaemic control in children and adolescents with type 1 diabetes mellitus. Cohort studies on risk predictors of glycaemic control in children and adolescents with type 1 diabetes were retrieved from CNKI, PubMed, Web of Science, Embase databases, etc. from the construction of the repository to 3 February 2023. Literature screening was conducted according to inclusion and exclusion criteria, then data extraction of region, sample size, age, follow-up time, risk predictors, outcome indicators, etc., and quality evaluation of The Newcastle-Ottawa Scale were conducted by two researchers while the third researcher makes decisions if there are disagreements. Finally, Revman5.4 and StataMP17 were used for meta-analysis. A total of 29 studies were included, and the results showed that insulin pump [Weighed mean difference (WMD) = -.48, 95% CI (-.73, -.24), p < .01], high-frequency sensor monitoring, early use of insulin pumps, prospective follow-up male, white race, large body mass index-standardised scoring, conscientiousness, agreeableness of mothers, eicosapentaenoic acid, leucine and protein (p < .05) were beneficial for reducing HbA1c levels in children and adolescents with diabetes. Ketoacidosis [WMD = .39, 95% CI (.28, .50), p < .01], selective admission, higher HbA1c level at one time (p < .01), higher glutamate decarboxylase antibody at 1 month after diagnosis, lower socio-economic status, non-living with biological parents, non-two-parent family, family disorder, family history of diabetes and high carbohydrate intake (p < .05) increased HbA1c levels in children and adolescents with diabetes. For children and adolescents with type 1 diabetes mellitus, the use of insulin pump, high-frequency sensor monitoring, prospective follow-up, good family support and reasonable diet are conducive to blood glucose control, while selective admission and DKA are not. Disease characteristics and demographic characteristics of children are closely related to subsequent blood glucose control, and the relationship between diagnosis age and blood glucose control needs to be further explored.

Investigating the impact of ultrasound-assisted cellulase pretreatment on the nutrients, phytic acid, and phenolics bioaccessibility in sprouted brown rice

This study aimed to elucidate the impact of ultrasound-assisted cellulase (UC) pretreatment on nutrients, phytic acid, and the bioavailability of phenolics during brown rice sprouting. It sought to unveil the underlying mechanisms by quantifying the activity of key enzymes implicated in these processes. The sprouted brown rice (SBR) surface structure was harmed by the UC pretreatment, which also increased the amount of γ-oryzanol and antioxidant activity in the SBR. Concurrently, the UC pretreatment boosted the activity of phytase, glutamate decarboxylase, succinate semialdehyde dehydrogenase, Gamma-aminobutyric acid (GABA) transaminase, chalcone isomerase, and phenylalanine ammonia lyase, thereby decreasing the phytic acid content and increasing the GABA, flavonoid, and phenolic content in SBR. In addition, UC-pretreated SBR showed increased phenolic release and bioaccessibility during in vitro digestion when compared to the treated group. These findings might offer theoretical direction for using SBR to maximize value.

GABA does not regulate stomatal CO2 signalling in Arabidopsis.

Optimal stomatal regulation is important for plant adaptation to changing environmental conditions and for maintaining crop yield. The guard cell signal γ-aminobutyric acid (GABA) is produced from glutamate by glutamate decarboxylase (GAD) during a reaction that generates CO2 as a by-product. Here, we investigated a putative connection between GABA signalling and the more clearly defined CO2 signalling pathway in guard cells. The GABA-deficient mutant Arabidopsis lines gad2-1, gad2-2, and gad1/2/4/5 were examined for stomatal sensitivity to various CO2 concentrations. Our findings show a phenotypical discrepancy between the allelic mutant lines gad2-1 and gad2-2-a weakened CO2 response in gad2-1 (GABI_474_E05) in contrast to a wild-type response in gad2-2 (SALK_028819) and gad1/2/4/5. Through transcriptomic and genomic investigation, we traced the response of gad2-1 to a deletion of full-length Mitogen-activated protein kinase 12 (MPK12) in the GABI-KAT line, thereafter renamed as gad2-1*. Guard cell-specific complementation of MPK12 in gad2-1* restored the wild-type CO2 phenotype, which confirms the proposed importance of MPK12 in CO2 sensitivity. Additionally, we found that stomatal opening under low atmospheric CO2 occurs independently of the GABA-modulated opening channel ALUMINIUM-ACTIVATED MALATE TRANSPORTER 9 (ALMT9). Our results demonstrate that GABA has a role in modulating the rate of stomatal opening and closing, but not in response to CO2per se.

