Gimmicks of gamma‐aminobutyric acid (GABA) in pancreatic β‐cell regeneration through transdifferentiation of pancreatic α‐ to β‐cells

In vivo regeneration of lost or dysfunctional islet β cells can fulfill the promise of improved therapy for diabetic patients. To achieve this, many mitogenic factors have been attempted, including gamma‐aminobutyric acid (GABA). GABA remarkably affects pancreatic islet cells’ (α cells and β cells) function through paracrine and/or autocrine binding to its membrane receptors on these cells. GABA has also been studied for promoting the transformation of α cells to β cells. Nonetheless, the gimmickry of GABA‐induced α‐cell transformation to β cells has two different perspectives. On the one hand, GABA was found to induce α‐cell transformation to β cells in vivo and insulin‐secreting β‐like cells in vitro. On the other hand, GABA treatment showed that it has no α‐ to β‐cell transformation response. Here, we will summarize the physiological effects of GABA on pancreatic islet β cells with an emphasis on its regenerative effects for transdifferentiation of islet α cells to β cells. We will also critically discuss the controversial results about GABA‐mediated transdifferentiation of α cells to β cells.     

Genetic manipulation of the γ‐aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ‐aminobutyric acid transaminase (GABA‐T) lead to sustained and high levels of GABA accumulation in rice kernels

Gamma‐aminobutyric acid (GABA) is a non‐protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA‐transaminase, GABA‐T), we attempted seed‐specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB‐1) or rice embryo globulin promoters (REG) and GABA‐T‐based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2–100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA‐T expression was relatively weak. In these two lines both with two T‐DNA copies, their starch, amylose, and protein levels were slightly lower than non‐transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75–350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.     

Metabolic pathways regulated by abscisic acid,salicylic acid and γ‐aminobutyric acid in association with improved drought tolerance in creeping bentgrass (Agrostis stolonifera)

Abscisic acid (ABA), salicylic acid (SA) and γ‐aminobutyric acid (GABA) are known to play roles in regulating plant stress responses. This study was conducted to determine metabolites and associated pathways regulated by ABA, SA and GABA that could contribute to drought tolerance in creeping bentgrass (Agrostis stolonifera). Plants were foliar sprayed with ABA (5 μM), GABA (0.5 mM) and SA (10 μM) or water (untreated control) prior to 25 days drought stress in controlled growth chambers. Application of ABA, GABA or SA had similar positive effects on alleviating drought damages, as manifested by the maintenance of lower electrolyte leakage and greater relative water content in leaves of treated plants relative to the untreated control. Metabolic profiling showed that ABA, GABA and SA induced differential metabolic changes under drought stress. ABA mainly promoted the accumulation of organic acids associated with tricarboxylic acid cycle (aconitic acid, succinic acid, lactic acid and malic acid). SA strongly stimulated the accumulation of amino acids (proline, serine, threonine and alanine) and carbohydrates (glucose, mannose, fructose and cellobiose). GABA enhanced the accumulation of amino acids (GABA, glycine, valine, proline, 5‐oxoproline, serine, threonine, aspartic acid and glutamic acid) and organic acids (malic acid, lactic acid, gluconic acid, malonic acid and ribonic acid). The enhanced drought tolerance could be mainly due to the enhanced respiration metabolism by ABA, amino acids and carbohydrates involved in osmotic adjustment (OA) and energy metabolism by SA, and amino acid metabolism related to OA and stress‐defense secondary metabolism by GABA.     

Ecological and genetic mechanisms of the dynamics of population abundance in silver crucian carp (Carassius auratus gibelio (Bloch, 1782)) from the Tsimlyansk Reservoir

Silver crucian carp is the dominant commercial fish of the Tsimlyansk Reservoir (the Don River). A retrospective analysis of the ecological and genetic factors determining the dynamics of the silver crucian carp population at different stages of reservoir functioning was performed. At the most recent stage, the lake-swamp environmental conditions suitable for crucian carp and a lack of any dependence on other cyprinid fishes during the reproduction of this species determine its domination in the reservoir??s fish community. The size, sex, and genetic structures of the crucian carp population, characterized by numerous age groups and low linear and weight growth rates, are presented.     

