Characterization of a gamma-aminobutyric acid transport system of rhizobium leguminosarum bv. viciae 3841

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

Supramolecular beta-sheet and nanofibril formation by self-assembling tripeptides containing an N-terminally located gamma-aminobutyric acid residue

Three terminally protected tripeptides Boc-gamma-Abu-Val-Leu-OMe 1, Boc-gamma-Abu-Leu-Phe-OMe 2 and Boc-gamma-Abu-Val-Tyr-OMe 3 (gamma-Abu = gamma-aminobutyric acid) each containing an N-terminally positioned gamma-aminobutyric acid residue have been synthesized, purified and studied. FT-IR studies of all these peptides revealed that these peptides form intermolecularly hydrogen bonded supramolecular beta-sheet structures. Peptides 1, 2 and 3 adopt extended backbone beta-strand molecular structures in crystals. Crystal packing of all these peptides demonstrates that these beta-strand structures self-assemble to form intermolecularly H-bonded parallel beta-sheet structures. Peptide 3 uses a side chain tyrosyl -OH group as an additional hydrogen bonding functionality in addition to the backbone CONH groups to pack in crystals. Transmission electron microscopic studies of all peptides indicate that they self-assemble to form nanofibrillar structures of an average diameter of 65 nm. These peptide fibrils exhibit amyloid-like behavior as they bind to a physiological dye Congo red and show a characteristic green-gold birefringence under polarizing microscope.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

m-aminobenzoic acid inserted beta-turn in acyclic tripeptides: a peptidomimetic design

X-ray diffraction studies show that peptides Boc-Leu-Aib-m-ABA-OMe (I) (Aib, alpha-aminoisobutyric acid; m-ABA, meta-aminobenzoic acid) and Boc-Phe-Aib-m-ABA-OMe, (II) adopt a type-II beta-turn conformation, solely stabilized by co-operative steric interactions amongst the amino acid residues. This type of U-turn without any intramolecular hydrogen bonding is generally referred to as an open turn. Although there are some examples of constrained cyclic peptides in which o-substituted benzenes have been inserted to mimic the turn region of the neurotrophin, a nerve growth factor, peptides I and II present novel two examples where m-aminobenzoic acid has been incorporated in the beta-turn of acyclic tripeptides. The result also demonstrates the first crystallographic evidence of a beta-turn structure containing an inserted m-aminobenzoic acid, which can be considered as a rigid gamma-aminobutyric acid with an all-trans extended configuration. (c) 2005 Elsevier Ltd. All rights reserved.

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized L-histidine, L-glutamine, L-proline, L-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.

Nerve terminal GABAA receptors activate Ca2+/calmodulin-dependent signaling to inhibit voltage-gated Ca2+ influx and glutamate release

-Aminobutyric acid type A (GABAA) receptors, a family of Cl-permeable ion channels, mediate fast synaptic inhibition as postsynaptically enriched receptors for -aminobutyric acid at GABAergic synapses. Here we describe an alternative type of inhibition mediated byGABAA receptors present on neocortical glutamatergic nerve terminals and examine the underlying signaling mechanism(s). By monitoring the activity of the presynaptic CaM kinase II/synapsin I signaling pathway in isolated nerve terminals, we demonstrate that GABAA receptor activation correlated with an increase in basal intraterminal [Ca2]i. Interestingly, this activation of GABAA receptors resulted in a reduction of subsequent depolarization-evoked Ca2 influx, which thereby led to an inhibition of glutamate release. To investigate how the observed GABAA receptor-mediated modulation operates, we determined the sensitivity of this process to the Na-K-2Cl cotransporter 1 antagonist bumetanide, as well as substitution of Ca2 with Ba2, or Ca2/calmodulin inhibition by W7. All of these treatments abolished the modulation by GABAA receptors. Application of selective antagonists of voltage-gated Ca2 channels (VGCCs) revealed that the GABAA receptor-mediated modulation of glutamate release required the specific activity of L- and R-type VGCCs. Crucially, the inhibition of release by these receptors was abolished in terminals isolated from R-type VGCC knock-out mice. Together, our results indicate that a functional coupling between nerve terminal GABAA receptors and L- or R-type VGCCs is mediated by Ca2/calmodulin-dependent signaling. This mechanism provides a GABA-mediated control of glutamatergic synaptic activity by a direct inhibition of glutamate release.