Microbial chassis design and engineering for production of gamma-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid which is widely applied in agriculture and pharmaceutical additive industries. GABA is synthesized from glutamate through irreversible α-decarboxylation by glutamate decarboxylase. Recently, microbial synthesis has become an inevitable trend to produce GABA due to its sustainable characteristics. Therefore, reasonable microbial platform design and metabolic engineering strategies for improving production of GABA are arousing a considerable attraction. The strategies concentrate on microbial platform optimization, fermentation process optimization, rational metabolic engineering as key metabolic pathway modification, promoter optimization, site-directed mutagenesis, modular transporter engineering, and dynamic switch systems application. In this review, the microbial producers for GABA were summarized, including lactic acid bacteria, Corynebacterium glutamicum, and Escherichia coli, as well as the efficient strategies for optimizing them to improve the production of GABA.

The potassium transporter TaNHX2 interacts with TaGAD1 to promote drought tolerance via modulating stomatal aperture in wheat.

Drought is a major global challenge in agriculture that decreases crop production. γ-Aminobutyric acid (GABA) interfaces with drought stress in plants; however, a mechanistic understanding of the interaction between GABA accumulation and drought response remains to be established. Here we showed the potassium/proton exchanger TaNHX2 functions as a positive regulator in drought resistance in wheat by mediating cross-talk between the stomatal aperture and GABA accumulation. TaNHX2 interacted with glutamate decarboxylase TaGAD1, a key enzyme that synthesizes GABA from glutamate. Furthermore, TaNHX2 targeted the C-terminal auto-inhibitory domain of TaGAD1, enhanced its activity, and promoted GABA accumulation under drought stress. Consistent with this, the tanhx2 and tagad1 mutants showed reduced drought tolerance, and transgenic wheat with enhanced TaNHX2 expression had a yield advantage under water deficit without growth penalty. These results shed light on the plant stomatal movement mechanism under drought stress and the TaNHX2-TaGAD1 module may be harnessed for amelioration of negative environmental effects in wheat as well as other crops.

Substrate specificity of a ketosynthase domain involved in bacillaene biosynthesis.

An isotopic labelling method was developed to investigate substrate binding by ketosynthases, exemplified by the second ketosynthase of the polyketide synthase BaeJ involved in bacillaene biosynthesis (BaeJ-KS2). For this purpose, both enantiomers of a 13C-labelled N-acetylcysteamine thioester (SNAC ester) surrogate of the proposed natural intermediate of BaeJ-KS2 were synthesised, including an enzymatic step with glutamate decarboxylase, and incubated with BaeJ-KS2. Substrate binding was demonstrated through 13C NMR analysis of the products against the background of various control experiments.

The metabolic reprogramming of γ-aminobutyrate in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy. The oncometabolites have been studied in OSCC, but the mechanism of metabolic reprogramming remains unclear. To identify the potential metabolic markers to distinguish malignant oral squamous cell carcinoma (OSCC) tissue from adjacent healthy tissue and study the mechanism of metabolic reprogramming in OSCC. We compared the metabolites between cancerous and paracancerous tissues of OSCC patients by 1HNMR analysis. We established OSCC derived cell lines and analyzed their difference of RNA expression by RNA sequencing. We investigated the metabolism of γ-aminobutyrate in OSCC derived cells by real time PCR and western blotting. Our data revealed that much more γ-aminobutyrate was produced in cancerous tissues of OSCC patients. The investigation based on OSCC derived cells showed that the increase of γ-aminobutyrate was promoted by the synthesis of glutamate beyond the mitochondria. In OSCC cancerous tissue derived cells, the glutamate was catalyzed to glutamine by glutamine synthetase (GLUL), and then the generated glutamine was metabolized to glutamate by glutaminase (GLS). Finally, the glutamate produced by glutamate-glutamine-glutamate cycle was converted to γ-aminobutyrate by glutamate decarboxylase 2 (GAD2). Our study is not only benefit for understanding the pathological mechanisms of OSCC, but also has application prospects for the diagnosis of OSCC.