Saprolegnia australis R. F. Elliott 1968 infection in Prussian carp Carassius gibelio (Bloch, 1782) eggs and its control with herb extracts

In order to control saprolegniosis in Prussian carp (Carassius gibelio (Bloch, 1782) eggs, it is important to screen herb extracts as potential anti‐Saprolegnia drugs in Prussian carp hatcheries. For this purpose, an oomycete water mould (strain SC) isolated from Prussian carp [Carassius gibelio (Bloch, 1782)] eggs suffering from saprolegniosis was characterised morphologically as well as from ITS rDNA sequence data. Initially identified as a Saprolegnia sp. based on its morphological features, the constructed phylogenetic tree using the neighbour joining method further indicated that the SC strain was closely related to Saprolegnia australis R. F. Elliott 1968 strain VI05733 (GenBank accession no. HE798564 ), and which could form biofilm communities as virulence factors. In addition, aqueous extracts from forty Chinese herbs were screened as possible anti‐Saprolegnia agents. Among them, a 1 g ml^?1 extract from Radix sanguisorbae was the most efficacious anti‐Saprolegnia agent, indicated by the minimum inhibitory concentration that was as low as 256 mg L^?1. Relative survival of 73 and 88% was obtained against the SC strain in fish eggs at concentrations of 256 and 1280 mg L^?1, respectively. This is the first known report of Saprolegnia australis R. F. Elliott 1968 infection in C. gibelio (Bloch, 1782) eggs involving the screening of R. sanguisorbae extracts as potential anti‐Saprolegnia agents.     

Multiplexed CRISPR/Cas9‐mediated metabolic engineering of γ‐aminobutyric acid levels in Solanum lycopersicum

In recent years, the type II CRISPR system has become a widely used and robust technique to implement site‐directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single‐site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited. Furthermore, we manipulated the γ‐aminobutyric acid (GABA) shunt in tomatoes using a multiplex pYLCRISPR/Cas9 system that targeted five key genes. Fifty‐three genome‐edited plants were obtained following single plant transformation, and these samples represented single to quadruple mutants. The GABA accumulation in both the leaves and fruits of genomically edited lines was significantly enhanced, and the GABA content in the leaves of quadruple mutants was 19‐fold higher than that in wild‐type plants. Our data demonstrate that the multiplex CRISPR/Cas9 system can be exploited to precisely edit tomato genomic sequences and effectively create multisite knockout mutations, which could shed new light on plant metabolic engineering regulations.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Phloem‐derived γ‐aminobutyric acid (GABA) is involved in upregulating nodule N2 fixation efficiency in the model legume Medicago truncatula

Nitrogen fixation in legumes is downregulated through a whole plant N feedback mechanism, for example, when under stress. This mechanism is probably triggered by the impact of shoot‐borne, phloem‐delivered compounds. However, little is known about any whole‐plant mechanism that might upregulate nitrogen fixation, for example, under N deficiency. We induced emerging N‐deficiency through partial excision of nodules from Medicago truncatula plants. Subsequently, the activity and composition of the remaining nodules and shifts in concentration of free amides/amino acids in the phloem were monitored. Furthermore, we mimicked these shifts through artificial feeding of γ‐aminobutyric acid (GABA) into the phloem of undisturbed plants. As a result of increased specific activity of nodules, N₂ fixation per plant recovered almost completely 4–5 d after excision. The concentration of amino acids, sugars and organic acids increased strongly in the upregulated nodules. A concomitant analysis of the phloem revealed a significant increase in GABA concentration. Comparable with the effect of nodule excision, artificial GABA feeding into the phloem resulted in an increased activity and higher concentration of amino acids and organic acids in nodules. It is concluded that GABA might be involved in upregulating nodule activity, possibly because of its constituting part of a putative amino acid cycle between bacteroids and the cytosol.     