Can a consecutive double turn conformation be considered as a peptide based molecular scaffold for supramolecular helix in the solid state?

Helices and sheets are ubiquitous in nature. However, there are also some examples of self-assembling molecules forming supramolecular helices and sheets in unnatural systems. Unlike supramolecular sheets there are a very few examples of peptide sub-units that can be used to construct supramolecular helical architectures using the backbone hydrogen bonding functionalities of peptides. In this report we describe the design and synthesis of two single turn/bend forming peptides (Boc-Phe-Aib-Ile-OMe 1 and Boc-Ala-Leu-Aib-OMe 2) (Aib: alpha-aminoisobutyric acid) and a series of double-turn forming peptides (Boc-Phe-Aib-IIe-Aib-OMe 3, Boc-Leu-Aib-Gly-Aib-OMe 4 and Boc-gamma-Abu-Aib-Leu-Aib-OMe 5) (gamma-Abu: gamma-aminobutyric acid). It has been found that, in crystals, on self-assembly, single turn/bend forming peptides form either a supramolecular sheet (peptide 1) or a supramolecular helix (peptide 2). unlike self-associating double turn forming peptides, which have only the option of forming supramolecular helical assemblages. (c) 2005 Elsevier Ltd. All rights reserved.

beta-Sheet mediated self-assembly of dipeptides of omega-amino acids and remarkable fibrillation in the solid state

Single crystal X-ray diffraction studies show that the extended structure of dipeptide I Boc-beta-Ala-m-ABA-OMe (m-ABA: meta-aminobenzoic acid) self-assembles in the solid state by intermolecular hydrogen bonding to create an infinite parallel P-sheet structure. In dipeptide II Boc-gamma-Abu-m-ABA-OMe (gamma-Abu: gamma-aminobutyric acid), two such parallel beta-sheets are further cross-linked by intermolecular hydrogen bonding through m-aminobenzoic acid moieties. SEM (scanning electron microscopy) studies reveal that both the peptides I and II form amyloid-like fibrils in the solid state. The fibrils are also found to be stained readily by Congo red, a characteristic feature of the amyloid fiber whose accumulation causes several fatal diseases such as Alzheimer's, prion-protein etc.

Nanozipper formation in the solid state from a self-assembling tripeptide with a single tryptophan residue

The self-assembly of a terminally protected tripeptide Boc-gamma-Abu(1)-Ala(2)-Trp(3)-OMe (gamma-Abu = gamma-aminobutyric acid) I results in the formation of a nanostructured supramolecular zipper through various non-covalent interactions in the crystal in which the indole side-chain of the Trp(3) residue plays a key role via N-H...pi interactions. (c) 2006 Published by Elsevier Ltd.

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

Peptidomimetic design of unusual turns by incorporating flexible and rigid omega-amino acids simultaneously

The tripeptides Boc-Gly-Aib-m-ABA-OMe (I), Boc-beta Ala-Aib-m-ABA-OMe (II) and Boc-gamma Abu-Aib-rn-ABA-OMe (III) (Aib: alpha-aminoisobutyric acid, beta Ala: beta-alanine, gamma Abu: gamma-aminobutyric acid, m-ABA: meta-aminobenzoic acid) with homologated amino acids at the N-terminus, the rigid gamma-amino acid m-ABA at the C-terminus and the helicogenic Aib at the central position have been chosen to create unusual turns. Single crystal X-ray diffraction studies, solvent dependent NMR titrations and 2D NMR analysis reveal that peptides II and III adopt unusual turns of 11- and 12-membered rings stabilized by modified 4 -> 1 type intramolecular hydrogen bonds. Solution phase studies indicate that peptide I exists in the beta-turn conformation stabilized by 10-membered intramolecular hydrogen bonding.