Autoantibodies in epileptic patients indicate the autoimmune epilepsy

Background Epilepsy is a multifaceted neurological condition marked by recurring seizures. Delving into the functions of VGKC (voltage-gated potassium channels), AMPA GluR3 (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 3), GAD65 (glutamate decarboxylase 65), and LGI1 (leucine-rich glioma-inactivated 1) in epilepsy yields significant perspectives into the fundamental immunological mechanisms of this disorder. Objective The study target was to assess the clinical significance of neural autoantibody biomarkers in patients with epilepsy and determine the types of autoimmune epilepsy. Materials and methods A case–control study that comprised 50 epilepsy patients (33 males and 17 females) and 40 controls (21 males and 19 females), the patients and controls attending Basrah Teaching Hospital between November 2022 and March 2023, Basrah City, Southern Iraq. The control age ranged from 2 to 62 years and patients’ age 2–68 years were considered the control group in this study. The VGKC, AMPA GluR3, GAD65, and LGI1 were measured using the sandwich ELISA technique. Results and conclusion There is no statistically significant difference between the patients and control group for VGKC, GAD65, and LGI1 (P=0.460, P=0.061, and P=0.440, respectively), while there is a statistically significant difference between the patients and the control group for AMPA GluR3 (P=0.012). GAD65 appeared an elevation in epilepsy patients but was not statistically significant. This upregulation may contribute to the hyperexcitability observed in the epileptic brain and potentially play a role in seizure generation and propagation.

Effect of Lactiplantibacillus plantarum cell-free supernatant on the physiology, quorum sensing, transcription, and enhanced GABA production of Enterococcus faecium

A co-culture strategy has been proposed for the production of desired products. However, the interaction effect on enhancing γ-aminobutyric acid (GABA) production requires further investigation. In this study, the mechanism by which Lactiplantibacillus plantarum BC112 cell-free supernatant (CFS) enhances GABA production in Enterococcus faecium AB157 was investigated, including physiological characteristics, quorum sensing (QS), and mRNA expression. The results showed CFS can significantly enhance the production of γ-aminobutyric acid (GABA) in E. faecium AB157, with a maximum increase of 4.6 times compared to the strain without CFS. CFS promoted the growth of E. faecium AB157, enhanced the activity of glutamate decarboxylase (GAD) and ATPase, and reduced the pH. The effect of CFS on QS was demonstrated through changes in Autoinducer-2 (AI-2) activity and luxS expression. Moreover, 16 amino acids were detected in CFS, with glutamic acid having the highest content. Additionally, 974 genes of E. faecium AB157 differed significantly in cultures with added CFS, which involved glycolytic, amino acid, and GABA biosynthetic pathways. Therefore, Lb. plantarum BC112 CFS enhances GABA production in E. faecium AB157 by affecting energy metabolism, the QS system, and the GABA biosynthesis system.

Participation of calcium-permeable AMPA receptors in the regulation of epileptiform activity of hippocampal neurons.

Epileptiform activity is the most striking result of hyperexcitation of a group of neurons that can occur in different brain regions and then spread to other sites. Later it was shown that these rhythms have a cellular correlate in vitro called paroxysmal depolarization shift (PDS). In 13-15 DIV neuron-glial cell culture, inhibition of the GABA(A) receptors induces bursts of action potential in the form of clasters PDS and oscillations of intracellular Ca2+ concentration ([Ca2+]i). We demonstrate that GABAergic neurons expressing calcium-permeable AMPA receptors (CP-AMPARs) as well as Kv7-type potassium channels regulate hippocampal glutamatergic neurons' excitability during epileptiform activity in culture. A combination of whole-cell patch-clamp in current clamp mode and calcium imaging microscopy was used to simultaneously register membrane potential and [Ca2+]i level. To identify GABAergic cell cultures were fixed and stained with antibodies against glutamate decarboxylase GAD 65/67 and neuron-specific enolase (NSE) after vital [Ca2+]i imaging. It was shown that CP-AMPARs are involved in the regulation of the PDS clusters and [Ca2+]i pulses accompanied them. Activation of CP-AMPARs of GABAergic neurons is thought to cause the release of GABA, which activates the GABA(B) receptors of other GABAergic interneurons. It is assumed that activation of these GABA(B) receptors leads to the release of beta-gamma subunits of Gi protein, which activate potassium channels, resulting in hyperpolarization and inhibition of these interneurons. The latter causes disinhibition of glutamatergic neurons, the targets of these interneurons. In turn, the CP-AMPAR antagonist, NASPM, has the opposite effect. Measurement of membrane potential in GABAergic neurons by the patch-clamp method in whole-cell configuration demonstrated that NASPM suppresses hyperpolarization in clusters and individual PDSs. It is believed that Kv7-type potassium channels are involved in the control of hyperpolarization during epileptiform activity. The blocker of Kv7 channels, XE 991, mimicked the effect of the CP-AMPARs antagonist on PDS clusters. Both drugs increased the duration of the PDS cluster. In turn, the Kv7 activator, retigabine, decreased the duration of the PDS cluster and Ca2+ pulse. In addition, retigabine led to deep posthyperpolarization at the end of the PDS cluster. The Kv7 channel is believed to be involved in the formation of PDS, as the channel blocker reduced the rate of hyperpolarization in the PDS almost three times. Thus, GABAergic neurons expressing CP-AMPARs, regulate the membrane potential of innervated glutamatergic neurons by modulating the activity of postsynaptic potassium channels of other GABAergic neurons.