The γ‐aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp. PCC 6803

A traditional 2‐oxoglutarate dehydrogenase complex is missing in the cyanobacterial tricarboxylic acid cycle. To determine pathways that convert 2‐oxoglutarate into succinate in the cyanobacterium Synechocystis sp. PCC 6803, a series of mutant strains, Δsll1981, Δslr0370, Δslr1022 and combinations thereof, deficient in 2‐oxoglutarate decarboxylase (Sll1981), succinate semialdehyde dehydrogenase (Slr0370), and/or in γ‐aminobutyrate metabolism (Slr1022) were constructed. Like in Pseudomonas aeruginosa, N‐acetylornithine aminotransferase, encoded by slr1022, was shown to also function as γ‐aminobutyrate aminotransferase, catalysing γ‐aminobutyrate conversion to succinic semialdehyde. As succinic semialdehyde dehydrogenase converts succinic semialdehyde to succinate, an intact γ‐aminobutyrate shunt is present in Synechocystis. The Δsll1981 strain, lacking 2‐oxoglutarate decarboxylase, exhibited a succinate level that was 60% of that in wild type. However, the succinate level in the Δslr1022 and Δslr0370 strains and the Δsll1981/Δslr1022 and Δsll1981/Δslr0370 double mutants was reduced to 20–40% of that in wild type, suggesting that the γ‐aminobutyrate shunt has a larger impact on metabolite flux to succinate than the pathway via 2‐oxoglutarate decarboxylase. ¹³C‐stable isotope analysis indicated that the γ‐aminobutyrate shunt catalysed conversion of glutamate to succinate. Independent of the 2‐oxoglutarate decarboxylase bypass, the γ‐aminobutyrate shunt is a major contributor to flux from 2‐oxoglutarate and glutamate to succinate in Synechocystis sp. PCC 6803.     

Variation in clones of the sperm-dependent parthenogenetic Carassius gibelio (Bloch) in Lake Pamvotis (north-west Greece)

Variation in three different clones of the invasive sperm-dependent, cyprinid fish Carassius gibelio were examined in Lake Pamvotis (north-west Greece). Differences between the clones were found in their proportion in the population, in their age structure, in the time of arrival to their spawning grounds and in the coefficients of the von Bertalanffy growth equation.     

Nitric oxide, γ‐aminobutyric acid,and mannose pretreatment influence metabolic profiles in white clover under water stress

Nitric oxide (NO), γ‐aminobutyric acid (GABA), and mannose (MAS) could be important regulators of plant growth and adaptation to water stress. The application of sodium nitroprusside (SNP, a NO donor), GABA, and MAS improved plant growth under water‐sufficient conditions and effectively mitigated water stress damage to white clover. The metabonomic analysis showed that both SNP and GABA application resulted in a significant increase in myo‐inositol content; the accumulation of mannose was commonly regulated by SNP and MAS; GABA and MAS induced the accumulation of aspartic acid, quinic acid, trehalose, and glycerol under water deficit. In addition, citric acid was uniquely up‐regulated by SNP associated with tricarboxylic acid (TCA) cycle under water stress. GABA specially induced the accumulation of GABA, glycine, methionine, and aconitic acid related to GABA shunt, amino acids metabolism, and TCA cycle in response to water stress. MAS uniquely enhanced the accumulation of asparagine, galactose, and D‐pinitol in association with amino acids and sugars metabolism under water stress. SNP‐, GABA‐, and MAS‐induced changes of metabolic profiles and associated metabolic pathways could contribute to enhanced stress tolerance via involvement in the TCA cycle for energy supply, osmotic adjustment, antioxidant defense, and signal transduction for stress defense in white clover.     

