Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: Evidence for an alcohol receptor?

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a “cutoff” is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the α subunit. We now demonstrate that these residues in the glycine α1 and the γ-aminobutyric acid ρ1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine α1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the γ-aminobutyric acid ρ1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: The proline glycine and methionine-rich motif

Pre-mRNA splicing requires the bridging of the 5′ and 3′ ends of the intron. In yeast, this bridging involves interactions between the WW domains in the splicing factor PRP40 and a proline-rich domain in the branchpoint binding protein, BBP. Using a proline-rich domain derived from formin (a product of the murine limb deformity locus), we have identified a family of murine formin binding proteins (FBP’s), each of which contains one or more of a special class of tyrosine-rich WW domains. Two of these WW domains, in the proteins FBP11 and FBP21, are strikingly similar to those found in the yeast splicing factor PRP40. We show that FBP21 is present in highly purified spliceosomal complex A, is associated with U2 snRNPs, and colocalizes with splicing factors in nuclear speckle domains. Moreover, FBP21 interacts directly with the U1 snRNP protein U1C, the core snRNP proteins SmB and SmB′, and the branchpoint binding protein SF1/mBBP. Thus, FBP21 may play a role in cross-intron bridging of U1 and U2 snRNPs in the mammalian A complex.

Modulation of N-methyl-d-aspartate receptor function by glycine transport

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-d-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.

The C9 methyl group of retinal interacts with glycine-121 in rhodopsin

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor–ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor–ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly121 was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark—i.e., without chromophore isomerization—was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly121 and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.

Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes

Na+/Cl−-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [35S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na+/Cl−-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.

Characteristics of glycine receptors expressed by embryonic rat brain mRNAs

A study was made of glycine (Gly) and γ-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-day-old rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 ± 35, 346 ± 28, and 389 ± 22 nA, whereas the GABA-currents (1 mM GABA) were all ≤40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC50 for Gly was 611 ± 77 μM for encephalon, 661 ± 28 μM for midbrain, and 506 ± 18 μM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all ≈2. Strychnine inhibited all of the Gly-currents, with an IC50 of 56 ± 3 nM for encephalon, 97 ± 4 nM for midbrain, and 72 ± 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca2+ concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription–PCR studies showed differences in the Gly receptor (GlyR) α-subunits expressed, whereas the β-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca2+ could play important functions during brain development.

Effects of Sulfanilamide and Methotrexate on 13C Fluxes through the Glycine Decarboxylase/Serine Hydroxymethyltransferase Enzyme System in Arabidopsis1

In C3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [α-13C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long half-life. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis.

The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.

The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site.

The guinea pig estrogen sulfotransferase gene has been cloned and compared to three other cloned steroid and phenol sulfotransferase genes (human estrogen sulfotransferase, human phenol sulfotransferase, and guinea pig 3 alpha-hydroxysteroid sulfotransferase). The four sulfotransferase genes demonstrate a common outstanding feature: the splice sites for their 3'-terminal exons are identically located. That is, the 3'-terminal exon splice sites involve a glycine that constitutes the N-terminal glycine of an invariably conserved GXXGXXK motif present in all steroid and phenol sulfotransferases for which primary structures are known. This consistency strongly suggests that all steroid and phenol sulfotransferase genes will be similarly spliced. The GXXGXXK motif forms the active binding site for the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate. Amino acid sequence alignment of 19 cloned steroid and phenol sulfotransferases starting with the GXXGXXK motif indicates that the 3'-terminal exon for each steroid and phenol sulfotransferase gene encodes a similarly sized C-terminal fragment of the protein. Interestingly, on further analysis of the alignment, three distinct amino acid sequence patterns emerge. The presence of the conserved functional GXXGXXK motif suggests that the protein domains encoded by steroid and phenol sulfotransferase 3'-terminal exons have evolved from a common ancestor. Furthermore, it is hypothesized that during the course of evolution, the 3'-terminal exon further diverged into at least three sulfotransferase subdivisions: a phenol or aryl group, an estrogen or phenolic steroid group, and a neutral steroid group.

Rizobactérias nativas da Caatinga com potencial para redução dos efeitos da seca em soja (Glycine max L.)

No semiárido brasileiro, a vegetação predominante é a Caatinga, bioma ainda pouco explorado, que apresenta plantas e micro-organismos com alta resistência aos períodos de seca imposto pelo clima. Os micro-organismos associados às plantas deste bioma, são capazes de desenvolver mecanismos de proteção celular contra o estresse hídrico, assim como proteção vegetal contra a dessecação. O presente estudo buscou compreender as rizobactérias associadas a Mimosa artemisiana a fim de selecionar bactérias tolerantes à seca com características de promover o crescimento de plantas sob condições de estresse hídrico, diminuindo assim, os efeitos adversos impostos pela seca. As amostras de solo rizosférico foram coletadas ao longo da Caatinga, englobando os estados da BA e PE, totalizando quatro pontos de coleta. Com o uso de metodologias dependentes de cultivo, foi isolado bactérias com algumas características de promoção de crescimento de plantas diretos e/ou indiretos, como produção de AIA e fixação de nitrogênio. Além disso, linhagens capazes de crescer em meio com reduzida atividade de água e com mecanismos de proteção contra a dessecação, como, produção de EPS, biofilme, produção da ACC deaminase e indução de resistência sistêmica através das enzimas peroxidase e polifenoloxidase. Uma linhagem de Paenibacillus sp. e outra de Bacillus sp. foram capazes de promover o crescimento de soja sob condições de estresse hídrico, aumentando alguns parâmetros vegetais como, parte aérea e sistema radicular analisados.

Synthesis of α,β-diamino acid derivatives via asymmetric Mannich reactions of glycine imino esters catalyzed by a chiral phosphoramidite·silver complex

AgOTf·phosphoramidite complexes efficiently catalyze the enantioselective Mannich-type reaction between benzophenone-imine glycine methyl ester and N-tosyl aldimines in the absence of a base. The corresponding syn-adducts, which are the direct precursors of α,β-diamino acids, are obtained with moderate to good syn-diastereoselectivities (up to 9:1) and high enantioselectivities (up to 99% ee).

Investigating the role of glycine in astrocyte signalling using calcium imaging of rat primary cultures of cortical astrocytes

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

LeProT1, a Transporter for Proline, Glycine Betaine, and γ-Amino Butyric Acid in Tomato Pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70 of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

A revision of the genus Glycine and its immediate allies /

Erratum inserted.

Tissue-specific gene expression in soybean (Glycine max) detected by cDNA microarray analysis

We have constructed cDNA microarrays for soybean (Glycine max L. Merrill), containing approximately 4,100 Unigene ESTs derived from axenic roots, to evaluate their application and utility for functional genomics of organ differentiation in legumes. We assessed microarray technology by conducting studies to evaluate the accuracy of microarray data and have found them to be both reliable and reproducible in repeat hybridisations. Several ESTs showed high levels (>50 fold) of differential expression in either root or shoot tissue of soybean. A small number of physiologically interesting, and differentially expressed sequences found by microarray analysis were verified by both quantitative real-time RT-PCR and Northern blot analysis. There was a linear correlation (r(2) = 0.99, over 5 orders of magnitude) between microarray and quantitative real-time RT-PCR data. Microarray analysis of soybean has enormous potential not only for the discovery of new genes involved in tissue differentiation and function, but also to study the expression of previously characterised genes, gene networks and gene interactions in wild-type, mutant or transgenic; plants.