Abstract 1873 The Synthesis of Neuromodulatory Molecules by A Gut Microbial Glutamate Decarboxylase: GABA, Taurine and its Analogs, and β-Alanine

Abstract 1873 The Synthesis of Neuromodulatory Molecules by A Gut Microbial Glutamate Decarboxylase: GABA, Taurine and its Analogs, and β-Alanine

Physiological role of genes involved in taurine biosynthesis in fishes and in silico approach to determine transcription factors in their promoters’ zone

Taurine, an amino sulfonic acid that is sometimes referred to as an amino acid, is endogenously synthesized by the action of the key genes/enzymes: cysteine dioxygenase (cdo), cysteine sulfonate decarboxylase (csad), glutamate decarboxylase (gad), and 2-amino ethanethiol dioxygenase (ado). The taurine transporter (taut) also distributes this taurine formation across the plasma. These genes have been identified as important in different physiological processes such as reproduction, digestion, olfactory, visual, circulatory, and muscular systems. Thus, a literature review of these genes in fish has been described in the present work. Moreover, there is null information regarding the study of regulatory elements such as transcription factors (TFs) in taurine biosynthesis and transportation genes of fishes. In this interest and taking advantage of the availability of different sequence databases, bioinformatics can be applied as a first approach for an in silico identification of the putative TFs and transcription factor binding sites (TFBS) that might play an important role in regulating these genes. The results showed that some are commonly shared, whereas TFs and TFBs vary among fish species. Hence, binding sites for homeobox protein BarH-like 1 (BARX1), brain-specific homeobox protein homolog (BSX), helicase-like transcription factor (HLTF), homeobox protein Hox-A7 (HOXA7), homeobox protein Hox-B3 (HOXB3), homeobox protein Hox-B6 (HOXB6), homeobox protein Meis1 (MEIS1), homeobox protein Meis3 (MEIS3), nuclear factor of activated T cells 1 (NFATC1), and homeobox protein Nkx-6.2 (NKX6-2) were commonly found in the promoter regions of genes involved in taurine transportation and biosynthesis. Additionally, our results suggested that the frequency of HOXB3, a transcription factor involved in development, has repetitive TFBS sites in the promoter region of all species analyzed in the present study. Although bioinformatics gives us an approach to determine putative TFs and TFBS, further work is needed to verify how the found regulatory elements play a key role in taurine biosynthesis and transportation.

Truncation of the calmodulin binding domain in rice glutamate decarboxylase 4 (OsGAD4) leads to accumulation of γ-aminobutyric acid and confers abiotic stress tolerance in rice seedlings.

The online version contains supplementary material available at 10.1007/s11032-024-01460-1.

Sucrose, cell wall, and polyamine metabolisms involve in preserving postharvest quality of ‘Zaosu’ pear fruit by L-glutamate treatment

‘Zaosu’ pear fruit is prone to yellowing of the surface and softening of the flesh after harvest. This work was performed to assess the influences of L-glutamate treatment on the quality of ‘Zaosu’ pears and elucidate the underlying mechanisms involved. Results demonstrated that L-glutamate immersion reduced ethylene release, respiratory intensity, weight loss, brightness (L*), redness (a*), yellowness (b*), and total coloration difference (ΔE); enhanced ascorbic acid, soluble solids, and soluble sugar contents; maintained chlorophyll content and flesh firmness of pears. L-glutamate also restrained the activities of neutral invertase and acid invertase, while enhancing sucrose phosphate synthetase and sucrose synthase activities to facilitate sucrose accumulation. The transcriptions of PbSGR1, PbSGR2, PbCHL, PbPPH, PbRCCR, and PbNYC were suppressed by L-glutamate, resulting in a deceleration of chlorophyll degradation. L-glutamate concurrently suppressed the transcription levels and enzymatic activities of polygalacturonases, pectin methylesterases, cellulase, and β-glucosidase. It restrained polygalacturonic acid trans-eliminase and pectin methyl-trans-eliminase activities as well as inhibited the transcription levels of PbPL and Pbβ-gal. Moreover, the gene transcriptions and enzymatic activities of arginine decarboxylase, ornithine decarboxylase, S-adenosine methionine decarboxylase, glutamate decarboxylase, γ-aminobutyric acid transaminase, glutamine synthetase along with the PbSPDS transcription was promoted by L-glutamate. L-glutamate also resulted in the down-regulation of PbPAO, PbDAO, PbSSADH, PbGDH, and PbGOGAT transcription levels, while enhancing γ-aminobutyric acid, glutamate, and pyruvate acid contents in pears. These findings suggest that L-glutamate immersion can effectively maintain the storage quality of ‘Zaosu’ pears via modulating key enzyme activities and gene transcriptions involved in sucrose, chlorophyll, cell wall, and polyamine metabolism.