Computationally designed β‐turn foldamers of γ‐peptides based on 2‐(aminomethyl)cyclohexanecarboxylic acid

The γ‐peptide β‐turn structures have been designed computationally by the combination of chirospecific γ 2 , 3 ‐residues of 2‐(aminomethyl)cyclohexanecarboxylic acid (γAmc₆) with a cyclohexyl constraint on the C^α?C^β bond using density functional methods in water. The chirospecific γAmc₆ dipeptide with the (2S,3S)‐(2R,3R) configurations forms a stable turn structure in water, resembling a type II′ turn of α‐peptides, which can be used as a β‐turn motif in β‐hairpins of Ala‐based α‐peptides. The γAmc₆ dipeptide with homochiral (2S,3S)‐(2S,3S) configurations but different cyclohexyl puckerings shows the capability to be incorporated into one of two β‐turn motifs of gramicidin S. The overall structure of this gramicidin S analogue is quite similar to the native gramicidin S with the same patterns and geometries of hydrogen bonds. Our calculated results and the recently observed results may imply the wider applicability of chirospecific γ‐peptides with a cyclohexyl constraint on the backbone to form various peptide foldamers. © 2012 Wiley Periodicals, Inc. Biopolymers 97:1018–1025, 2012.     

Metabolic regulation of polyamines and γ‐aminobutyric acid in relation to spermidine‐induced heat tolerance in white clover

• Heat stress decreases crop growth and yield worldwide. Spermidine (Spd) is a small aliphatic amine and acts as a ubiquitous regulator for plant growth, development and stress tolerance.
• Objectives of this study were to determine effects of exogenous Spd on changes in endogenous polyamine (PA) and γ‐aminobutyric acid (GABA) metabolism, oxidative damage, senescence and heat shock protein (HSP) expression in white clover subjected to heat stress. Physiological and molecular methods, including colorimetric assay, high performance liquid chromatography and qRT‐PCR, were applied.
• Results showed that exogenous Spd significantly alleviated heat‐induced stress damage. Application of Spd not only increased endogenous putrescine, Spd, spermine and total PA accumulation, but also accelerated PA oxidation and improved glutamic acid decarboxylase activity, leading to GABA accumulation in leaves under heat stress. The Spd-pretreated white clover maintained a significantly higher chlorophyll (Chl) content than untreated plants under heat stress, which could be related to the roles of Spd in up‐regulating genes encoding Chl synthesis (PBGD and Mg‐CHT) and maintaining reduced Chl degradation (PaO and CHLASE) during heat stress. In addition, Spd up‐regulated HSP70, HSP70B and HSP70‐5 expression, which might function in stabilizing denatured proteins and helping proteins to folding correctly in white clover under high temperature stress.
• In summary, exogenous Spd treatment improves the heat tolerance of white clover by altering endogenous PA and GABA content and metabolism, enhancing the antioxidant system and HSP expression and slowing leaf senescence related to an increase in Chl biosynthesis and a decrease in Chl degradation during heat stress.

     

Accumulation patterns of endogenous β‐aminobutyric acid during plant development and defence in Arabidopsis thaliana

• We recently discovered that β‐aminobutyric acid (BABA), a molecule known for its ability to prime defences in plants, is a natural plant metabolite. However, the role played by endogenous BABA in plants is currently unknown. In this study we investigated the systemic accumulation of BABA during pathogen infection, levels of BABA during plant growth and development and analysed mutants possibly involved in BABA transport or regulation.
• BABA was quantified by LC‐MS using an improved method adapted from a previously published protocol. Systemic accumulation of BABA was determined by analysing non‐infected leaves and roots after localised infections with Plectosphaerella cucumerina or Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpt2. The levels of BABA were also quantified in different plant tissues and organs during normal plant growth, and in leaves during senescence. Mutants affecting amino acid transport (aap6, aap3, prot1 and gat1), γ‐aminobutyric acid levels (pop2) and senescence/defence (cpr5‐2) were analysed.
• BABA was found to accumulate only locally after bacterial or fungal infection, with no detectable increase in non‐infected systemic plant parts. In leaves, BABA content increased during natural and induced senescence. Reproductive organs had the highest levels of BABA, and the mutant cpr5‐2 produced constitutively high levels of BABA.
• Synthetic BABA is highly mobile in the receiving plant, whereas endogenous BABA appears to be produced and accumulated locally in a tissue‐specific way. We discuss a possible role for BABA in age‐related resistance and propose a comprehensive model for endogenous and synthetic BABA.