Nano structures through self-assembly of protected hydrophobic amino acids: encapsulation of rhodamine B dye by proline-based nanovesicles

The development of novel molecules for the creation of nanometer structures with specific properties has been the current interest of this research. We have developed a set of molecules from hydrophobic omega- and alpha-amino acids by protecting the -NH(2) with Boc (t-butyloxycarbonyl) group and -CO(2)H with para-nitroanilide such as BocHN-Xx-CONH-(p-NO(2))center dot C(6)H(4), where Xx is gamma-aminobutyric acid (gamma-Abu), (L)-isoleucine, alpha-aminoisobutyric acid, proline, etc. These molecules generate various nanometer structures, such as nanofibrils, nanotubes and nanovesicles, in methanol/water through the self-assembly of bilayers in which the nitro benzene moieties are stacked in the middle and the Boc-protected amino acids parts are packed in the outer surface. The bilayers can be further stacked one over the other through hydrophobic interactions to form multilayer structure, which helps to generate different kinds of nanoscopic structures. The formation of the nanostructures has been facilitated through the participation of various noncovalent interactions, such as hydrophobic interactions, hydrogen bonding and aromatic p-stacking interactions. Fluorescence microscopy and UV studies reveal that the nanovesicles generated from pro-based molecule can encapsulate dye molecules which can be released by addition of acid (at pH 2). These single amino acid based molecules are both easy to synthesize and cost-effective and therefore offer novel scaffolds for the future design of nanoscale structures.

Ketamine, propofol, and the EEG: A neural field analysis of HCN1-mediated interactions

Ketamine and propofol are two well-known, powerful anesthetic agents, yet at first sight this appears to be their only commonality. Ketamine is a dissociative anesthetic agent, whose main mechanism of action is considered to be N-methyl-D-aspartate (NMDA) antagonism; whereas propofol is a general anesthetic agent, which is assumed to primarily potentiate currents gated by γ-aminobutyric acid type A (GABAA) receptors. However, several experimental observations suggest a closer relationship. First, the effect of ketamine on the electroencephalogram (EEG) is markedly changed in the presence of propofol: on its own ketamine increases θ (4–8 Hz) and decreases α (8–13 Hz) oscillations, whereas ketamine induces a significant shift to beta band frequencies (13–30 Hz) in the presence of propofol. Second, both ketamine and propofol cause inhibition of the inward pacemaker current Ih, by binding to the corresponding hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1) subunit. The resulting effect is a hyperpolarization of the neuron’s resting membrane potential. Third, the ability of both ketamine and propofol to induce hypnosis is reduced in HCN1-knockout mice. Here we show that one can theoretically understand the observed spectral changes of the EEG based on HCN1-mediated hyperpolarizations alone, without involving the supposed main mechanisms of action of these drugs through NMDA and GABAA, respectively. On the basis of our successful EEG model we conclude that ketamine and propofol should be antagonistic to each other in their interaction at HCN1 subunits. Such a prediction is in accord with the results of clinical experiment in which it is found that ketamine and propofol interact in an infra-additive manner with respect to the endpoints of hypnosis and immobility.

Genetic variation in GABRB3 is associated with Asperger syndrome and multiple endophenotypes relevant to autism.

BACKGROUND: Autism spectrum conditions (ASC) are associated with deficits in social interaction and communication, alongside repetitive, restricted, and stereotyped behavior. ASC is highly heritable. The gamma-aminobutyric acid (GABA)-ergic system has been associated consistently with atypicalities in autism, in both genetic association and expression studies. A key component of the GABA-ergic system is encoded by the GABRB3 gene, which has been previously implicated both in ASC and in individual differences in empathy. METHODS: In this study, 45 genotyped single nucleotide polymorphisms (SNPs) within GABRB3 were tested for association with Asperger syndrome (AS), and related quantitative traits measured through the following tests: the Empathy Quotient (EQ), the Autism Spectrum Quotient (AQ), the Systemizing Quotient-Revised (SQ-R), the Embedded Figures Test (EFT), the Reading the Mind in the Eyes Test (RMET), and the Mental Rotation Test (MRT). Two-loci, three-loci, four-loci haplotype analyses, and one seven-loci haplotype analysis were also performed in the AS case--control sample. RESULTS: Three SNPs (rs7180158, rs7165604, rs12593579) were significantly associated with AS, and two SNPs (rs9806546, rs11636966) were significantly associated with EQ. Two SNP-SNP pairs, rs12438141-rs1035751 and rs12438141-rs7179514, showed significant association with variation in the EFT scores. One SNP-SNP pair, rs7174437-rs1863455, was significantly associated with variation in the MRT scores. Additionally, a few haplotypes, including a 19 kb genomic region that formed a linkage disequilibrium (LD) block in our sample and contained several nominally significant SNPs, were found to be significantly associated with AS. CONCLUSION: The current study confirms the role of GABRB3 as an important candidate gene in both ASC and normative variation in related endophenotypes.