The transcription factor ThDOF8 binds to a novel cis-element and mediates molecular responses to salt stress in Tamarix hispida.

Salt stress is a common abiotic factor that restricts plant growth and development. As a halophyte, Tamarix hispida is a good model plant for exploring salt-tolerance genes and regulatory mechanisms. DNA-binding with one finger (DOF) is an important transcription factor (TF) that influences and controls various signaling substances involved in diverse biological processes related to plant growth and development, but the regulatory mechanisms of DOF TFs in response to salt stress are largely unknown in T. hispida. In the present study, a newly identified Dof gene, ThDOF8, was cloned from T. hispida, and its expression was found to be induced by salt stress. Transient overexpression of ThDOF8 enhanced T. hispida salt tolerance by enhancing proline levels, and increasing the activities of the antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD). These results were also verified in stably transformed Arabidopsis. Results from TF-centered yeast one-hybrid (Y1H) assays and EMSAs showed that ThDOF8 binds to a newly identified cis-element (TGCG). Expression profiling by gene chip analysis identified four potential direct targets of ThDOF8, namely the cysteine-rich receptor-like kinases genes, CRK10 and CRK26, and two glutamate decarboxylase genes, GAD41, and GAD42, and these were further verified by ChIP-quantitative-PCR, EMSAs, Y1H assays, and β-glucuronidase enzyme activity assays. ThDOF8 can bind to the TGCG element in the promoter regions of its target genes, and transient overexpression of ThCRK10 also enhanced T. hispida salt tolerance. On the basis of our results, we propose a new regulatory mechanism model, in which ThDOF8 binds to the TGCG cis-element in the promoter of the target gene CRK10 to regulate its expression and improve salt tolerance in T. hispida. This study provides a basis for furthering our understanding the role of DOF TFs and identifying other downstream candidate genes that have the potential for improving plant salt tolerance via molecular breeding.

Neurotoxicant effects of bisphenol A, nonylphenol, and tert‑butyl phenol in the Nile tilapia (Oreochromis niloticus)

Worldwide production of alkyl phenols and ethoxylated alkyl phenols is high due to their broad industrial uses. It has been widely documented that they are endocrine disruptors, and it has been suggested that they could exert neurotoxic effects. However, a lack of information about the neurotoxic effects of APs and APEs prevails. In this study, the bisphenol A (BPA), 4-nonylphenol (NP), and 3‑tert-butylphenol (tertBP) effects on brain and spinal cord of Nile tilapia exposed to environmental concentrations were evaluated by assessing acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and carboxylesterases (CES) activities, and γ-aminobutyric acid (GABA) levels and their effects were evaluated by molecular docking. BPA and NP, tertBP behave as agonists and antagonists of AChE, BuChE, CES, and GABA, with notable differences among organs. However, none of these compounds or their metabolites interact with the enzymes' catalytic triad, suggesting an indirect alteration of enzymatic activities. While inhibiting these enzymes stand out hydrophobic interactions with the peripheral anion site, contacts with the inner face of the active site and blocking the mouth of the gorge of the active site, and steric hindrance in the enzyme pocket of glutamate decarboxylase (GAD). In contrast, inductions probably are by homotropic pseudo-cooperative phenomenon, where APEs behave as anchors favoring the active site to remain open and interactions that confer a conservative stabilization of the regulatory domain. Although the results of this study are complex, with notable differences between organs and toxicants, they are some of the first evidence of the neurotoxicity of alkylphenols and their ethoxylated derivatives.

The high level food-grade expression of glutamate decarboxylase in Bacillus subtilis through combination of genomic integration and free plasmid

The high level food-grade expression of glutamate decarboxylase in Bacillus subtilis through combination of genomic integration and free plasmid