     

Conformation of poly(γ‐glutamic acid) in aqueous solution

Local conformation and overall conformation of poly(γ‐DL‐glutamic acid) (PγDLGA) and poly(γ‐L‐glutamic acid) (PγLGA) in aqueous solution was studied as a function of degree of ionization ε by ¹H‐NMR, circular dichroism, and potentiometric titration. It was clarified that their local conformation is represented by random coil over an entire ε range and their overall conformation is represented by expanded random‐coil in a range of ε > ε^*, where ε^* is about 0.3, 0.35, 0.45, and 0.5 for added‐salt concentration of 0.02M, 0.05M, 0.1M, and 0.2M, respectively. In a range of ε < ε^*, however, ε dependence of their overall conformation is significantly differentiated from each other. PγDLGA tends to aggregate intramolecularly and/or intermolecularly with decreasing ε, but PγLGA still behaves as expanded random‐coil. It is speculated that spatial arrangement of adjacent carboxyl groups along the backbone chain essentially affects the overall conformation of PγGA in acidic media. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 191–198, 2016.     

Role of microRNA‐155 in modifying neuroinflammation and γ‐aminobutyric acid transporters in specific central regions after post‐ischaemic seizures

In the central nervous system, interleukin (IL)‐1β, IL‐6 and tumour necrosis factor (TNF)‐α have a regulatory role in pathophysiological processes of epilepsy. In addition, γ‐aminobutyric acid (GABA) transporter type 1 and type 3 (GAT‐1 and GAT‐3) modulate the levels of extracellular GABA in involvement in the neuroinflammation on epileptogenesis. Thus, in the current report we examined the effects of inhibiting microRNA‐155 (miR‐155) on the levels of IL‐1β, IL‐6 and TNF‐α, and expression of GAT‐1 and GAT‐3 in the parietal cortex, hippocampus and amygdala of rats with nonconvulsive seizure (NCS) following cerebral ischaemia. Real time RT‐PCR, ELISA and Western blot analysis were used to examine the miR‐155, proinflammatory cytokines (PICs) and GAT‐1/GAT‐3 respectively. With induction of NCS, the levels of miR‐155 were amplified in the parietal cortex, hippocampus and amygdala and this was accompanied with increases of IL‐1β, IL‐6 and TNF‐α. In those central areas, expression of GAT‐1 and GAT‐3 was upregulated; and GABA was reduced in rats following NCS. Intracerebroventricular infusion of miR‐155 inhibitor attenuated the elevation of PICs, amplification of GAT‐1 and GAT‐3 and impairment of GABA. Furthermore, inhibition of miR‐155 decreased the number of NCS events following cerebral ischaemia. Inhibition of miR‐155 further improved post‐ischaemia‐evoked NCS by altering neuroinflammation‐GABA signal pathways in the parietal cortex, hippocampus and amygdala. Results suggest the role of miR‐155 in regulating post‐ischaemic seizures via PICs‐GABA mechanisms.     

Intracellular chloride and calcium transients evoked by γ‐aminobutyric acid and glycine in neurons of the rat inferior colliculus

Microfluorometric recordings showed that the inhibitory neurotransmitters γ‐aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl⁻ concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin‐perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO and characterized by a continuous shift of their reversal potential (E_GABA/gly) in the positive direction. In HCO‐buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca²⁺ concentration. Ca²⁺ transients occurred only with large depolarizations and were blocked by Cd²⁺, suggesting an activation of voltage‐gated Ca²⁺ channels. However, in the absence of HCO, only a small rise, if any, in the intracellular Ca²⁺ concentration could be evoked by GABA or glycine. We suggest that the activation of GABA_A or glycine receptors results in an acute accumulation of Cl⁻ that is enhanced by the depolarization owing to HCO efflux, thus shifting E_GABA/gly to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca²⁺ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 386–396, 1999